A Quantitative Proteomics Study of the Additive Effect of Inflammatory Cytokines and Injurious Compression on Cartilage Damage

Home , ADAMTS2, Chondroitin ABC lyase, Proteoglycan 4, Thymosin, Type XVIII collagen

A QUANTITATIVE PROTEOMICS STUDY OF THE ADDITIVE EFFECT OF INFLAMMATORY CYTOKINES AND INJURIOUS COMPRESSION ON CARTILAGE DAMAGE

JMASSACHUSniTS INSTITUTE OF TEcr-, Krishnakumar Swaminathan JUL 142011 B.Tech Biotechnology Indian Institute of Technology Madras, 2006

ARCHIVES Submitted to the Department of Biological Engineering in Partial Fulfillment of the Requirements for the Degree of

Masters of Science in Biological Engineering at the

Massachusetts Institute of Technology February 2011

V( Signature of Author Krishnakumar Swaminathan Department of Biological Engineering

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Certified by -r - 1 4r Alan J. qrdznsky, th7TAdvs r Professor of Biological, Electrical and Mechanical Engi ~g

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Accepted by Forest M. White, Graduate Program Committee Department of Biological Engineering ABSTRACT

Objectives: 1) To perform a quantitative comparison of proteins released to media on combination with cytokine (IL-1p or TNF-a) and Injury as compared to either treatment alone, and to thus identify proteins which may be responsible for the synergism seen between cytokine and injury in causing catabolism of cartilage in vitro. 2) To identify proteins which contribute most to some commonly observed phenotypes on treatment of cartilage with cytokine or injury or both.

Methods : Cartilage explants from calves were treated with (i)IL-1 (10 ng/ml), (ii)TNF- a (100 ng/ml), (iii)Injurious compression (50% strain at 100%/sec) and IL-1p (10 ng/ml) or (iv)Injurious compression (50% strain at 100%/sec) and TNF-a(10 ng/ml), cultured for 5 days post treatment, and the pooled media collected, labeled with one of four iTRAQ labels and subjected to nano-2D-LC/MS/MS on a quadrupole time of flight instrument. Peptides were identified and quantified using Protein PilotTM, and MATLAB scripts used to obtain protein ratios. These results were analyzed using different statistical techniques. Data from two iTRAQ experiments were combined to generate data for all possible injury and cytokine treatment conditions, and proteins on which injury and cytokines acted synergistically identified. PLSR analysis was performed using Unscrambler@X software with the combined data set to determine which proteins are most relevant to some observed phenotypes. The phenotypes chosen were sGAG released to media in 5 days post treatment, proline and sulfate incorporation rates on day 6 post treatment, and nitrite accumulation in media in 5 days post treatment

Results and Discussion: TNF-a+injury and IL-1p +injury treatment conditions show a very high correlation with each other. Most cytosolic, ER lumen and nuclear protein levels are highly elevated with both cytokine+injury conditions, while ECM proteins are either highly down regulated or marginally elevated. Many collagen telopeptides are down regulated, possibly indicating reduced anabolism. However, attempts at repair exist, as shown by increased levels of TGF- and activin A, and reduced levels of LTBP1. Also, biglycan and lumican, SLRPs known to be involved in early development are significantly increased, possibly indicating repair attempts. Other SLRPs such as PRELP and chondroadherin are also highly elevated, with one or both injury+cytokine treatments. While MMPs are mildly down regulated or remain the same, ADAMTS1 increases with TNF-a+injury, indicating increased catabolism. Among ECM structural proteins, COMP shows high down regulation with TNF-a+injury, possibly due to reduced synthesis. Proenkephalin, a signaling molecule possibly involved in tissue/repair and apoptosis, AIMPI, a multifunctional proapoptotic, inflammatory and pro-repair cytokine and Annexin A5, a protein indicating mineralization and apoptosis are all highly elevated with cytokine+injury indicating heightened apoptosis and/or repair.

When results of two 4-plex iTRAQ experiments are combined to obtain data for all possible combinations of injury and cytokine, we again find a very high correlation between TNF-a+injury and IL-1 +injury (-95%), slightly higher than the correlation between TNF-c alone and IL-1$ alone (-90%), and much higher than the correlation of either cytokine+injury condition with cytokine alone (-70%) or injury alone (-75%). This shows that IL-1P and TNF-a in combination with injury act through very similar pathways in chondrocytes to produce their effect on cartilage tissue. TNF-a and injury were seen to act synergistically in a positive fashion on aggrecan, CILP-2, COL6A3 and histone H4, and in a negative fashion on SPARC and IGFBP7, suggesting that these proteins may be involved in causing synergism between injury and cytokine in releasing sGAG to the media. A PLSR analysis shows that SPARC and IGFBP7 project close to proline and sulfate incorporation, and far away from sGAG, indicating that SPARC and IGFBP7 may be proteins involved in anabolism. The highest phenotype-protein positive correlations obtained using PLSR are sGAG with Perlecan, SAA3, Complement factor B, CILP-2 and pleiotropin, indicating that all these 5 proteins are associated strongly with catabolism and can serve as markers of catabolism. The correlation of inflammatory proteins SAA3 and complement factor B with sGAG indicates the role of inflammation with catabolism.

Conclusion: The combination of injury and cytokine affects tissue differently at a molecular level as compared to either chemical or mechanical stresses alone. Increased catabolism and increased attempts at tissue repair are observed due to a combination of injury and cytokine, and a combination of injury and cytokine may thus serve as a useful model to study OA in vitro. ACKNOWLEDGEMENTS

I am grateful to the Biological Engineering Department and MIT for giving me the best of education, and teaching me what true scientific research and thinking is all about. My sincere thanks to Linda Bragman and Han-Hwa Hung for help in many small ways and to

Dalia Fares for taking care of administrative issues. Dr. Anna Stevens performed the cartilage explantation experiments, and Dr.Ramesh Indrakanti ran the Mass Spec, my grateful thanks to them. My heartfelt thanks to Anna, who guided me into this work, and was always there for any questions I had (and there were many!). And most important of all, I am indebted to Prof. Alan Grodzinsky who stood by me through thick and thin, and was a constant source of motivation and inspiration. TABLE OF CONTENTS

A bstract...... 2

Acknowledgements...... 4

L ist of Figures...... 7

L ist of T ables...... 8

Chapter 1: Introduction ...... 9 1.1 Arthritis - An Overview...... 9 1.2 Osteoarthritis : Pathology, Symptoms and Treatment...... 9 1.3 A rticular Cartilage...... 10 1.4 Cartilage Components...... 10 1.5 Post-traumatic GA...... 18 1.6 Cytokines in GA ...... 20 1.7 In vitro models combining injury and cytokine...... 23 1.8 Thesis Objectives...... 23

Chapter 2: M ethods...... 25 2.1 O verview ...... 25 2.2 Cartilage explantation, Mechanical Injury and Cytokine T reatm ent...... 25 2.3 sGAG removal, trypsin digestion and iTRAQ labeling.....26 2.4 SCX chromatography and LC/MS/MS...... 28 2.5 Data Analysis...... 29 2.6 Comparison of biological duplicates and final protein list com pilation...... 31 2.7 Global proteomic analysis and clustering...... 31 2.8 Merging data between two iTRAQ experiments...... 32 2.9 Finding proteins on which injury and cytokines act synergistically...... 33 2.10 Partial Least Square Regression (PLSR)...... 33

Chapter 3: R esults...... 35 3.1 Reproducibility between biological replicates...... 35 3.2 Global proteomic analysis...... 36 3.3 Effect of combination of cytokine and injury on specific p rotein s...... 37 3.4 Effect of combination of injury and cytokine as opposed to either treatment alone...... 40 3.5 Partial Least Square Regression (PLSR) between protein ratios and phenotypes...... 43

Chapter 4: Discussion 4.1 Methodology for correcting for multiple comparisons - The Benjamini Hochberg Procedure...... 63 4.2 Variation between biological replicates...... 63 4.3 A global proteomic analysis of the effect of cytokine+injury...... 64 4.4 Effect of cytokine+injury on specific protein classes...... 66 4.5 Effect of combination of cytokine and injury as opposed to either treatment alone...... 86 4.6 Partial Least Square Regression (PLSR) Analysis...... 92 4.7 Limitations of this study...... 97

Chapter 5: Conclusion...... 99

References...... 103

A ppendix ...... 124 LIST OF FIGURES

Figure 1: Histograms of biological replicate comparisons...... 45

Figure 2: Projection plot of log transformed protein ratios with the axes as (IL-iI+injury/IL- 1P) and (TNF-a+injury/IL- I1P) as the axes...... 47

Figure 3: 3D plot and 2D projections of log transformed protein ratios, clustered by K-means clustering with (TNF-a/IL-11), (IL-1 +injury/LL-lp) and (TNF-a+injury/IL- 11P) as the axes...... 48

Figure 4: Profile of means of log transformed protein ratios sorted by clusters identified by K-means clustering...... 49

Figure 5: Profile of means of log transformed protein ratios sorted by cellular location...... 50

Figure 6: Behavior of different protein classes on cytokine+injury treatm ent...... 5 1

Figure 7: Histograms of comparison of log transformed protein ratios obtained by two iTRAQ experiments...... 54

Figure 8: PLSR plots : Explained variance in protein ratios and phenotypes by the latent vectors, projection plots of treatment conditions, log transformed protein ratios and log transformed phenotype ratios on the first tw o latent vectors...... 57 LIST OF TABLES

Table 1: Matrix of correlation coefficients between TNF-ac alone, IL-1p +injury and TNF-x+injury treatment conditions...... 46

Table 2: Matrix of correlation coefficients between IL-lIP alone, TNF-a alone, injury alone, IL- I+injury and TNF-a+injury conditions...... 55

Table 3: Proteins on which TNF-a and injury act in a positive or negative synergistic fashion...... 56

Table 4: List of protein-phenotype pairs showing the top 5 positive and negative correlations...... 60

Table 5: Effect of injury or cytokine or both on different phenotypes...... 60 INTRODUCTION

1.1 Arthritis: An Overview

The word "Arthritis" literally means inflammation of joint, and encompasses more than 100 different rheumatic diseases and conditions. It is the leading cause of disability in the US 1. Arthritis affects around 21.6% of the US population, and limits the activity of -40% of people having the disease 2 . It is projected to affect nearly 67 million people by 2030 3. Osteoarthritis (OA) is the most common form of arthritis and is estimated to affect nearly 27 million people currently 4. It poses an immense societal and economic burden, with OA alone estimated to cost $185.5 billion annually 5.

1.2 Osteoarthritis: Pathology, Symptoms and Treatment

Osteoarthritis is a condition representing an imbalance between degradation and repair involving the whole joint and its component parts, with secondary inflammatory changes in the synovium 6. Pathological features of OA include degradation of hyaline cartilage leading to loss of joint integrity, elevation in bone density at areas of cartilage loss (bony sclerosis), subchondral cyst formation and endochondral ossification

(osteophyte formation). The synovium, ligaments and neuromuscular apparatus may also show pathological changes. Symptoms of OA include joint pain, swelling and stiffness of joint and loss of mobility. Several factors increase the risk of OA, including body weight,

age, gender, physical activity, bone mineral density, injury and genetic factors 6

Pharmacological treatment of OA is usually symptomatic, with analgesics, NSAIDs and neutraceuticals like glucosamine and chondroitin sulfate used to combat inflammation and pain. Non-pharmacological interventions including rest, physical therapy and lifestyle changes seem to help, however, surgery is usually the only option in end stage disease. Common surgical procedures are osteotomy (joint realignment), arthroscopy

(removal of loose bodies and inflamed synovium, and smoothing irregular joint surfaces), arthrodesis (joint fusion) and arthroplasty (joint replacement). Total joint replacement is the recommended procedure for older patients experiencing significant pain and highly impaired mobility, however, the life time of the joint may not be more than 10-15 years.

No permanent cure currently exists for OA.

1.3 Articular Cartilage

Articular cartilage is a thin, smooth and stiff layer covering bone surfaces in synovial joints. It consists of an extensive hydrated extracellular matrix (ECM), maintained by a population of specialized cells, the chondrocytes, which can occupy as little as 2% of the total cartilage volume. It serves as a low-friction surface between the bones of the diarthrodial joint, and is responsible for load support, load distribution and joint motion. Cartilage is avascular, alymphatic and aneural, and the major source of nutrition for chondrocytes in adult articular cartilage is through diffusion from the synovial fluid 6

1.4 Cartilage Components

1.4.1 The Collagens

The major components of the ECM in cartilage are collagens and the large aggrecating proteoglycan aggrecan. Collagen accounts for two-third of the dry weight of cartilage, while collagen type II fibrils are responsible for the tensile strength of cartilage, extensive cross linking of the collagen fibrils is responsible for the mechanical strength of cartilage (for review 7). Collagen fibers also help counteract the swelling caused due to hydration of proteoglycans. Several different collagen types have been identified. In articular cartilage, Collagen types II, IX and XI form a fibrillar network which constitutes the structural framework of cartilage, in the form of an anisotropic and inhomogeneous mesh of fibers. While collagen type II is the predominant fibrillar collagen, collagen IX molecules have several cross-linking sites through which they interact with collagen type

II and other collagen type IX molecules. Collagen Type XI on the other hand is believed to form a template which constrains the lateral growth of type II collagen heterofibrils 8

Two predominant classes of proteinases are known to degrade collagens, the matrix metalloproteinases (MMPs) and the cathepsins, and cleavage of collagen by MMPs is an important feature of OA 9. Collagen type II synthesis is much lower in adult tissue as compared to immature cartilage; however, synthesis is seen to significantly increase after injury 10

Cartilage can be divided into four different depth dependent zones, superficial or tangential, intermediate, deep or radial and calcified, all of which have different orientations of their collagen fibrils. The inherent inhomogeneous nature of collagen fibrils along with depth dependent variation in their orientation is responsible for the anisotropic and non linear properties of cartilage in compression and tension

1.4.2 Aggrecan

Proteoglycans are a family of molecules with a central core protein, to which one or more glycosaminoglycan (GAG) chains are added post-translationally. GAG chains are large extended structures with highly charged sulfate and carboxylate groups, which dominate the properties of the proteins to which they are attached. Common GAG chains include Keratan sulfate (KS) and Chondroitin sulfate (CS). (for review 12). Aggrecan is the major proteoglycan in articular cartilage, where it associates with hyaluran and link protein to form large aggregates. The large fixed negative charge of these aggregates (due to the large number of GAG chains on aggrecan) causes an osmotic imbalance, and draws water from the surrounding areas, thus keeping cartilage hydrated. This hydration of cartilage by aggrecan influences nutrient and solute transport in cartilage. The high negative charge of aggrecan aggregates is the main reason for the compressive stiffness of cartilage (for review 13)

1.4.3 Other ECM Molecules

Besides the collagens and aggrecan, several other molecules are present in the

ECM, including thrombospondins, matrilins, cartilage intermediate layer proteins (CILP), fibulins, laminins, SPARC, fibronectin and members of the Small leucine rich proteins/proteoglycans (SLRP) family. Some of these proteins such as matrilins and laminins have structural roles, while others such as some thrombospondins and SPARC are involved in modulating cell function, tissue development and remodeling. The latter set of proteins are termed as matricellular proteins 1

Thrombospondins (TSPs) are multidomain proteins containing either three (TSP

1,2) or five (TSP 3,4 and 5) identical subunits. The thrombospondins identified in cartilage are TSP-1,3 and 5, of which TSP-5 (COMP) is dominant (for review 15). TSP-1,

TSP-2 and TSP-5 (COMP) function at the interface of the ECM and cell surface, thus regulating cellular behavior and hence can be said to be members of the class of 16,17 matricellular proteins 1,. To accomplish their roles in ECM remodeling as matricellular proteins, TSPs 1,2 and 5 interact with a wide variety of proteins and proteoglycans. TSP-

1 is the only thrombospondin which can activate TGF-, which leads to synthesis of collagens and other molecules. It is known to bind to heparin, perlecan, syndecans-1,3 and 4, versican, Collagen type V, fibrinogen and decorin. It also inhibits several proteases including MMPs and Cathepsin G 17. Cutaneous wounds in TSP-1 null mice showed delayed wound healing, reduced levels of active TGF-3, decreased collagen and reduced inflammatory response 18.

TSP-3 is expressed in early proliferative zone and hypertrophic zone in cartilage.

Its function has not yet been elucidated, while single knockouts of TSPs does not affect stature, mice that lack TSP-3,TSP-5 and Type IX collagen have 20% lesser limb length, suggesting a role for TSP-3 in ECM function 119

COMP, made of five identical domains, is one of the major non collagenous proteins composing cartilage ECM and comprises up to 1% cartilage wet weight.

Mutations in the COMP gene can cause inherited osteochondrodysplasias such as seudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) 20. COMP has been shown to bind to Collagen type I and II 2 and aggrecan 22. It is also known to interact with Type IX collagen 23 , fibronectin 24 and matrilins 25. Its interactions with several ECM structural molecules may serve to organize cartilage matrix and regulate the load bearing function of cartilage. COMP interacts with BMP-2 to regulate mesenchymal chondrogenesis in vitro 26. It aids chondrocyte attachment through interaction with integrins 27. Thus, it has roles in chondrocyte attachment, differentiation and ECM assembly. SPARC is a matricellular protein expressed highly in developing tissue compared to mature, and increased SPARC production is associated with adult tissue remodeling. It can bind to several different collagens, including collagens I, III, IV and V, and is possibly involved in ECM assembly and turnover 28

Perlecan is another molecule which plays an important role in the organization of the ECM through its interaction with many ECM molecules including fibronectin, nidogen-1 and nidogen-2, PRELP, collagen types XIII and XVIII (for review 29). It also plays a crucial role in cartilage development, possibly by interacting with and altering distributions of several growth factors 30

A family of non collagenous ECM proteins which may be involved in structural roles are the matrilins. There are four matrilins known currently, of which Matrilin-1 and

3 are almost exclusively present in cartilage (for review 31). In the adult knee, all matrilins are present in the growth plate and peripheral areas of articular cartilage.

Matrilin-1 binds to aggrecan and associates with collagen type II containing fibrils.

Matrilins -1, -3 and -4 form complexes with SLRPs biglycan and decorin 32 and bind to

COMP 25 . Matrilin-3 additionally interacts with collagen type IX 33. Matrilin-3 mutations have been associated with skeletal disorders such as Multiple epiphseal dysplasia (MED)

34 and bilateral hereditary micro-epiphseal dysplasia (BHMED) 35

Cartilage intermediate layer proteins 1 and 2 (CILP-1 and CILP-2) are two related structural proteins found in articular cartilage. CILP-1 is a monomeric glycoprotein predominantly expressed in the intermediate zone of cartilage 36. CILP-1 forms a functional feedback loop with TGF-, with TGF-p inducing CILP-1 mRNA expression, and CILP-1 in turn binding TGF- 37. CILP-2 is a protein which exhibits -50% homology with CILP-1, and its function is not yet clear 38.

Another set of proteins involved in collagen fibril formation are members of the

Small leucine rich proteoglycan (SLRP) family (for review 39). Studies by Vogel et al 40 were among the earliest to show that small dermatan sulfate proteoglycans inhibited fibrillogenesis in both Type I and Type II collagen from tendon and cartilage respectively in vitro. Since then, various SLRPs have been identified, including biglycan, decorin,

PRELP, chondroadherin, osteoadherin, fibromodulin and lumican, and probably have unique functions in collagen assembly, as knockout of any SLRP cannot be compensated by other SLRPs. SLRPs can also be categorized as matricellular proteins as different members of this family interact with different molecules in the ECM, and thus help in remodeling the ECM 41. Recent studies have identified that MMPs can cleave some

SLRPs at precise sites, and it has been postulated that the metabolites thus released may serve as bio-markers to monitor the progression of OA 42

1.4.4 Matrix proteases

Degradation of the ECM by various proteolytic enzymes is a primary feature of

OA. These proteinases can be classified based on the chemical group which participates in hydrolysis of the peptide bond. The primary extracellular proteinases are the metalloproteinases and the serine proteinases, both of which act at neutral pH. The metalloproteinases are distinguished by a highly conserved motif containing three histidines that bind zinc at the catalytic site, and a conserved methionine turn beneath the active site zinc. Metalloproteinases can further be divided into different groups based on structure, of which MMPs and the ADAMTSs are the most active in the cartilage ECM

The Matrix metalloproteinases (MMPs) are a family of enzymes that digest components of the ECM. Most MMPs have three domains, a prodomain which must be removed to activate the enzyme, a Zn** containing active domain where the substrate is catalyzed, and a hemopexin-like C-terminal domain which determines the substrate specificity of the enzyme 44. Members of the MMP family can be broadly divided into four groups based on their substrates and cellular location: (i) The collagenases (MMP-1,

-8, -13) cleave major fibrillar triple helical collagens into characteristic %/ and 1/4 length fragments. (ii) The gelatinases (MMP-2, -9) have high activity against gelatins (denatured component chains of collagen) and Type IV collagen, the predominant constituent of basement membrane. However, they have broad substrate specificities and can also cleave fibrillar collagens and other ECM molecules such as fibronectin and decorin (iii)

The stromelysins (MMP-3,10,11) cleave a wide variety of ECM and non ECM substrates including aggrecan, laminin, fibronectin, etc and (iv) The membrane type MMPs (for review 9). It is highly likely that MMPs are responsible for digestion of collagens in vivo in OA, as MMPs digest collagens in vitro, are expressed in OA tissue at the correct time necessary to cause degradation, and activities altering expression of MMPs alter the progression of OA 44. However, MMPs may have more complex roles than pure catabolism in normal and OA cartilage, as they are known to regulate matrix biology by means of varied functions including release of growth factors, enhancing/inhibiting angiogenesis and modification of the cell-ECM interface 45 A second important group of proteinases involved in cartilage remodeling are the

ADAMTSs (A disintegrin and metalloproteinase with thrombospondin repeats) (for review 46). ADAMTS proteins can be categorized into different subgroups based on sequence similarities and substrates, if known. ADAMTS-1,4,5,8 and 15 form a subgroup called "aggrecanases" as they all cleave aggrecan, though with different efficiencies.

Some aggrecanases can cleave other proteoglycans such as versican47 and brevican48 as well. ADAMTS-4 and 5 are the predominant aggrecanases in human OA4 9 , these cleave aggrecan at the Glu 373-Ala 374 bond in the IGD region, and also at four other distinct sites between the G2 and G3 domainssosi. Aggrecanases may also have roles besides aggrecan degradation, with ADAMTS-1 being implicated in growth and development, organ morphology and fertility 52 . While aggrecanase mediated degradation is probably more destructive to cartilage than MMP mediated degradation 53, cartilage may have the ability to recover better from aggrecanase degradation than from MMP degradation 54

Another major group of ADAMTS proteins involved in cartilage remodeling are the procollagen N-proteinases (ADAMTS-2,3,14) . As the name suggests, these are involved in the removal of N-terminal propeptides of procollagen 55-57, and hence are anabolic in nature.

Regulation of metalloproteinases can happen at different levels. MMP activity in vivo can be controlled by regulation of gene expression, activation of the pro-enzyme form and inhibition of active enzymes by inhibitors 58. Tissue inhibitor of metalloproteinases (TIMP) refer to the family of proteins which inhibit active MMPs by binding to them (for review 59). Four TIMPs (TIMP 1-4) have currently been identified in vertebrates. Mammalian TIMPs have two domains, a larger N-terminal domain and a smaller C-terminal domain, each having three disulfide bonds. All active MMPs are inhibited by TIMP, however, the four TIMPs differ in affinity for different MMPs.

TIMPs may also form non-inhibitory complexes with proMMPs, with TIMP-2 known to form a tight complex with proMMP-2 to activate it 60 . ADAMTS proteins are also inhibited by TIMPs. TIMP-3 seems to be the strongest inhibitor of the ADAMTS family, inhibiting ADAMTS-4 and 5 with Ki values in the sub nanomolar range 61. TIMP-1 shows some inhibitory activity against ADAMTS-4 62 and TIMP-2 and TIMP-3 against

ADAMTS-1 63. TIMPs have also been shown to be involved in regulation of cell growth, migration and apoptosis, either through inhibiting metalloproteinases, or by directly binding to cell surface receptors 64

1.4.5 Other molecules

Several signaling molecules, such as annexins A2 and A5, proenkephalin, pleiotropin and many others play important roles in tissue morphogenesis and differentiation, and their expression levels are significantly altered in diseased state 65-67

1.5 Post-traumatic OA

OA is broadly classified into two forms, primary and secondary. Primary, or idiopathic OA, is the common age-related onset of the disease, with no obvious cause. In contrast, secondary OA has clear identifiable causes, such as trauma or a congenital or developmental abnormality 6. Post-traumatic OA, a type of secondary OA, refers to joint degeneration, dysfunction and pain which develop after joint injuries. Unlike primary OA which affects aged people, post traumatic OA affects younger individuals primarily, as they are more prone to joint injury 68. A study by Gelber et al 69, for example, found that cumulative incidences of knee OA by 65 years was 13.9% for participants who had an injury during adolescence and young adulthood, and 6% for those who did not. Joint impact loading; tears in the meniscus, ligament and joint capsule, intraarticular fractures and joint dislocations all increase the risk of posttraumatic OA 70. In vivo joint injury causes immediate mechanical damage to cartilage, followed by an acute inflammatory phase which can last up to two months, and a chronic phase which eventually leads to

OA like symptoms 7 1

1.5.1 In Vitro Cartilage Injury Models

The stress distribution of articular cartilage in vivo depends on several factors, including contact force, surface geometry, tissue properties, and cartilage thickness 72, thus making it extremely difficult to quantify and study the effect of mechanical injury on cartilage biochemical properties. In vitro models enable precise application of load, and accurate measurements of the stress and strain profiles thus generated, which can then be correlated with cellular and biochemical changes in cartilage. Usually, high stress/stress rate mechanical compression is applied, and the resulting maximal strain measured or vice versa. Also, mechanical compression injury can either be confined or unconfined.

The extent of damage to cartilage by blunt trauma in vitro depends on the magnitude of stress applied 73 , the orientation of load relative to the articular surface 74 and the rate of loading 7. Injury immediately affects the collagen network, which in turn reduces the tensile load carrying capacity of cartilage 76, affects compressive and shear stiffness 77 and causes increase in matrix water content 73 and cartilage swelling because of reduced ability of damaged collagen to withstand swelling pressure of the GAGs. Rapid GAG loss to the media post injury occurs, possibly due to mechanical disruption of the ECM78 Kurz et al showed that injury lowers biosynthetic rates of chondrocytes immediately, and

biosythesis remains impaired even after 3 days post injury '7. Within 24 hours of a single injurious compression, gene expression of MMP -1,-3,-9,-13; TIMP-1 and ADAMTS -

4,5 increase significantly 79. Pooled media for 5 days post injury show that MMP -1,-3,-

13 are highly up regulated, and MMP-9,TIMP-1 and TIMP-2 mildly up regulated at the protein level 80 . Injurious compression also causes chondrocyte cell death by a combination of necrosis and apoptosis, with necrosis occurring earlier at the site of injury and apoptosis responsible for subsequent spread of chondrocyte death beyond the initial impact area 81,71 The extent of apoptosis is directly related to the load applied, and apoptosis seems to occur at stresses lower than that required for matrix degradation . it is however unclear if apoptosis drives matrix degradation or whether matrix degradation causes apoptosis in vivo. Apoptosis of chondrocytes subsequently reduces the ability of cartilage to repair itself and contributes to the evolution of an OA like phenotype.

1.6 Cytokines in OA

Cytokines such as IL-1$ and TNF-a are key mediators in the post traumatic inflammatory phase. These cytokines are produced by the synovial membrane, diffuse through the synovial fluid into cartilage, and activate chondrocytes, which in turn produce pro-inflammatory cytokines and proteases 83. Therefore, these cytokines act in an autocrine and a paracrine fashion. IL-1p and TNF-a are present in OA cartilage, but not in normal 84. Also, both IL-1$ and TNF-a have been isolated from the synovial fluid of patients undergoing ACL rupture, with the concentrations of IL-1 and TNF-a proportional to the severity of chondral damage 85,86 1.6.1 IL-1p in cartilage damage

IL-16 is synthesized as a 31 kDa precursor, which is then cleaved to form the active 17.5 kDa protein 87. IL-1$ acts upon cells through specific cell surface receptors, type I and type II, of which type I IL-1 receptor is elevated in OA cells, and is mainly responsible for signal transduction by IL-16 in OA 87. Direct injections or gene transfection of IL-IRa, a soluble receptor antagonist of IL-1, reduce the progression of experimental OA in animal models 88.

IL-1 promotes collagen degradation 89 by stimulating metalloproteases 90. IL-iP causes strong up regulation of MMP-1, MMP-3 and MMP-13 at both the mRNA 91 and the protein levels 80 in vitro. It causes aggrecan degradation by stimulating aggrecanases in vitro, but it is unclear whether ADAMTS-4 or ADAMTS-5 is the predominant aggrecanase responsible for this. Species specific differences may be possible, with

ADAMTS-4 the predominant aggrecanase in cultures of human OA chondrocytes stimulated by IL-la 92, and ADAMTS-5 in murine cartilage explants stimulated by IL-la

. IL-1 also decreases proteoglycan and collagen synthesis in cartilage in vitro 4

Exposure of cartilage to IL-1f also affects physical properties of cartilage, causing an increase in hydraulic permeability and decrease in streaming potential and equilibrium modulus, possibly due to the degradative activities of MMPs and aggrecanases 96

1.6.2 TNF-a in cartilage damage

TNF-a is synthesized as a type II transmembrane protein arranged as homotrimers, which is then released by proteolytic cleavage to form the homotrimeric soluble form. It activates cells through two kinds of receptors, TNF-R1 and TNF-R2, of which TNF-R1 is the type present in several kinds of tissues including cartilage. TNF-R1 is responsive to both the soluble and membrane trimeric forms of TNF- 97 , and is over expressed in OA cartilage on the surface of chondrocytes located near sites of high GAG loss 98. Like IL-1P, TNF-a also causes aggrecan degradation by stimulating aggrecanases

99,100, and suppresses proteoglycan synthesis, though less efficiently than IL-1 01. TNF-a also highly up regulates several MMPs including MMP-1, MMP-3, MMP-13 and MMP-9

80. Additionally, TNF-a stimulates IL-6 production 101, which in combination with its soluble receptor can contribute to further proteoglycan catabolism 102

1.6.3 Comparison between IL-1 and TNF-a in cartilage damage

TNF-a exerts effects similar to IL-1, and can also act synergistically to IL-1 103 however, animal models of RA indicate that TNF-a and IL-1p may have distinct functions, with TNF-a important in initiating joint swelling, and IL-1 playing a role in subsequent inflammation and cartilage erosion 104. Chondrocytes stimulated by IL-1 alone or IL-1 in combination with TNF-a produce inflammatory molecules such as inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2) and phospholipase

A2 (sPLA2). Molecules such as nitric oxide and E-series prostaglandins produced by the activity of these enzymes have further anabolic/catabolic roles downstream 105

1.6.4 Anti-Cytokine Therapies for OA treatment

In spite of the fact that IL-1 and TNF-a seem to be key molecules involved in the pathogenesis of OA, the inhibition of IL-1 and TNF-a using IL-iRa and anti-TNF alpha

therapies in OA patients is not very successful 106 , generating the need for further

investigation of the roles of these cytokines and better therapies. 1.7 In Vitro Models combining injury and cytokine

Therefore, to get a more realistic in vitro model of post traumatic OA, we need to capture the effect of cytokines as well. Patwari et al used an in vitro model where cartilage explants from newborn bovine and human knee tissue were subjected to a single injurious compression followed by incubation with exogenous cytokines IL-la and TNF- a for three days 107. They found that incubation of injured bovine cartilage with cytokines increased sGAG loss to the media greatly, from a mean of -7% sGAG loss from injured cartilage without exogenous cytokine, to a mean of 60% and 54% sGAG loss for injured cartilage incubated with 10 ng/ml IL-la, and 100 ng/ml TNF-a respectively. Interaction between injury and cytokine in both cases was highly significant, with P<0.001 by two way anova. IL-la and TNF-a showed synergism with injury for human knee tissue explants too, though the magnitude of synergism was far lower. Sui et al found that anti-

IL-6 Fab reduced the amount of sGAG loss caused by synergism between TNF-a and injury in newborn bovine cartilage explants, but did not completely eliminate it, suggesting that the synergism between TNF-a and injury is at least partly mediated by

IL-6 produced by chondrocytes 108. Further, addition of exogenous IL-6 along with its soluble receptor (sIL6R) increased the synergism between exogenous TNF-U and mechanical injury in newborn bovine and in human knee and ankle cartilage explants.

1.8 Thesis Objectives

Injury and cytokines both play a significant role in the progression of post

traumatic OA, and interplay between these two factors possibly drives OA progression in

vivo. Therefore, an in vitro model system in which injured cartilage explants are incubated in IL-16 or TNF-a is used to try to elucidate the mechanism by which injury interacts with cytokines to drive OA. A proteomic profile of media released from cartilage under different treatment conditions (injury, cytokine and combination of injury and cytokine) is generated using a quantitative iTRAQ 2D-LC-MS-MS approach. The levels of several different proteins under different treatment conditions are compared, and proteins on which injury and cytokine act synergistically, in a positive or a negative fashion are identified. This approach thus helps understand the mechanism by which injury and cytokine interact with each other in driving the pathogenesis of OA.

The next part of our study uses a partial least square regression (PLSR) approach to relate protein ratios across different treatment conditions with measured phenotypes across the treatment conditions. This approach removes redundant data, and determines which proteins contribute most to specific observable phenotypes. Using this approach, we are able to see hidden associations between proteins, between phenotypes and between proteins and phenotypes.

Thus, using a simplistic in vitro model, the overall goal of this project is to determine molecules which may be important to OA disease progression, and may serve as biomarkers or possible therapeutic targets for OA. METHODS

2.1 Overview

Equal volumes of cartilage were treated in equal medium volumes with one of the following treatments (i) 10 ng/ml IL-iI3 (ii) 100 ng/ml TNF-a (iii) unconfined mechanical compression to 50% strain at 100%/sec followed by incubation with 10 ng/ml IL-l $3(iv) unconfined mechanical compression to 50% strain at 100%/sec followed by incubation with 100 ng/ml TNF-a. Medium was pooled, deglycosylated by chondroitinase ABC and digested with trypsin. A 4-plex iTRAQ labeling strategy was used, with sample from each treatment condition labeled with one of the four iTRAQ labels as follows: IL-1 : iTRAQ 114; TNF-a : iTRAQ 115; IL-1f +injury : iTRAQ 116 and TNF-a +injury : iTRAQ 117. The samples were then combined, fractioned by SCX chromatography, subjected to RP-LC/MS/MS and analyzed using ProteinPilot TM (ABI). Matlab scripts were used to generate protein ratios from the peptide summary reports of protein pilot.

These data were subjected to subsequent analysis. To study the effect of biological variance, all steps are performed on biological replicates, replicate 1 consisting of cartilage from a set of three animals, and replicate 2 from a different set of three animals.

2.2 Cartilage Explantation, Mechanical Injury and Cytokine Treatment

Cartilage explants were obtained from the femoropatellar groves of 2-3 week old calves as described previously 109. Briefly, cartilage was cored and punched to obtain disks (1 mm thick X 3 mm diameter). Between 8-12 disks (explants) from each of 3 animals were used for each treatment condition for biological replicate 1, and the same procedure followed for biological replicate 2 with 3 different animals. These disks were equilibrated for 5 days prior to start of treatment in 1% ITS (high glucose (25 mM))

DMEM medium supplemented with 10 mM HEPES, 0.1 mM non essential amino acids,

115 uM ascorbic acid, 400 uM L-proline, 100 U/ml penicillin, 100 ug/ml streptomycin,

0.25 ug/ml amphotericin B powder (PSA for tissue culture) and 1 mM sodium pyruvate.

On treatment day, location matched explants were placed in four different groups and cultured in 2 ml medium without ITS supplementation.

Cartilage explants meant to be treated with cytokines alone were placed in medium without ITS containing 10 ng/ml IL-1p or 100 ng/ml TNF-a. The explants were then cultured for 120 hours with 10% medium removal and supplementation every 24 hours. Cartilage explants meant to be treated with cytokine+injury were individually injured first by mechanical compression in a custom designed, incubator housed apparatus as described previously 110,79,108 . Explants were subjected to a single unconfined compression at a strain of 50% and strain rate of 100%.second. After injury, they were placed in medium without ITS containing 10 ng/ml IL-1IP or 100 ng/ml TNF-a, and cultured for 120 hours, with medium removal and supplementation as before. After treatment, explants and cartilage were stored at -80 C

2.3 sGAG Removal, Trypsin digestion and iTRAQ labeling

3 ml of the medium from each condition (for biological replicates 1 and 2 each) was supplemented with 5 mM EDTA, 100 ug/ml PMSF and 5 mg/ml iodoacetamide. The samples were dialyzed for 3 hours against a buffer with 10 mM Tris acetate, 40 mM

NaCl and 5 mM EDTA, mixed with 70 milliunits of Chondroitinase ABC (the deglycosylation agent and the internal standard) and left to dialyze and deglycosylate overnight at 37 C. The samples were further dialyzed against 2 mM TEAB and 0.5 mM

EDTA for 8 hours, and then against 500 mM TEAB. The samples were then frozen, concentrated 10 fold, and then further concentrated to a final volume of 150 ul. Samples were then reduced by 2 mM TCEP (in 100 mM TEAB) for 2 hours, and alkylated by 5 mM iodoacetamide (in 100 mM TEAB) for 2 hours. The samples are then precipitated by

6 volumes of ice cold acetone and then centrifuged at 15000 x g for 30 min at 4C. The pellets were re-suspended in 25 ul of 50 mM TEAB containing 0.1% SDS. Trypsin (0.1 mg/ml in 50 mM TEAB) was added to each sample in the ratio 1:37.5. Sample volume was increased by addition of water and acetonitrile to 40 ul, following which samples were incubated at 37 C overnight, speedvae'd to dryness, and resuspended in 500 mM

TEAB.

The peptides were then iTRAQ labeled according to manufacturer's instructions.

Briefly, iTRAQ labels were equilibrated at room temperature, diluted with ethanol, vortexed and centrifuged at 16000 x g for 1 minute. The iTRAQ reagent was then added to 100 ug peptides from each sample. The samples were then vortexed for 1 minute, sonicated for 5 minutes, centrifuged at 16000 x g for 1 min, and equilibrated at room temperature for 55 minutes. Following this, samples were diluted to 200 pl total volume with water and combined in equal proportion according to volume equivalents. The combined sample volume was reduced to 50 ptl, further combined with 1% phosphoric acid and 1 ml of strong cation exchange buffer A (10 mM Potassium phosphate pH 2.8,

25% acetonitrile), made to pH 3.5 and diluted with 3 ml SCX buffer A. 2.4 SCX Chromatography and LC/MS/MS

The combined labeled sample was injected onto a 2.1 mm X 100 mm strong cation exchange column on an Agilent 1100 HPLC at a flow-rate of 250 ul/min; with

100% Buffer A for 20 minutes, 0 to 40% gradient SCX buffer B (10 mM phosphate buffer pH 2.8, 400 mM KCl, 25% acetonitrile) for the next 40 minutes, 40 to 90% gradient SCX buffer B for the next 5 minutes and finally 95% buffer B for the next 5 minutes before returning to starting conditions (Total run time ~ 80 minutes). Peptide elution was monitored by UV absorption at 214 nm. Fractions were collected every 0.5 minutes and stored at -80 C. Fractions were then concentrated by speedvac and desalted by zip tip. Desalted factions were loaded onto a 75 um x 160 mm column with 10 um tip packed in house with Vydac protein/peptide C18 packing material. Peptides were loaded at flow-rate 250 nl/min at 2% buffer B (1.2% acetic acid in 90% acetonitrile) for 12.5 minutes, eluted with 2 to 40% buffer B gradient for 147.5 minutes and washed out with

40 to 60% buffer B gradient for 20 minutes. This was followed by holding at 60% B for 5 minutes, returning to 2% B over 15 minutes and equilibrating at 2% B for 60 minutes.

The Buffer A is 1.2% acetic acid in water. Following LC, mass spectrometry was performed using QStar, a Quadrupole time of flight mass spectrometer (ABI) with a nanospray source. Precursor ions between m/z 400 and 1600 with a charge state between two and four were selected with Analyst 1.1. Each cycle (10 sec) consisted of an MS scan followed by 3 data dependent MS/MS scans (m/z 100-1600), with ions excluded for 100 seconds after a single spectra. All fractions from the first replicate were run first, before running samples from the second replicate. 2.5 Data Analysis

Data were analyzed using the Paragon and Pro Group algorithms of

ProteinPilot TM Software 2.0.1 (ABI). The variable modifications allowed were NH 2 terminal iTRAQ labeling, iTRAQ labeled lysine, iTRAQ labeled tyrosine and oxidized methionine. The number of missed trypsin cleavages was restricted to 1. The search was conducted against the NCBInr bovine database assembled in June 2008.

The peptide summary report generated is then exported into excel, and scripts are written using Matlab (Mathworks) to calculate mean protein ratios, standard deviation and standard error, and p values for the null hypothesis that the protein ratios are different from 1. The Matlab script also makes sure that only peptides which are identified by

Protein Pilot with confidence greater than 90%, which are quantified well for both labels in the ratio under consideration (i.e. the ratios generated from protein pilot are not 0 or

9999), and whose spectra are manually verified are chosen for calculating protein information. All proteins identified by less than two peptides satisfying all the above criteria are discarded. All mathematical operations for calculating mean, standard deviation, standard error, p value, etc are done using log transformed ratios to make sure that ratios lesser than and greater than one are treated identically. The means and standard

1 deviation are calculated using ( ) listed by protein pilot as weights. The weighted %Error mean, the weighted standard deviation and the weighted standard error in the log space are calculated using the following formulas N

__ _Wii___ Sd_ X= SE =

Sd NN Wi (N -1)w N

Where X w is the weighted mean of the log transformed peptide ratios Xi , Wg is the

1 weight of the peptide i (calculated as ), N refers to the number of well quantified %Error manually verified peptides identified with confidence > 90%, Sdw is the weighted standard deviation, and SEW is the weighted standard error.

Normalization of the protein ratios thus obtained was done using the weighted mean of chondroitinase ABC, the enzyme used for glycosylation. This protein is a non mammalian protein and can thus serve as an internal standard 80. Also, the ratio of this protein is almost 1 in all cases, thus illustrating that the levels of this protein do not get affected by treatment, as expected. To obtain the p values for the null hypothesis that log transformed protein ratios were different from zero, the normality of log transformed peptide ratios was first checked using Lilliefors test for normality in MATLAB, if the log transformed ratios were normal, a paired t test was used, if not, the Wilcoxon sign rank test was used. To correct for multiple comparisons, the Benjamini Hochberg procedure

m was used and false discovery rate (FDR) controlled at 0.1. The Benjamini Hochberg procedure was performed for each ratio (i.e. 115:114, 116:114 and 117:114) separately. 2.6 Comparison of biological duplicates and final protein list compilation

To test whether there exists significant variation between biological replicates, for proteins identified in common between biological replicate 1 and 2, histograms of the difference between log transformed ratios from biological replicates 1 and 2 are plotted, and the mean and standard deviation of this difference computed, for each ratio.

A final protein list is then compiled for use in further analysis. To do so, a list of proteins which satisfy Benjamini Hochberg procedure across all three ratios from the first biological replicate is selected. This is supplemented with proteins identified from the second biological replicate alone which satisfy Benjamini Hochberg procedure across all three ratios, after correcting for any major inter experimental bias. This correction is done using the difference between the means of log transformed ratios from replicates 1 and 2 obtained by comparing the biological replicates in the previous step. A total of 199 proteins are thus chosen for the final compiled list, for each of which we have ratios for three conditions, TNF-ax:IL-1p, IL-1f +injury:IL-1 and TNF-a +injury:IL-1p.

2.7 Global Proteomic Analysis and Clustering

A correlation matrix is generated using MATLAB between different ratios (TNF- a:IL-1p, [IL-1f+inj]:IL-1p and [TNF-u+inj]:IL-1p) using the compiled protein list. K- means clustering analysis was done using the statistics toolbox in MATLAB on the compiled protein data set. This methodology partitions the 199 proteins into k clusters, by minimizing the sum of distances between each protein and the centroid of the cluster the protein is partitioned into. The number of clusters is chosen such that the mean of the silhouette values is the maximum. The Mann Whitney U test or the Unpaired t test is used to check for the uniqueness of each cluster.

2.8 Merging data between two iTRAQ experiments

We seek to combine data from two iTRAQ experiments, an earlier experiment 80 and this experiment. The earlier experiment has ratios (IL-1IP:untreated), (TNF- a:untreated) and (injury:untreated), while the current experiment has ratios (TNF-a:IL-

1p), (IL-1p +injury:IL-1p) and (TNF-a +injury:IL-1p). By merging data from both experiments, we hope to obtain data for all possible ratio combinations with respect to untreated control (i.e. IL- 13 :untreated, TNF-a:untreated, Injury:untreated, IL-l$

+injury:untreated and TNF-a +injury:untreated). Before doing so, we need to compare data for proteins found in common between these two experiments to ensure that there is no significant inter experimental error. The comparison is done only between proteins which satisfy Benjamini Hochberg procedure across all ratios at FDR 0.1 in both experiments. 74 proteins are thus found in common between both these experiments, a histogram of the difference between the log transformed (TNF-a /IL- 10) ratios from both these experiments is plotted, and the mean and standard deviation of this difference computed.

If there is no significant inter experimental error, we merge data from both these experiments to obtain data for all five treatment conditions for 74 proteins. A correlation matrix is generated using Matlab to ascertain how these five conditions (IL-1p, TNF-a, injury, IL- 11 +injury and TNF-a +injury) correlate to each other. 2.9 Finding proteins on which injury and cytokines act synergistically

We want to identify proteins on which injury and cytokine act synergistically in either a positive or a negative fashion. To do so, if the ratio of (cytokine:untreated) were

RI, the ratio of (injury:untreated) R2 and (cytokine+injury:untreated) R3 for a given protein, if all the three conditions R3>R1, R3>R2 and R3>R1*R2 are satisfied for any protein, injury and cytokine are determined to exert positive feedback on that protein. On the other hand, if the three conditions R3

2.10 Partial Least Square Regression (PLSR)

We want to find out which protein ratios contribute most to observed phenotypes.

We take the compiled protein list consisting of log transformed protein ratios for 74 proteins across 5 conditions, and eliminate two proteins because they act as outliers and add noise to the PLSR model. We thus have an independent 72*5 data matrix (X) consisting of log transformed protein ratios across five treatment conditions. For the dependent Y matrix, we choose phenotypes for which data are already available for all the five treatment conditions. The phenotypes thus chosen are amount of sGAG loss to the medium during the 5 day treatment regime, amount of nitrite accumulation in medium during the 5 day treatment regime, proline incorporation rate on the 6 th day post start of treatment and sulfate incorporation rate on the 6th day post start of treatment 11

We thus create a (4*5) matrix Y, with each element of the matrix = log -Ilj , where P is the value of the phenotype i on receiving treatment j, and Pe is the value of the

phenotype i on receiving no treatment. (For example, Pj may be sGAG released to the

medium on treatment with TNF-a, with PI, the sGAG released to the medium on absence

of treatment)

PLSR 114 is used to find a relation between Y and X, by choosing dimensions

(latent vectors) such that superfluous information in X and Y is eliminated, and the

maximum possible co-variation between X and Y is captured in the first few latent

vectors. Both the X and Y matrices are mean centered and set to unit variance along the

columns (i.e. proteins and phenotypes respectively) before performing PLSR.

PLSR is performed using the NIPALS algorithm 115 with The Unscrambler @ X Version

10.0.1 (Camo) software. The PLSR model is trained and validated by cross-validation,

where portions are omitted from the input matrix in turn, omitted data predicted from the

remaining data in the matrix, and predicted data compared to actual omitted data. Once

the latent vectors are determined, the proteins, phenotypes and treatments are projected

on the first few latent vectors, this enables one to reduce the dimensionality of data and

see relationships between proteins, phenotypes and treatments which were previously hidden. RESULTS

3.1 Reproducibility between biological replicates

We want to check whether the methodology and our data are sufficiently robust as to show reproducibility between biological duplicates (Unlike technical replicates which are designed to show variability in the process, biological replicates capture variability in both the target population and the process) . For this, only protein ratios whose p values satisfy Benjamini Hochberg procedure at FDR 0.1 are chosen, for each ratio separately.

Proteins identified in common between the two biological replicates are compared by dividing the geometric mean of the peptide ratios for any protein in the first biological replicate (ratiol) by the geometric mean of the peptide ratios for the same protein in the second biological replicate (ratio2). These ratios of ratios (ratiol/ratio2) are log transformed and plotted as histograms for each treatment condition (Figure 1) We expect to see a mean of zero and negligible standard deviation for these histograms if biological replicates were perfectly reproducible. We note that the mean +/- standard deviation is

0.1010 +/- 0.3015 for log2(ratiol 115:114/ratio2 115:114), 0.0807 +/- 0.3134 for log 2(ratiol 116:114/ratio2 116:114), and 0.4317 +/- 0.4292 for log2(ratio1

117:114/ratio2 117:114). The huge error (43.17%) between 117:114 ratios is probably due to incomplete protein digestion in the sample from biological duplicate 2 containing the 117.1 m/z label (a white precipitate was seen in this particular sample). To correct for this error, 0.4317 was added to log 2(ratio2 117:114) for all protein readings obtained from the second biological replicate. The % variation observed in the other two cases

(10.1% and 8.07%) is well within the 25% variation found between biological replicates and the 23% variation between experimental replicates (variation due to different iTRAQ

experiments) in the study by Gan et al 116

3.2 Global proteomic analysis

A total of 199 proteins identified by either iTRAQI or iTRAQ2 or both and which

satisfies Benjamini Hochberg procedure at FDR 0.1 across all ratios were used to perform

a systems level analysis of the effect of the combination of injury and cytokine on the cartilage sample. A matrix of correlation coefficients between the log transformed protein ratios is shown in Table 1. (All ratios are expressed with respect to IL-1P because a

4-plex iTRAQ analysis (using 4 labels) were chosen). A high correlation of ~ 91.4% was seen between the two (Cytokine+Injury) combination conditions indicating either that the proteins were affected similarly by the two cytokines in combination with injury or that the effect of injury was much higher than the effect of cytokines , thus dominating the overall (Cytokine+Injury) response. This high correlation between the two

(Injury+Cytokine) conditions is verified by a plot between log2(IL-1p +inj/IL-1p) and

2 log 2(TNF-a+inj/IL- 1p ) (Figure 2) , which shows a linear relation with R equaling

0.8353.

3.2.1 K-means clustering analysis

K-means clustering was used to cluster the proteins into groups which would enable better understanding of how groups of proteins varied with treatment. Silhouette plots were used to determine that 3 clusters yielded optimum inter-cluster variation as compared to intra-cluster variation. Figures 3a-d represent the proteins in 3D space and their projections on 2D planes. The proteins are determined to be well separated into three clusters by means of unpaired t test (All p values are << 0.001). Figure 4 shows

how the centroid of each cluster varies with treatment. Broadly, cluster 2 proteins are

depressed by all conditions wrt IL- 13, and additionally depressed in the (TNF-a +injury)

condition. Cluster 1 proteins are almost unchanged with all treatments, showing a mild

increase on treatment with injury and cytokine. Cluster 3 proteins are in general elevated,

and show increased elevation on treatment with injury and cytokine.

3.2.2 Effect of Cytokine+injury on proteins based on cell location

Proteins are sorted by location and average ratios are found out for all proteins belonging to a particular cellular location. We observe that proteins inside the cell

(Including the cytosol, ER lumen and nucleus) all show highly elevated levels in response to both (TNF-a +injury) and (IL-1P +injury) (p<0.05) (Figure 5). ECM proteins and membrane protein levels remain almost the same on treatment with (cytokine+injury).

Secreted proteins are down regulated to a greater extent with TNF-a alone than with

(cytokine+injury).

3.3 Effect of combination of cytokine and injury on specific proteins

In cartilage, a few collagens are synthesized in a procollagen form and require processing by removal of N- and C-terminal propeptides to form mature collagen. The release of C-terminal propeptides is hence an indicator of collagen synthesis. From

Figure 6a, we see that C-terminal propeptide levels of many collagen chains are mildly to moderately down regulated with IL-1 +injury, and moderately to highly down regulated with TNF-a +injury, indicating reduced synthesis. When we examine other major structural proteins found in the ECM, we find that fibronectin is highly down regulated with TNF-a treatment alone or (TNF-ct +injury) and COMP is highly down regulated with (TNF-a + injury) (Figure 6b). All the Matrilins are down regulated with

TNF-a or (IL-1 p + injury), but elevated with (TNF-a + injury), with Matrilin-3 showing the most significant down regulation (Figure 6b). Aggrecan shows marginal down regulation with all three treatments with respect to IL- 1P. Members of the Small leucine rich proteoglycan (SLRP) family respond significantly to the combination of treatment and injury, with chondroadherin, biglycan, lumican and PRELP all showing significant up regulation with (TNF-a + injury) (Figure 6c). Biglycan also shows up regulation with

(IL-IP + injury).

We next look at proteases which are known to be important in ECM degradation

(Figure 6d). Both MMP 2 and MMP3 are highly suppressed with TNF-a compared to

IL- 1p, but only very mildly so on treatment with a combination of cytokine and injury

MMP 13 is down regulated with (IL-1P + injury) treatment. ADAMTS-1 is the only

ADAMTS protein found in our study, it is up regulated with (IL- 13 + injury) and more significantly with (TNF-a + injury). The metalloproteinase inhibitor TIMP-2 is marginally up regulated on treatment with IL-1I and injury, but not so with TNF-a and

injury.

The next figure (Figure 6e) shows the effect of the combination of cytokine and

injury on members of the TGF- super family and their inhibitors. TGF- family

members are known to be anabolic and to stimulate proteoglycan synthesis by

chondrocytes. While TGF-p2 itself is up regulated significantly with (Cytokine + injury)

for either cytokine and especially for (TNF-a + injury), inhibin PA shows extremely high

elevation with (Cytokine + injury) treatment for both IL-1 and TNF-a. Among the LTBP's , proteins known to help in TGF-p secretion, LTBP1 and LTBP2 are both down regulated with all three treatments, LTBP1 down regulated more with (TNF-a + injury) and (IL-1I3 +injury) than with TNF-a; and LTBP2 more with TNF-a. Connective Tissue

Growth Factor (CTGF) is significantly down regulated with (TNF-a +injury) , while (IL-

1f +injury) does not seem to have much effect. TGF-p inhibitor vasorin is almost unchanged with all treatments, while BMP inhibitor chordin-like 2 is highly down regulated with cytokine+injury.

IGF is also an important anabolic agent. IGF binding proteins (IGFBP) show varied response to (Cytokine+Injury) (Figure 6f). While IGFBP7 is down regulated on treatment with both (IL-1 B3+injury) and (TNF-a +injury), IGFBP5 is up regulated with

(TNF-a +injury) but not with (IL-1 +injury). IGFBP3 is elevated with (IL-1 P +injury) and (TNF-a +injury), and IGFBP6 is marginally down regulated with (IL-I P +injury) and

(TNF-a +injury)

Figure 6g looks at proteins involved in the innate immune response. Complement factors C3, Cis, Clq and C1r all show mild down regulation in the presence of (IL- 1

+injury). C3 and Cir show significant down regulation in the presence of (TNF-a

+injury), while Clq is mildly up regulated. C1 inhibitor and PGLYRP2 show significant down regulation with (IL-1p +injury), but remain almost the same with (TNF-a +injury).

Lactotransferrin shows mild down regulation with all treatments, while

Lipopolysaccharide binding protein (LBP) is highly down regulated with TNF-a alone, and moderately with (IL-1p +injury) and (TNF-a +injury). CD14 shows significant down regulation with (IL- 1P +injury), and mild down regulation with (TNF-a +injury) We next look at some proteins involved in signaling including cytokines (Figure

6h). Proenkephalin shows the highest response to the combination of injury and cytokine, with eight and thirteen fold elevation to (IL-1f +inj) and (TNF-a +inj) respectively.

Calcium binding proteins annexin A2 and annexin A5 are also elevated significantly by injury and cytokine. Angiopoietin-like 7, on the other hand, is depressed by both the injury+cytokine conditions. Pleiotropin shows mild elevation with (IL-I P +inj) and

(TNF-a +inj) treatments. Granulin shows a more than two fold depression with (TNF-a

+inj) alone. The membrane protein CD44 is significantly depressed by (IL-1p +inj), and mildly by (TNF-a +inj).

3.4 Effect of combination of injury and cytokine as opposed to either treatment alone

Data from a previous iTRAQ experiment in our lab 80 are used in combination with data from this experiment to generate all the information necessary to perform this study. To combine data from both the experiments, we first verify that there is no significant experimental error/bias between both these experiments.

3.4.1 Comparison between two iTRAQ experiments

We want to check whether there are no major sources of variation between the results of both these iTRAQ experiments. As earlier, this is done by first identifying proteins found in common between experiment A (the current experiment) and experiment B 8. (The final data set used for comparison from experiment A is the set of

199 proteins identified by merging proteins identified by either or both of the biological duplicates and which satisfy Benjamini Hochberg procedure at FDR 0.1; similarly, the final data set used from experiment B consists of 136 proteins found in either or both technical duplicates combined which satisfy Benjamini Hochberg procedure across all ratios at FDR 0.1). 74 proteins are thus found in common between Experiments A and B.

We compare reproducibility between exp A and B by taking the ratio of (TNF-c /IL-1 ) ratios for proteins in both experiments ((TNF-a /IL- 1P) is the only ratio in common between experiment A and experiment B). This ratio of (TNF-a /IL- 1P) ratios is log transformed and a histogram is plotted (Figure 8). The mean+/- standard deviation is seen to be 0.1067 +/- 0.2191. There thus seems to be good reproducibility between experiments A and B with variation of around 10.7%.

3.4.2 Correlation coefficient matrix between different conditions

Since variance between experiment A and B is around 10.7% only, the data for the 74 proteins identified in common between both these experiments are fused.

Experiment A data which are ratios wrt IL-iI are converted to ratios wrt untreated control by multiplying with (IL- 1P:untreated) data from experiment B. Thus, all possible ratios i.e. (IL- I :untreated), (TNF-a:untreated), (injury:untreated),(IL- 13

+injury):untreated and (TNF-a +injury):untreated are obtained for these 74 proteins. A matrix of correlation coefficients is generated from log transformed ratios (Table 2).

Around 90.3% correlation between TNF-a and IL-1$, and around 95.3% correlation between (TNF-a +injury) and (IL- 1P +injury). There is very little correlation between

injury and cytokine treatment, and both injury and cytokine show almost equal

correlations with (cytokine+injury). Curiously, TNF-a shows greater correlation with (IL-

1P +injury) than with (TNF-a +injury). 3.4.3 Proteins on which injury and cytokine act independently

If the effect of injury and cytokine were almost independent on a protein, we would expect that the effect of the combination of injury and cytokine would be a product of the effect of injury and the effect of cytokine. Therefore, if the ratio of

(cytokine:untreated) were R1 and the ratio of (injury:untreated) were R2 for a given protein, if cytokine and injury acted independently, the ratio of

(cytokine+injury:untreated) (R3) would be R1*R2. We therefore check for all proteins for which

log 2(R1*R2) -0.1 <= log 2(R3) <= log2(R1*R2) + 0.1

The addition and subtraction of 0.1 is to specify error range.

Under this criteria, we observe that IL-13 and injury act almost independently on aggrecan, CILP-2 and SPARC. TNF-ax and injury act almost independently on Collagen

VI alpha II, MMP-2 and C-type lectin domain family 3 member A.

3.4.4 Positive and negative feedback by cytokine and injury

If the effect of combination of cytokine and injury is greater than that of cytokine alone, injury alone and the effect obtained if cytokine and injury acted independently, cytokine and injury positively reinforce each other, in other words, exert a positive feedback on the protein. Similarly, if effect of combination of cytokine and injury is lesser than that of cytokine alone, injury alone and the effect obtained if cytokine and injury acted independently, cytokine and injury exert a negative feedback.

To formulate the above statements as equations, if the ratio of

(cytokine:untreated) were R1, the ratio of (injury:untreated) R2 and

(cytokine+injury:untreated) R3 for a given protein, If R3 > R1, R3 > R2 and R3 > R1*R2, a positive feedback is seen.

If R3 < R1, R3 < R2 and R3 < R1*R2, a negative feedback is seen.

Under these criteria, TNF-a and injury are seen to show positive feedback for aggrecan, collagen VI alpha III, histone H4 and CILP-2. TNF-a and injury show negative feedback for SPARC and IGFBP-7.We do not see any protein for which IL-l $3and injury show positive or negative feedback.

3.5 Partial least square regression (PLSR) between protein ratios and phenotypes

We want to find out how protein ratio under different treatment conditions (the independent variables) causes changes in various observed phenotypes under the same treatment conditions (the dependent variables). The phenotypes available to us for the treatment conditions IL-iI3, TNF-a, injury, IL-1P +injury and TNF-a +injury are: amount of sGAG released into medium at the end of five days post treatment, amount of nitrite accumulation at the end of five days, and rate of proline and sulfate incorporation measured on the sixth day (Table 5). We thus want to find out a simple relation between the protein ratio matrix (a matrix consisting of log transformed protein ratios as rows and treatment conditions as columns) and the phenotype matrix (a matrix consisting of log transformed phenotype ratios wrt control as rows and treatment conditions as columns).

Since there exists a lot of redundancy in the protein ratio matrix, we perform PLSR in order to find a set of components (called latent vectors) which explain as much as possible of the co-variation between the protein ratio matrix and the phenotype matrix. First, from Figures 8a and 8b, we see that two latent vectors (factors) seem to explain most of the variation in both X (the protein matrix) and Y (the phenotype matrix).

Therefore, a projection of the treatment conditions on the first two latent vectors is shown in Figure 8c. From this, we see that both the cytokine only conditions are relatively close, both the (injury+cytokine) combination are also close to each other, whereas the injury alone condition lies away from the other conditions. This tallies with the correlation matrix in Table 2. Figure 8d shows the projection of the phenotypes and proteins on the first two latent vectors. Proline and sulfate are seen to project nearby, corresponding to the fact that protein and proteoglycan synthesis are related.

Complements C3,B and C1r cluster together in the second quadrant. Cytoskeletal proteins such as beta actin, vimentin, actinin alpha 4, alpha actinin and fascin are all found close together in the fourth quadrant. Both the histones H2B and H4 are in the third quadrant. A matrix containing the correlation shown by each protein with each phenotype is generated.

In Table 4, the protein-phenotype pair showing the strongest positive and negative correlations are listed. Perlecan-sGAG, pleotropin-sGAG, serum amyloid A3- sGAG, CILP2-sGAG and complement B-sGAG are the pairs showing the highest positive correlation. The pairs which show the highest negative correlation are Perlecan- sulfate, perlecan-proline, pleiotropin-sulphate, pleiotropin-proline and cathepsin B- sGAG. Figure 1: Comparison of the first and second biological replicates. Proteins are identified in common between the first and second biological replicates and their mean ratios after internal standard correction compared. Only ratios whose respective p values satisfy Benjamini Hochberg procedure at FDR 0.1 are chosen for the respective plot. Histograms of log 2 transformed ratio of ratios (i.e. log2(ratiol/ratio2) ) are plotted for each of the ratios 115:114 (A), 116:114 (B) and 117:114 (C). The Mean +/- standard deviation is 0.1010 +/- 0.3015, 0.0807 +/- 0.3134 and 0.4317 +/- 0.4292 for A, B and C respectively.

40-

35-

25-

20-

10-

01 2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 Lom(ratio1 115:114 /ratio2 115:114) Lo&(ratiol 116:114 /ratio2 116:114)

-2 -15 1 -u.5 U U.0 I 1.b 2 2.5 Log(ratio1 117:114 / ratio2 117:114) Table 1: Matrix of correlation coefficients. Correlation coefficients are found between TNF-a only, combination of IL- 1s and injury and combination of TNF-a and injury treatments. Log transformed protein ratios obtained from iTRAQ experiments for the corresponding treatments are used as vectors to calculate these coefficients. Only proteins, the p values of all whose ratios satisfy Benjamini Hochberg procedure at FDR 0.1 are chosen.

log 2(TNF-a/L-1 p) Iog 2(IL-1 P+inj/L-1 p) log 2(TNF-a+inj/L-1 p) log2(TNF-a/IL-1 P) 1 0.6786 0.6957 log2(IL-1 p+inj/IL-1 p) 0.6786 1 0.9139

1og 2 (TNF- a+iinj/IL-1p) 0.6957 0.9139 1 ......

Figure 2: Expresses the relation between effect of cytokines IL-1$ and TNF-a in combination with injury on proteins. Log 2(IL- 1p +Injury/IL- 1p) (x axis) is plotted against Log2(TNF-a +injury/IL-1 [) (y axis). Figure 2 has been fitted to a line with a slope of 1.0729, y-intercept of 0.1353 and an R2 value of 0.8353. This indicates a highly correlated relation between the two (Injury+Cytokine) conditions in their effect on protein expression.

-2-

LL z .. I- -2 0

log2( IL- 1 A4i nj/IL- 10) ......

Figure 3: Figure 3A is a plot of proteins in three dimensions with log transformed ratios as axes, the x, y and z axis representing log 2(TNF-a /IL- 1$), log2(IL- 1p +Injury/IL- 1p) and log2(TNF-a +injury/IL- 1p) respectively. Colors represent protein clusters as computed by k-means clustering. Figures 3B-D represent projections of the 3D plot on different axes pairs.

K-i O K-2 LK=3 2

1 2K= EU U. * 10 * * M dP now 0 8..

-4

-1 log2(L1+inj:L1) -4 Iog2(INF:M~) log2(rNF-uAL-1p)

2 2.

-14 4:.. *P~*4~ * K=I IK=3 2 KL=J 2** A -2-2 7- an -2 U - -3

og L-1pSi L1) log2(TNF-AL-10) Figure 4: Profiles of the means of the three clusters identified by K-means clustering. Error bars represent standard error of the mean. The clusters are well separated (p<0.001, significance measured by Mann Whitney U test). Clusters 1, 2 and 3 contain 59, 55 and 85 proteins respectively.

2.5

1.5

00.-+- K=12

K=3

0. TNF-a/J*4P UIP4k*H /i*4-1 nij

-0.5-

-15- -- Figure 5: Profiles of means of log 2 transformed ratios of proteins sorted by location. Error bars represent standard error of the mean. Asterisks indicate p<0.05, the null hypothesis being that means of log transformed ratios are 0. Significance is measured by Mann Whitney U test or Two Sample T test, depending on whether distribution is normal or not.

2.5

OTNF-a:IL-lb * L-1b-inj:L-1b 0.5 . 0 TNF-a+inj-IL-1 b 0> -9 T T1 Figure 6: Figure 6 shows the behavior of different sets of proteins to the combination of Injury and Cytokine. All data represent mean log transformed ratios of the respective treatments wrt IL-1p +/- standard error. The proteins whose effects are measured are (A) C-terminal propeptides of Collagens. (B) Key structural proteins belonging to the ECM. (C) Members of the SLRP family, key structural molecules in the ECM. (D) Proteases including members of the MMP family and ADAMTS 1, and MMP inhibitors. (E) Members of the TGFp super-family and their inhibitors. (F) IGF binding proteins. (G) Proteins involved in the innate immune response. (H) Key signaling molecules. Asterisks represent ratios whose p values are significant. Significance is measured by Wilcoxon sign rank test or paired t-test under the null hypothesis that protein expression does not change under treatment. Multiple comparisons are corrected for using Benjamini Hochberg procedure at FDR 0.1

0.5

0. T

-0.5 .TNF-a.IL-lb .L-1b+inj :L-1b -- TNF-e+inj:IL-1b

-1.5 -- - - -

-2

-2.5 -

B 1.5

*TNF-a: IL-lb *L-ib+!rn: L-lb o TNF-a+inj :L-1b 14 0

0. MTNF-a: IL-lb ML-lb+inj: IL-lb E3TW-a-Hnj: IL-lb

0.5 0 mTW-a: IL-lb mL-lb+inj: IL-lb 2-0.5 c3TW-a4jnj: IL-lb el -

STNF-a: IL-lb mIL-lb+ini: IL-lb c3TNF-a+ini: IL-lb 1.5

1 ci 0.5

0 STM--a: IL-1b * L-1b+inj: IL-1b -0.5 E TM--a+inj: IL-lb

-1.5

-2

C- 1

0 mTNF-a: IL-lb m IL-1b+inj: L-1b o TiF-a+inj: IL-lb .- 2

-3

-4

*TNF-a:IL-lb * L-lb+inj:IL-lb 0 TNF-a+inj:IL-lb Figure 7: Comparison of iTRAQ data generated from this experiment (experiment A) with iTRAQ data generated from previous experiment performed in our lab (experiment B) 80. Proteins are identified in common experiments A and B and mean (TNF-a /IL-1p) ratios from experiment A compared with mean (TNF-a /IL-1p) ratios from experiment B. Only ratios whose respective p values satisfy Benjamini Hochberg procedure at FDR 0.1 are chosen for the respective plot. Histogram of log 2 transformed ratio of ratios (i.e. log2(exp B (TNF-a IL- 1p) / exp A (TNF-a /IL-i ) is plotted.The Mean +/- standard deviation is 0.1067 +/- 0.2191

-3 -2 -1 0 1 2 3 log2( expB(TNFIL1) / expA(TNFAL1)) Table 2: Matrix of correlation coefficients. Correlation coefficients are found between IL-1p only, TNF-a only, injury only, combination of (IL-l p and injury) combination of (TNF-a and injury) conditions. Log transformed protein ratios obtained from combining data from experiments A and B and converting to untreated basis are used as vectors to calculate correlation coefficients. Only ratios whose p values satisfy Benjamini Hochberg procedure at FDR 0.1 are used in this calculation

log 2(IL-1 P/untreated) log2(TNF-a/untreated) log2(inj/untreated) log 2(IL-1 P+inyuntreated) Iog2(TNF-a+inyuntreated) log2(IL-1p/untreated) 1 0.9028 0.1416 0.6839 0.5944 log2(TNF-a/untreated) 0.9028 1 0.3473 0.7707 0.7046 log2(inyuntreated) 0.1416 0.3473 1 0.7581 0.7486 log2(IL-1 P+inyuntreated) 0.6839 0.7707 0.7581 1 0.9574 Iog2(TNF-a+inj/untreated) 0.5944 0.7046 0.7486 0.9574 1 Table 3. List of proteins showing positive and negative synergism. The columns are protein name, log 2(TNF-a /untreated), log2(injury/untreated), expected log2(TNF-a +injury/untreated) if injury and cytokine acted independently, actual log2(TNF-a +injury/untreated), and percentage synergism (A). Lists proteins showing positive synergism. If the maximum ratio among log 2(TNF-a/untreated), log2(injury/untreated) and expected log2(TNF-a+injury/untreated) were called R1, the percentage positive synergism is calculated as (( actual log2(TNF-a+injury/untreated) - R1)/R1 )*100 (B). Lists proteins showing negative synergism. If the minimum ratio among log2(TNF-a/untreated), log2(injury/untreated) and expected log2(TNF-a+injury/untreated) were called R2, the percentage negative synergism is calculated as (( actual log2(TNF-a+injury/untreated) - R2)/R2 )*100

log2(1NF-a/untreated) log2(injury/untreated) Expected Actual log2(TNF- % hi gher than log2(TNF-+ a+injury/untreated) maximum of injury/untreated) if [TNF-a, injury, independent TNF-m+injury if indep endent] Aggrecan 1.014 -0.111 0.903 1.059 4.377%

Col6A3 0.468 0.684 1.152 1.389 20.588%

Histone H4 0.931 0.941 1.873 2.379 27.032/o replacement-like

CLP-2 0.406 -0.147 0.259 0.735 81.115%

log2(TNF-aluntreated) log2(injury/untreated) Expected Actual 1og2(TNF- % lower than log2(TNF-m+ a+injury/untreated) minimum of injury/untreated) if [TNF-a, injury, independent TNF-a+irnjury if independent]

SPARC -0.765 -1.084 -1.849 -3.599 94.631%

IGFBP7 -0.644 -1.110 -1.754 -2.141 22.025% Figure 8: Figure 8 is obtained by performing Partial Lease Square Regression (PLSR) between protein ratios and phenotypes (sGAG loss into medium in five days post treatment, nitrite accumulation in five days post treatment, rate of proline incorporation and rate of sulfate incorporation on the 6 h day post treatment). X represents matrix of log transformed protein ratios under five different treatment conditions, and Y matrix of log transformed phenotype ratios (wrt control) under the same conditions. The matrices X and Y are mean centered and set to unit variance before PLSR (A) Percent variation in the protein ratio matrix (X) explained by 1,2 and 3 Principal components (factors). Each vertical line represents a single protein (B) Percent variation in phenotypes explained by 1,2 and 3 principal components. The blue line is the training data set, and the red line the validation data set. In both (A) and (B), two factors explain the variation very well. (C) Projection plot of the five treatment conditions on the first two principal components. (D) Projection plot of proteins (blue) and the phenotypes (red) on the first two principal components. Points close to each other are positively correlated, and in opposite quadrants are negatively correlated. The Unscrambler @ X Version 10.0.1 used for generating plots A-D

Explained Vaniance Each line represents one protein 100

(D 40

130 - 20

-10.

-20

-30 Factor-0 Factor-i Factor-2 Factor-3 Factor-4 Factors Explained Variance 100

80,

60 so

-10

-20 - Factor-0 Factor-2 Factor-4 Factors

7- 6

4 3.1 2 -

15 injury -2 -3 L1 +inj

-5 -6 -7 TNF+inj -8 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 Factor-i (31%. 74%) 0.2-

MMP2 IGFBP7 SPARC Iaminin A4. nn~f?~ EDIL3 0.1 lactoran 4 proine

cathepsin B CILP2

COL6Al .COLGA3 FK8P 10

*iea Cnnexin A8 u sema 3

Factor-i (31%, 74%) Table 4: List of protein-phenotype pairs showing (A) The top five positive correlations and (B) The top five (in magnitude) negative correlations, as generated by PLSR.

Top 5 positive correlations Protein phenotype

Perlecan sGAG release

pleiotropin sGAG release

serum amyloid A3 sGAG release

CILP-2 sGAG release

compl B sGAG release

Top 5 negative correlations

Protein phenotype

pleiotropin proline incorporation

cathepsin B sGAG release

perlecan proline incorporation

pleiotropin sulfate incorporation

perlecan sulfate incorporation

Table 5: Effect of injury or cytokine treatment or both on different phenotypes. Rows represent treatment conditions and columns represent phenotypes. This data matrix forms the Y matrix in the PLSR analysis.

nmol proline microg sGAG per set nmol sulphate incorporation lincorporation / nmol Nitrite of 4 explants (in 120 day (Measured at the 6th day (Measured accumulation (in Experimental Condition hrs) day) at the 6th day) 120 hrs) Control 194.7035484 139.4045 68.32229 9.397892 injury 199.7683616 85.85213 50.74262 10.31592 L-1p 1045.690457 58.85496 37.03414 71.23021 L-1p+ injury 1164.124135 36.51318 28.51041 34.39866 TNF-a 829.7448476 66.59978 41.7247 104.7924

TNF-a + injury 2080.996078 34.77035 32.56093 152.8823 DISCUSSION

The aim of this study is to understand the molecular basis behind the synergism seen between cytokine and injury in causing sGAG loss to medium from cartilage explants as observed by Patwari et al' . With this aim in mind, we generate a quantitative profile of proteins released to medium on treatment of cartilage explants with cytokine (IL-1p or TNF-a), injury or a combination of cytokine and injury. By doing so, we hope to identify those proteins whose expression values differ most on combination of injury and cytokine, as compared to either treatment alone. Since we used a 4-plex iTRAQ labeling strategy, we could analyze only four treatment conditions at a time.

Therefore, in the first part of this study, we divided cartilage explants into four groups, and subjected each group to one of the treatments: IL-1f alone, TNF-a alone, IL-1p and injury and TNF-a and injury. Medium from each group was collected, iTRAQ labeled and a 2D-LC/MS/MS analysis performed. iTRAQ ratios from this experiment were specified with respect to IL-1p alone values and proteins whose levels differed significantly on treatment with cytokine and injury, as compared to IL- 13 alone screened.

Clustering and correlation analyses were performed to observe which proteins and which treatment conditions behaved similarly.

The next part of this study combined data from this experiment with earlier iTRAQ experiments in our lab 80 which contained treatment conditions not present in the first experiment (ie injury alone, and a control without any treatment). Data from both these experiments were combined after suitably correcting for inter experimental bias.

This helped generate all possible ratios for several proteins with respect to control (ie IL-

1p:control, TNF-a:control, Injury:control, IL-1f +injury:control and TNF-a +injury:control). These ratios were used to determine the proteins on which injury and cytokine acted synergistically, in either a positive or a negative fashion. These proteins thus identified are probably the proteins involved in causing synergistic sGAG loss to the medium at a macro level.

Since we have protein levels for several proteins across 5 treatment conditions with respect to control (IL-1:control, TNF-a:control, Injury:control, IL-1

+injury:control and TNF-a+injury:control), we have a huge data matrix in which we expect to see a lot of redundant information. To get the core non redundant information from this highly "collinear" data matrix, we next use a PLSR (partial least square regression) analysis, to determine those combinations of protein ratios (latent vectors) which contribute most to some observed phenotypes. The phenotypes used in our PLSR model are sGAG loss to the media in 5 days post treatment, rate of proline incorporation on day 6 post treatment, rate of sulfate incorporation on day 6 post treatment, and total nitrite accumulation in 5 days post treatment. Since redundant information is eliminated by this analysis, PLSR helps unearth non-obvious groupings of proteins which behave similarly on treatment, and proteins which are most responsible for an observed phenotype. For example, because sGAG loss to media is directly indicative of catabolism, and proline and sulfate incorporation rates of anabolism, the choice of these phenotypes in the PLSR model helps to identify proteins which closely correlate with catabolism and anabolism. 4.1 Methodology for correcting for multiple comparisons - The

Benjamini Hochberg procedure

Instead of correcting for multiple comparisons by controlling the Family wise error rate (FWER) using Bonferroni correction, we opt to adopt the procedure adopted by

Benjamini and Hochberg 112 to control the False Discovery Rate (FDR). The advantage of this methodology over the Bonferroni correction is that Bonferroni correction is exceedingly stringent and hence a lot of protein ratios which represent real differences may not be counted. Using the Benjamini - Hochberg procedure thus ensures fewer false negatives and more "hits" than the Bonferroni correction 117 . A FDR of 0.1 is chosen for our analysis.

4.2 Variation between biological replicates

The LC/MS/MS analysis was done on biological replicates. The source of variation between any two experiments can be broadly classified as technical, experimental and biological 116. Briefly, technical variation measures the variation observed by repeated tests on the same test subjects under identical conditions. In the iTRAQ experiment, this includes all steps from protein extraction till quantification.

Experimental variation in the context of iTRAQ experiments refers to variation caused due to repetition of an identical iTRAQ experiment. Biological variation measures biological variability in the test subject. Since the biological replicates in our experiments are also subjected to separate iTRAQ experiments using a different set of labels, the source of variation between these replicates is biological as well as experimental. We observe that log(ratiol/ratio2) for proteins identified in common between experiments 1 and 2 (only proteins which satisfy Benjamini Hochberg procedure are used in this comparison) has a mean of 10.1 % for the 115 label and 8.07 % for the 116 label (Figure

1). Both these are lesser than the 23 % average variation between experimental replicates and 25 % average variation between biological replicates as reported in an earlier iTRAQ study with a different model system 116.16 The log(ratiol/ratio2) for the 117 label however shows a difference of 43.17 % . This huge difference is possibly due to incomplete digestion in the iTRAQ2 117 label sample, where a white precipitate was observed after trypsin digestion. This implies that only a part of all proteins released to medium is subjected to LC/MS/MS analysis, and hence quantitation of this sample is naturally inaccurate. To correct for this difference, for all proteins identified in biological duplicate

2 alone and not in biological duplicate 1, 0.4317 is added to log transformed 117:114 ratios alone. For proteins identified by biological duplicate 1 alone or in common by both experiments, experiment 1 values are used for further analysis.

4.3 A global proteomic analysis of the effect of Cytokine+injury

4.3.1 Correlation between IL-1p +injury and TNF-a+injury treatment conditions

It is highly interesting that a global proteomic analysis between different treatments yields a very high correlation between the two cytokine+injury treatments, with a correlation coefficient of 0.914 (Table 1). (Only proteins whose p values suggest good quantification across all ratios are chosen for all global analysis). This implies one of two things, either that TNF-a and IL-1 interact similarly with injury and activate similar pathways in the cell, or that the effect of injury dominates over the effect of cytokine. To confirm which of these two hypothesis is right, we require data representing the effect of injury alone on cartilage, if the injury alone condition were to show a high correlation with both the (cytokine+injury) conditions, it would imply that injury dominates over cytokine; if not, it would imply that both the cytokines act on the cell through very similar pathways, thus causing very similar change in protein levels. This study is performed later in this discussion when we combine results from two iTRAQ experiments.

4.3.2 K-means clustering of proteins

K-means clustering is performed on the data matrix, comprising 199 proteins with

3 conditions, and the optimum number of clusters was chosen as three. Figure 3a shows a 3D plot of log transformed protein ratios, with the three treatment conditions as three axes, and clusters differentiated by color. The centroids of these three clusters are plotted versus treatment in Figure 4, to better illustrate the differences between the clusters.

Broadly, cluster 1 proteins show a mild elevation with (cytokine+injury), cluster 2 proteins are significantly depressed and cluster 3 proteins are highly elevated. A closer examination of the proteins present in each cluster reveals that about 73% of the proteins present in cluster 3 are cytoplasmic proteins. Therefore, most of the cytoplasmic proteins are highly elevated on treatment with (cytokine+injury). This is presumably because of lysis of the cell membrane caused by mechanical injury, and subsequent release of proteins in the cytosol to the medium .This is consistent with the study by Stevens et al

118 which also finds a high proportion of cytosolic proteins to be released to the medium on treatment with injury alone. We also find that all ECM proteins identified are present in either cluster 1 or 2 (i.e. slightly increased or highly depressed with (cytokine+injury)).

The structural proteins aggrecan, matrilin-1, matrilin-2, matrilin-3; the SLRPs chondroadherin, fibromodulin, epiphycan and biglycan all cluster together in cluster 1.

While collagens III, VI, XI and XII cluster together in cluster 1, collagens I and II cluster together in cluster 2. MMPs 2, 3 and 13 also cluster together in cluster 2, and are depressed by cytokine+injury.

4.4 Effect of Cytokine + Injury on specific protein classes

We next look at specific classes of proteins in greater detail, to find out how these key proteins vary with (cytokine+injury), to find out how (cytokine+injury) affects catabolism and synthesis. From this, we try to see if this in vitro model is representative of OA.

4.4.1 The Collagen Family

Collagen accounts for two-thirds of the dry weight of adult articular cartilage, and collagen fibers provide cartilage with the ability to withstand tensile and shear forces.

Fibrillar collagen chains are generally synthesized as procollagens, and formation of mature collagen fibrils requires cleavage of the N and C terminal propeptides of procollagens (for review 119). The cleavage and release of Collage C-terminal propeptides to the medium is thus indicative of the ability of cartilage to synthesize new collagen, and is a useful marker of the anabolic state of cartilage. In Figure 6a, we see that both

(cytokine+injury) treatments inhibit collagen synthesis of several collagen chains, with

TNF-a+injury inhibiting collagen synthesis to a greater extent than IL-1 +injury. This suggests that the combination of injury and cytokine reduces anabolic activity in the cell, and impairs the ability of cartilage to recover from damage. 4.4.2 Aggrecan

Aggrecan is the major proteoglycan present in hyaline cartilage, and is responsible for compressive stiffness of cartilage (for review 13). The core protein of aggrecan has a modular structure, with three disulfide-bonded globular domains G1, G2 and G3 and an extended central portion between G2 and G3 for GAG attachment

(chondroitin sulfate (CS) and keratan sulfate (KS)). G1 and G2 are separated by an interglobular domain (IGD) 120. Depletion of aggrecan due to its degradation is an important characteristic of OA. This occurs due to increased proteolytic cleavage of the core protein by MMPs'21 123 and primarily aggrecanases 2. In our study (Figure 6b), we find that

aggrecan levels remain almost constant on treatment with (injury + cytokine) with very mild down regulation (<0.5 difference on log 2 scale)

4.4.3 Other structural and matricellular molecules

Besides collagen and aggrecan, several other molecules are present in the ECM.

These either aid in structural roles, such as collagen fibril formation and collagen cross-

linking, or in remodeling of ECM. Thrombospondins are multi-domain, calcium binding

ECM proteins which have a high affinity to collagen (for review' 5 ). There are five

thrombospondins currently discovered. We identified all five in our study (Appendix),

but only Thrombospondins 1 and 5 were quantified well across ratios (Figure 6b). We

see that while thrombospondin-1 is only slightly elevated with (TNF-a + injury), COMP

(Thrombospondin-5) is highly down-regulated with TNF-a + injury. COMP is cleaved

by ADAMTS-4 and MMP-19 and 20 1'1 and elevated levels of COMP/COMP

fragments in the serum and the synovial fluid have been correlated with OA severity and joint injury 12,127 . However, a decreased amount of COMP remains in OA cartilage and the distribution also changes, with higher COMP in the fibrillated superficial zones, and almost no COMP in the middle and deeper zones 128. Since we are measuring proteins released to medium from cartilage explants, we would expect elevated levels of

COMP/COMP fragments on treatment with injury + cytokine, due to diffusion of newly synthesized COMP unanchored to the matrix due to proteoglycan loss; and cleavage with proteases respectively. Hence, it is surprising to see that (TNF-a + injury) decreases

COMP released to the medium significantly compared to IL-1 alone. One possible reason is that synthesis of new COMP is reduced. Since we did not observe ADAMTS-4 among our proteases (Figure 6d) , it is possible that the COMP we observe released to the medium is not due to degradation of existing COMP, but due to synthesis of new

COMP. Hence, if synthesis were reduced, we would observe reduction in COMP levels released to medium. Further studies have to be undertaken to find out if the COMP released is due to synthesis (i.e. whether the COMP released to medium represent intact

COMP) , and if so, reason out why COMP synthesis is reduced on treatment with (TNF- a+injury).

Among the matrilins, Matrilin-1 and 2 are almost unchanged with (injury + cytokine) (< 0.5 on the log scale). Matrilin-3 is moderately down regulated with (IL-1 + injury) alone. Matrilins are proteins thought to play a role in the formation of filamentous networks in the ECM through interacting with collagen and other molecules 31. A mutation in Matn3 gene was found to be linked to hand OA in a genome wide linkage analysis 129. While Ko et al 130 found Matrilin-3 deficient mice to have no skeletal malformations and normal life spans , Van der Weyden et al 131found an expanded hypertrophic zone of the growth plate in the embryonic stage due to premature chondrocyte differentiation and a higher predisposition for severe OA in one year old animals. An examination of the collagen architecture of Matn3- knock-out mice with electron-microscope showed mild abnormalities with slightly increased collagen fibril diameter and collagen volume densities 132. In view of these studies, the decrease in

Matrilin-3 we observe with (IL-1p +injury) may indicate premature differentiation of chondrocytes, with impaired collagen fibril network structure resulting in altered mechanical properties.

4.4.4 Small leucine rich proteins (SLRP)

Since SLRPs have been postulated to serve as biomarkers of OA 42, we next look at how these ECM proteins respond to treatment in our in vitro model. SLRPs are currently classified into four classes. Our study (Figure 6c) identifies decorin and biglycan (class

I); fibromodulin, lumican, PRELP and osteoadherin (class II); epiphycan and osteoglycin

(class III); and chondroadherin (class IV).

Decorin shows mild up regulation with IL-1 +injury (not attaining statistical significance) and moderate down regulation with TNF-a+injury. Disruption of the decorin gene in mice leads to fragile skin with markedly reduced tensile strength, with collagen fibrils thicker than wild type mouse, indicating that decorin may have a role in inhibiting fibrillogenesis. 133 . Decorin has a role in connecting fibrillar collagens such as collagens I or II with minor collagens such as collagens VI, XII and XIV 32,134,135

As opposed to decorin, Biglycan is highly over expressed with IL-1P +injury and moderately with TNF-a+injury. This is similar to the result obtained by Robbins et al 136 who found that mechanical injury up regulated biglycan in tendons. The precise interaction of biglycan with collagen is unclear; with some studies claiming that biglycan binds collagen 1137 and others that biglycan does not 138. Besides connecting collagen VI to collagen II, biglycan also appears to organize collagen VI microfibrils into a hexagonal network in vitro 139,32. Biglycan is known to be cleaved by MMP-13 at a specific location

140, but this is not the reason for increased biglycan levels with (cytokine + injury) in our study, as MMP-13 levels are seen to decrease with (cytokine + injury) (Figure 6d)

Biglycan and decorin differ in their pattern of temporal expression in corneal stroma development. While decorin was expressed at high levels in all stages of development, biglycan was expressed highly in early stages of development, decreasing until expressed at very low levels in mature cornea 141

Looking at Class II SLRP's, fibromodulin and osteoadherin does not change much with treatments. Lumican is slightly over expressed with IL-1p + injury, and highly with TNF-at + injury. PRELP is moderately over expressed with IL-1 + injury, and highly with TNF-ca + injury. Fibromodulin has two sites of different affinities at which it binds to collagen, and lumican has one site. The lumican site and the low affinity fibromodulin sites are homologous and compete in binding collagen I, but fibromodulin has a higher affinity to collagen I because of the presence of an additional high affinity site 12. During tendon development, lumican is expressed earlier and fibromodulin later, with lumican deficient mice showing distorted fibril morphology earlier, and fibromodulin deficient mice later. This could possibly be because lumican aids in forming early thinner fibrils, and once fibromodulin levels rise later in development, it displaces lumican, and helps join the thin fibrils to form thicker fibrils 39.

It is highly interesting that both lumican and biglycan, both of which are expressed earlier in development show high expressions with cytokine and injury. This possibly indicates an attempt to repair tissue damage by development of new collagen fibrils.

Also, our results for decorin, biglycan, fibromodulin and lumican are identical to the results obtained by Young et al in a sheep model of OA (143. They found increased mRNA levels of lumican and biglycan, decreased decorin levels and unchanged

fibromodulin levels, identical to what we observe at a protein level in our in vitro model

of injury. This indicates that the changes in protein levels we observe are probably the

result of new protein synthesis of SLRPs which again suggests an attempt to repair

cartilage. This also shows that the combination of injury and cytokine shows a

remarkably similar SLRP profile as compared to OA tissue, and hence this in vitro

treatment condition approximates OA reasonably well.

PRELP is highly up regulated with cytokine+injury in our study. While most

SLRPs have amino terminal domains with acidic properties, PRELP has a basic amino

terminal region. It also has no GAG chains. PRELP is capable of binding heparin and

heparin sulfate containing proteoglycans such as perlecan through its amino terminal

region, and collagen type I and type II through its Leucine rich repeat region. Hence, it

possibly connects the basement membrane (rich in perlecan) to the underlying connective

144 145 tissue . PRELP is expressed highly in cartilage 4. Over expression of PRELP in skin,

where it is normally present at low levels, results in a structural change, with decrease in

collagen fiber bundle content and size, and decreased thickness of hyperdermal fat layer

146 . However, the collagen fibrils obtained are of normal structure. It is not clear what the

high up regulation of PRELP with (cytokine+injury) means, and further knowledge about

PRELP and its functions is necessary to interpret this result. Among class III SLRPs, epiphycan shows moderate up regulation with (IL-1p + injury), and is not changed much with (TNF-a + injury). Epiphycan-deficient mice generated by disrupting the epiphycan gene in mouse embryonic stem cells developed

OA, and male epiphycan-deficient mice had shorter femurs than wild type mice. Also, epiphycan-biglycan double deficient mice developed OA earliest suggesting possible interaction between both in vivo 47 Osteoglycin does not show statistically significant change with (cytokine + injury).

The class IV proteoglycan chondroadherin shows high up regulation with TNF- a+injury, and is not changed much with IL-1p + injury. Chondroadherin is particularly enriched in cartilage, but also found in bone. It binds to collagen type II 148 , and also to N and C terminal globular domain of collagen type VI 32. It binds to integrins on the surface of cells, and causes intracellular signaling through tyrosine phosphorylation of factors like ERK 149. Like PRELP, chondroadherin too has a heparin binding domain via which it can bind to perlecan and cell surface receptors carrying heparan sulfate proteoglycans 15.

The elevation of chondroadherin with (TNF-a+injury) may thus indicate an attempt to repair the collagen type II network. Chondroadherin elevation may also cause changes in synthesis of other proteins through intracellular signaling via cell surface receptors.

4.4.5 Matrix proteases and their inhibitors

A healthy cartilage lies in equilibrium between synthesis and degradation. Matrix degrading proteases such as collagenases and aggrecanases play a crucial role in maintaining this equilibrium 151; however, aberrant regulation accompanied by over expression of these proteases result in a shift of the cartilage away from equilibrium towards degradation resulting in degenerative diseases such as OA 9. It is believed that aggrecanases act in early stage OA, and collagenases in late stage OA. A study by

Karsdal et al 54 shows that aggrecanase mediated aggrecan degradation in cartilage can be reversed by anabolic stimuli, provided catabolism has not been too severe. However,

MMP mediated collagen and aggrecan degradation impaired the repair capacity of cartilage, and was irreversible.

MMP-13 is thought to be the major collagenase in OA 152 and is highly over expressed in

OA cartilage. Mice knockout experiments showed significant protection against OA in

ADAMTS-5 single knockout and ADAMTS-4/ADAMTS-5 double knockout mice, but lack of protection in ADAMTS-4 single knockout mice 153,154. A study by Kevorkian et al

155 profiling several MMP, ADAMTS and TIMP (Inhibitors of MMP and ADAMTS) in end stage hip OA cartilage found increased genetic expression of the gelatinases MMP2 and MMP9, MMP13, ADAMTS2 and TIMP3 and decreased expression of MMP1, MMP3,

ADAMTS1, TIMP1, TIMP4 and ADAMTS5 at various levels of statistical significance.

However, MMP1 was increased in knee cartilage indicating differences in regulation of

MMP1 between hip and knee. Another global study by Swingler et al 156 which profiled expression levels of several genes in hip OA and compared them to normal cartilage found an increase in expression of MMP13, MMP3, MMP9, ADAMTS2, MMP2 and a decrease in expression of ADAMTS9, ADAMTS1, MMP1 among others at P<0.01

Figure 6d shows the response of members of the MMP, ADAMTS and TIMP families to the combination of cytokine and injury. Several MMPs identified in our study

(MMP's-1,2,3,9) show very mild down regulation with (IL-1 +injury) as compared to

IL-13 alone, with MMP-2 alone showing moderate down regulation with (TNF- a+injury). The gelatinases MMP-2 and MMP-9 seem to be almost unchanged .MMP-13 alone shows high down regulation with (IL-1 +injury), and a mild down regulation with

(TNF-a+injury), both showing statistical significance. This is contradictory to studies which show that MMP-13, whose main substrate is Collagen 1I, is the major collagenase involved in OA 1.57 It is surprising that the collagenases (MMP-1, MMP-13), the gelatinases (MMP-2, MMP-9) and stromelyin-1 (MMP-3) all mostly mildly down regulated with (cytokine+injury) with respect to IL-1. With the mild down regulation possibly due to apoptosis of some cells due to injury, this means that addition of injury to cytokine does not change the expression of MMP as compared to cytokine in general.

Therefore, addition of injury to cytokine does not increase catabolism due to MMP.

However, ADAMTS-1, the only ADAMTS identified in our study, shows significant up

regulation with (cytokine+inury) for both IL-1p and TNF-a. ADAMTS-1 does not cleave

63 any member of the collagen family, but cleaves aggrecan and versican

Among the TIMPs, both TIMP-1 and TIMP-2 were identified. TIMP-1 shows

decrease with TNF-a+injury, without reaching statistical significance. TIMP-2 on the

other hand is moderately up regulated with IL-1 and injury, and not changed with TNF-

a+injury. TIMP-1 has previously been shown to be significantly down regulated and

TIMP-2 up regulated in OA hip cartilage 155. The up regulation of TIMP-2 might be an

attempt to inhibit ADAMTS-1, which is seen to be up regulated, and against which

TIMP-2 has inhibitory activity.

4.4.6 Members of the TGF-p superfamily and their inhibitors

The TGF-P superfamily refers to a large family of growth factors which are

predominantly anabolic in nature and includes the TGF-3 subfamily, the activin/inhibin subfamily, bone morphogenetic proteins (BMP) and some other members 158 . TGF-p causes net synthesis of the ECM by inhibiting collagenases and up regulating TIMP 159.

Treatment of chondrocytes with TGF-p1 is known to down regulate mRNA levels of

MMP-1 and MMP-13 and also reduce mRNA levels of IL-1 and TNF receptors 160, thus moving the chondrocyte towards an anabolic state. TGF-p also increases net proteoglycan synthesis as measured by sulfate incorporation in vivo 161. TGF-1 and TNF-a are known to increase their own mRNA levels and reduce each others', resulting in an autocrine feedback loop shifting between anabolism and catabolism.

Connective tissue growth factor (CTGF) is a growth factor, known to be inducible by TGF-, which synthesizes collagen and fibronectin in fibroblasts 162. It is involved in fibrotic diseases, characterized by excessive collagen deposition. CTGF null mutant mice develop cartilage which has impaired mechanical properties due to reduced aggrecan and link protein production 163. CTGF has previously been identified in human osteoarthritic cartilage 164 , but at very low levels. CTGF synthesis as measured by mRNA levels has been found to reduce in a sheep model of OA 143

Cartilage derived morphogenetic protein - 2 (CDMP-2) is a bone morphogenetic

protein belonging to the TGF-3 super-family. Along with CDMP-1 and CDMP-3, they

form a distinct subgroup within the BMP family 165 Expression patterns of CDMP-2 in

bovine cartilage indicate that it is involved in terminal differentiation of chondrocytes and

at the earliest stages of endochondral bone formation, including angiogenesis and

osteoblast differentiation. CDMP-2 implantation subcutaneously or intramuscularly is

known to induce bone or cartilage; or tendon, depending on the degree of mechanical

stimulus, higher mechanical stimulus leads to bone formation, and lower, tendon'166 Activin and inhibin are two closely related members of the TGF- superfamily which have opposing functions. While activin enhances FSH biosynthesis and has roles in cell proliferation, differentiation, apoptosis, inflammation, fibrosis and wound repair 167,168. inhibin down regulates FSH synthesis 169. Activin consists of inhibin P chains alone, while inhibin contains both inhibin a and inhibin p 1. Proinhibin PA and mature inhibin

PA were detected in all OA samples but only in half the control samples in a study by

Hermannson et al 164 indicating presence of activin A in OA cartilage. Hermannson et al also found that activin A was induced in chondrocyte monolayers by IL-13, TGF-31,

FGF-2, PDGF,EGF and activin A itself. Also, it is seen to induce TIMP-1 production in culture medium, and thus acts as an anabolic factor.

Members of the TGF-$ superfamily such as TGF-, BMPs and activin are regulated at several different levels (for review 1. LTBP (Latent TGF-p binding proteins) are proteins, which as the name suggests, bind to TGF-p and another protein, called Latency associated proteins (LAP), thus maintaining TGF-3 in an inactive form.

Cleavage of LTBP is necessary for TGF- to be active. LTBPs are also involved in assembly, secretion and targeting of TGF-p to its site of action. (for review 172). MMP-2,

MMP-3 and MMP-9 have been shown to cleave LTBP, thus releasing active TGF-p 41

LTBP-3 null mice develop craniofacial abnormalities and eventually develop osteoarthritis. 73

Looking at other inhibitors of the TGF- superfamily, the ECM proteins decorin and biglycan have been seen to inhibit TGF- activity 174175. Chordin and gremlin are soluble extracellular inhibitors of BMPs 176. Chordin-like 2 is a protein structurally similar to chordin which inhibits BMP in vitro and in vivo. It is specifically up regulated in the middle zone of OA cartilage and may delay chondrocyte hypertrophy, thereby ameliorating cartilage degeneration7 Vasorin is a type 1 membrane protein which is observed to directly bind to TGF- and inhibit its action 178. Follistatin is an autocrine glycoprotein whose primary function is the inhibition of activin 79. Follistatin-like protein

1 is a secreted glycoprotein sharing a characteristic structural molecule (the FS domain) with follistatin. It has a role in inflammation and has been shown to both be highly over expressed in early arthritis, leading to up regulation of pro-inflammatory cytokines like

IL-1 and TNF-a 180; and contradictorily to ameliorate arthritis severity in a mouse model' 81 .

In our results, looking at members of the TGF-P superfamily (Figure 6e), we see that TGF-2 itself is highly elevated with TNF-a + injury, and moderately with IL-1f + injury. LTBP-1 is highly down regulated for both the (cytokine + injury) conditions.

Down regulation of LTBP implies that TGF-p is activated from its latent form, thus increasing net synthesis and moving towards anabolism. Inhibin BA is extremely elevated with the (cytokine + injury) treatments. Only inhibin PA is found and not inhibin a, which implies that activin A which is a homodimer of inhibin PA is up regulated with

cytokine+injury. This again indicates a shift towards anabolism, and tallies with the fact

that activin A is found to be upregulated in OA samples 164. Follistatin-like 1 is also

moderately down regulated with (TNF-a + injury), which could mean reduced

inflammation, this tallies with the high levels of activin A observed.

CTGF is extremely down regulated with (TNF-a + injury), indicating cartilage with

impaired mechanical properties. However, its levels don't change much with (IL-1p +

injury). One possible reason why CTGF is inhibited is the sequestering of TGF- by SLRPs like biglycan which show increased levels with (cytokine+injury) (Figure 6c).

However, it is unclear why CTGF levels are unchanged with (IL-1p + injury) if this be the case, as biglycan levels are increased both with (IL-1P + injury) and (TNF-a + injury). Other inhibitors of the TGF-6 superfamily such as chordin and gremlin remain almost the same with (cytokine + injury) treatment.

4.4.7 Insulin-like Growth Factor Binding Proteins

IGF (Insulin-like growth factors) are ubiquitously expressed mitogens showing high sequence similarity to insulin and which affect cell growth and metabolism. They are predominantly anabolic in nature and known to stimulate DNA synthesis, protein synthesis, proteoglycan synthesis and glycosaminoglycan synthesis 1 82 . There are two forms of IGF, IGF-1 and IGF-2. In cartilage, IGF-1 acts as an enhancer of matrix

synthesis and a growth promoter, and IGF-2 acts as a growth stimulator in a non-

differentiated state 183. Their actions are mediated by IGF receptors. IGFBP (IGF-binding

proteins) are soluble proteins which bind to IGF in biological fluids. They have higher

affinity for IGF than IGF receptors, and thus act as carriers of IGF and also modulators of

IGF availability and activity (for review182,184). IGFBPs also have actions independent of

IGF. For example, IGFBP-5 administration increased bone formation parameters both in

vitro and in vivo with a magnitude comparable to IGF-1185 and IGFBP-7 plays a potential

186 tumor suppressor role in colorectal carcinogenesis186

In our study (Figure 6f), four IGFBP's were identified, IGFBP-3,5,6 and 7.

IGFBP-3 is moderately up regulated with (IL-13 +injury), and up regulated with (TNF-a

+ injury) without approaching statistical significance. This represents a move towards

catabolism, as IGFBP-3 will sequester IGF and prevent IGF mediated anabolism. IGFBP- 3 has previously been found to be higher in severe OA cartilage as compared to mild OA in a statistically significant manner, but difference between mild and intermediate or intermediate and severe does not approach statistical significance 187. A regression done in the same study between OA score and IGFBP-3 levels show a very weak but statistically significant dependency. The up regulation seen in IGFBP-3 may be because of increase in IGFBP-3 in either matrix or in cytoplasm or nucleus of chondrocytes, where it is seen to localize 188. This might be because of a role of IGFBP-3 independent of IGF.

IGFBP-5 is highly up regulated with (TNF-ct + injury) in our study, but the level remains the same with (IL-1 + injury). This possibly represents an attempt to repair cartilage as increase in IGFBP-5 has previously been seen to increase IGF-1 and thus improve joint architecture during OA development 89. IGFBP-5 is also increased in chondrocytes and bone matrix during arthritic joint deterioration 90. IGFBP-5 is degraded by complement component Cis. Figure 6g shows that complement component Cis is mildly down regulated with both the (cytokine + injury) conditions. This mild down

regulation might partly explain the elevation of IGFBP-5 in the (TNF-a + injury)

condition, but it isn't known why the same down regulation of Complement Cis in the

(IL-13 + injury) condition does not cause an increase in IGFBP-5.

IGFBP-6 is one of the predominant IGFBP's found in bovine cartilage, and not

found much in human 191,192. We find IGFBP-6 to be mildly down regulated in both the

(injury + cytokine) conditions.

IGFBP-7 is moderately down regulated with (IL-1 +injury), and highly with

(TNF-a + injury). This protein is discussed in greater detail in a later section. It is difficult to say with certainty how change in levels of IGFBPs are affected/affect any roles of IGFBP independent of IGF, as these roles are yet unclear.

4.4.8 Proteins associated with the innate immune response

The complement system has traditionally been associated with the innate immune response, functioning in host defense against invading pathogens (for review 19. It does this through the release of inflammatory mediators and has been implicated in the pathogenesis of several autoimmune, ischemic and vascular diseases 194 '195. The complement system also serves as a vital link between innate and acquired immunity196

Complement component C1 triggers the classical component pathway. It is a complex

formed by association of the recognition protein Clq with two copies of two proteases

Cir and Cis. Clq binds the target, thus triggering self-activation of C1r, which converts

pro-enzyme Cis into a protease which cleaves C4 and C2, thus activating the classical

component pathway197 . The component Clq is also known to bind to, and induce

clearance of apoptotic cells198 .

The complement system is also involved in functions very different from its

traditional role in the immune response. It may be involved in the replacement of

199 cartilaginous matrix by endochondral bone in early fetal development' . The

complement component C3 may be involved in osteoclast development by potentiating

M-CSF dependent proliferation of bone marrow cells and induction of osteoclasts 200

Complement proteins C3, B, C5, C9 and properdin are localized to distinct areas in the

developing endochondral bone, suggesting that complements proteins are involved in the

cartilage cell death, vascularization and hence the transformation of cartilage to bone 201

A significant role for the complement system exists in rheumatoid arthritis (for review 202). Complement component C3 and C4 are drastically reduced relative to total protein in the synovial fluid of patients with RA. Genetic deletion of complement component C5, C3 or factor B in arthritis susceptible DBA/1 mice resulted in each case

203 204 in mice resistant to collagen induced arthritis (CIA) , . Wu et al 205 found complement components such as complement factor B, complement component 4 binding protein, complement 9 and complement factor H-related protein 5 to be over expressed in OA

cartilage compared to normal cartilage.

We identify complement factors B, I, components C3, C4-A, and subcomponents

C1r, Cis, and the A, B and C subunits of subcomponent Clq in our study (Figure 6d).

Cl-inhibitor is also present. Factor B is moderately down regulated with (IL-1P + injury)

and almost unchanged with (TNF-a + injury). Factor C3 is highly suppressed with both

(cytokine + injury) conditions. Injurious compression of cartilage is known to induce

apoptosis82. Since both factor C3 and B are known to be increased with inflammatory

cytokines such as TNF-a20 6 , it is likely that the combination of injury with these

cytokines results apoptosis of some cells, thus causing reduced levels of complement B

and C3 production. Subcomponents Cis, Cir and Clq are mildly downregulated with

(IL-13 + injury), and CIr moderately down regulated with (TNF-a + injury). C1 inhibitor

is highly down regulated with IL-1 + injury, but almost remains the same with (TNF-a +

injury). Deficiency of C1 inhibitor is associated with autoimmune diseases such as

systemic lupus erythematosus207

Peptidoglycan recognition proteins (PGLYRP) are innate immunity proteins

which recognize bacterial peptidoglycan and function in antibacterial immunity and

inflammation (for review 208). Four mammalian PGFLYRP's (PGLYRP 1-4) have been identified. While human PGLYRPs have the highest affinity for peptidoglycans, bovine

PGLYRP1 has the highest affinity for LPS (Lipopolysaccharide) and LTA (Lipoteichoic acid). PGLYRP2 plays a pro inflammatory role in a model of peptidoglycan-induced arthritis in mice, with PGLYRP2- mice resistant to peptidoglycan induced arthritis and inflammation209 PGLYRP1 on the other hand has an anti inflammatory effect. In our

study, we identify both PGLYRP1 and PGLYRP2. PGLYRP1 is down regulated mildly with IL-13 + injury and moderately with TNF-a + injury compared to IL-1 alone.

PGLYRP2 on the other hand remains almost the same with (TNF-a + injury) (without

attaining statistical significance) and is highly down regulated with (IL-1B + injury). It is

curious that both PGLYRP1 and PGLYRP2 are down regulated with (IL-1P + injury),

since they have anti and pro inflammatory activities respectively. Since PGLYRP2 is

down regulated to a greater extent than PGLYRP1, this might imply an attempt to reduce

inflammation and repair tissue. Since IL-1 and TNF-a are pro-inflammatory cytokines,

we would expect that IL-1 alone/TNF-a alone would increase PGLYRP2 and decrease

PGLYRP1 compared to [IL-13 + injury]/[TNF-ax + injury], unless mechanical injury

increases chondrocyte access to the cytokines in the combination treatment. Some as yet

undiscovered function of the PGLYRPs may account for the way their levels change with

(cytokine + injury).

CD14, a membrane protein commonly found on monocytes/macrophages, is

critical for activation of the innate immune system by lipopolysaccharides2".

Lipopolysaccharide binding protein (LBP) binds to LPS on bacteria and transfers it to

CD14 on macrophages. CD14 and LBP are necessary and sufficient for the activation of

the Toll-like receptor system211. CD14 is moderately down regulated with IL-1P + injury and very mildly with TNF-a + injury. LBP is moderately down regulated with both the

(cytokine + injury) conditions. The down regulation of CD14 and LBP with both the

(cytokine + injury) conditions as compared to IL-1 alone signifies that the inflammation and macrophage activation caused by cytokine alone conditions is reduced on addition of

injury.

4.4.9 Signaling molecules

Proenkephalin is one of the proteins whose expression changes the most with

(cytokine + injury) in our study, showing -8 fold increase and -13 fold increase with IL-

1p + injury and TNF-a + injury respectively as compared to IL-1P alone (Figure 6h).

Proenkephalin is the precursor of enkephalin, opioid peptides which are

neurotransmitters, neurohormones and neuromodulators. Initially associated with the

nervous and neuroendocrine systems, proenkephalin was found to be highly elevated in

non-differentiated cells of diverse mesodermal lineages in the process of development

into adult tissues, including cartilage mm. It was, however, almost undetectable in

differentiated tissue, suggesting a role in cell proliferation/differentiation. Proenkephalin

expression was closely associated with cell proliferation and stimulated by TGF- in

chondrocytes. While very few primary chondrocytes express proenkephalin mRNA, a

high proportion of subcultured cells do . Rosen et al hypothesize that a small proportion

of undifferentiated cells in differentiated tissue retain the ability to synthesize

proenkephalin, and do so in situations demanding repair 66. Recent studies by McTavish et

a12 15 show that proenkephalin assists stress activated apoptosis by transcriptional

repression of p53 and NF-KB gene targets. Therefore, the high elevation of proenkephalin with cytokine+injury could indicate an attempt to repair damaged tissue. It could also

help assist in apoptosis of cells, which has been observed with mechanical injury8 2

AIMP1 (p43) is another signaling protein highly elevated with (cytokine+injury).

AIMPI was first identified as a factor associated with a macromolecular tRNA synthetase

complex in mammalian systems 216. It was later found to be secreted and acts on diverse

target cells such as endothelial cells, macrophages and fibroblasts to control

angiogenesis, inflammation and dermal regeneration respectively. It is seen to activate

MAPK and NF-KB, and activates cytokine and chemokine genes involved in

inflammation such as TNF-a, IL-1p, IL-8, macrophage inflammatory proteins MIP-la

(CCL3), MIP-2a, MIP- 13 (CCL4) and RANTES (CCL5)m. It promotes endothelial cell

death by apoptosis at high dose 218, but also promotes fibroblast proliferation and wound

repair2. AIMPI has not been identified in cartilage in any study so far. The up

regulation of AIMPI with cytokine+injury might indicate inflammatory response, but it

is doubtful that cytokine+injury might have such highly elevated levels of inflammation

compared to cytokine alone (as the cytokines IL-1 and TNF-ax are highly pro

inflammatory themselves, and addition of injury would not increase inflammation). It

could represent an attempt to repair tissue, in line with its role in dermal wound repair, or

it could be involved in apoptosis caused by injury. Since AIMP1 seems to be a highly

multi-functional protein with different domains contributing to different functions220, it

could play any of these roles here, and further studies are needed to clarify the role of

AIMPI in stressed cartilage.

Annexins A2 (II) and A5 (V) show high elevation with TNF-a + injury. Annexin

A5 is also highly elevated with IL-1 + injury, and Annexin A2 moderately. Annexins are a family of proteins with the ability to bind to acidic phospholipids in the presence of

Ca*. Three annexins, 1I, V and VI are known to be highly expressed in calcifying cartilage and bone 2. Annexins II, V and VI are major components of matrix vesicles

(MV), particles released from plasma membrane of hypertrophic chondrocytes or osteoblasts, which initiate mineralization of cartilage. These annexins form calcium channels in MV's and mediate rapid influx of Ca** into the vesicles which serves as the start of the mineralization process. Annexin mediated Ca"* influx also leads to terminal differentiation and apoptosis of growth plate chondrocytess,222 . The up regulation of

Annexins II and V in our study with (cytokine+injury) therefore indicates the onset of mineralization, terminal differentiation and apoptosis in cartilage, which are features of

OA cartilage as well. SPARC (osteonectin) shows high down regulation with (TNF-a + injury), and is discussed in greater detail later in this discussion.

4.5 Comparing the effect of combination of cytokine and injury as opposed to either treatment alone

4.5.1 Combining data from two iTRAQ experiments

Since we used a four-plex iTRAQ experimental design, we chose iTRAQ

114,115,116 and 117 labels to represent IL-1 alone, TNF-a alone, IL-1 + injury and

TNF-a + injury treatments respectively. Since we had only four labels available to us, we could not introduce an untreated control, and a sample for injury alone treatment.

However, for a true study of whether there exists synergy between injury and either cytokine in their effect on protein levels, we require data for six treatment conditions, namely untreated control, IL-1 alone, injury alone, IL-1 + injury, TNF-a alone and TNF-a + injury. Therefore, we combine data from this experiment with a previous

experiment done in our lab (which had untreated control, IL-13 alone, TNF-a alone and

Injury alone treatment sample data8 0) to generate all possible ratios with respect to

control.

4.5.2 Variation between the two iTRAQ experiments

We first check for reproducibility between the two iTRAQ experiment data, by

looking at the only common ratio between the two experiments, TNF-a/IL-1. To do this,

we first identify proteins which are found in common between experiment A (this

experiment) and experiment B 80, and which are quantified well across all ratios. We

identify 74 such proteins. The log of ratio of (TNF-a/IL-1P) ratios from experiment A

and B for these proteins has a mean of 0.1067. This variation of 10.67% is again lesser

than the 23% and 25% variation seen between experimental and biological replicates in

an earlier iTRAQ study1 1 6. Therefore, for these 74 proteins, data from both experiments

are merged and converted to ratios with respect to untreated controls.

4.5.3 Correlation between cytokine alone, injury alone and cytokine+injury

treatment conditions

A matrix of correlation coefficients is thus generated for these 74 proteins for the

five ratios (IL-1I:control), (TNF-a:control), (injury:control), (IL-1IP+injury:control) and

(TNF-a+injury:control) (Table 2) . We see that while IL-1 alone and TNF-a alone have

a correlation of -90.2%, (IL-1 + injury) and (TNF-a + injury) have a 95.7% correlation.

It is highly interesting that in the presence of injury, the two cytokines show an even

higher similarity in their effect on proteins than in the absence of injury. This shows that while IL-1 and TNF-a themselves have very similar effects on cells globally, the

addition of injury further augments this similarity. This high similarity between the two

(cytokine+injury) conditions is not due to the effect of injury masking the effect of

cytokine, as the correlation between injury alone and (cytokine+injury) (-75%) is much

less than the 95% correlation between the two (cytokine+injury) conditions. One possible

hypothesis to explain higher correlation the (cytokine+injury) treatments (as compared to

correlation between cytokine alone treatments) is that the combination of injury with IL-

1p and TNF-a may activate some new pathways in the chondrocyte, in addition to

pathways activated by cytokine alone and injury alone, and that these new pathways

activated are common for both (IL-1 + injury) and (TNF-at + injury).

4.5.4 Proteins on which injury and cytokine act in a synergistic fashion

To identify proteins on which injury and cytokine act synergistically, we look at

proteins for which the effect of (injury + cytokine) is greater than that caused by injury

alone or cytokine alone. If the ratio of (injury + cytokine) for a protein is greater than the

injury alone ratio, the cytokine alone ratio, and the product of injury alone and cytokine

alone ratios (the ratio expected if injury and cytokine act independently), injury and

cytokine are identified as exerting a positive feedback on each other's action (positive

synergism) for that protein. On the other hand, if the ratio if (injury + cytokine) is lesser

than the injury alone ratio, the cytokine alone ratio, and the product of these ratios, injury

and cytokine are said to exert a negative feedback on each other's action for that protein. By having such a stringent criterion, we make sure that we pick only proteins on which injury and cytokine truly act synergistically, and avoid artifacts due to quantitation error, and any error in combining data from the two experiments.

When we thus look at proteins which satisfy this criterion, we find that among our

list of 74 proteins, for no protein does IL-1P and injury exhibit positive or negative

feedback. However, we identify a few proteins for which TNF-a and injury act

synergistically. TNF-a and injury exhibit positive feedback for Aggrecan, Histone H4,

CILP-2 and COL6A3 ; and negative feedback for SPARC and IGFBP7 (Table 3).

Aggrecan is almost doubled with (TNF-a) alone, and slightly down regulated with

injury alone, but when injury and TNF-aL are combined, the level rises to more than that

by TNF-ax alone. The percentage synergism is 4.3%. This small synergism may be due to

enhanced transport in the ECM caused by injury, which allows better access of aggrecan

to ADAMTS and MMP, thus causing higher cleavage and greater release of cleaved

fragments to medium, resulting in increased levels.

CILP-2 shows the highest percent positive synergism (81%) among these four

proteins. Cartilage intermediate layer protein 2 (CILP-2) is an isoform of the ECM

protein CILP-1. CILP-1 and CILP-2 are 50.6% identical and around -66% of their amino

acids are conserved 38. CILP-1 has been found to bind to TGF- and act as a negative

regulator of TGF-p in chondrocytes, thus preventing TGF-p induction of aggrecan and

Collagen type I 223. It is also seen to block the ability of IGF-1 to decrease extracellular

inorganic pyrophosphate; however, CILP-2 has no such effect 38. A protein profile of

human OA cartilage yielded CILP-2 fragments, suggesting possible CILP-2 cleavage in

OA 224. The increase in CILP-2 levels we observe due to synergism between injury and TNF-a may thus be due to cleavage of CILP-2. Further studies on the function of CILP-2 may help in interpreting these results better.

The alpha III chain of Type VI collagen (COL6A3) shows 20.6% positive synergism between TNF-a and injury. Collagen VI is structurally composed of three different peptide chains, al(VI), a2(VI) and a3(VI); a3(VI) (260-300 kDa) is much larger than al(VI) and a2(VI) (both around 140 kDa). It is an ECM protein which forms a microfibrillar network around or in between collagen type II fibers in cartilage; it is concentrated in areas surrounding chondrocytes225 226 . It interacts with several matrix constituents including collagens type Im and type II228 ; SLRP's biglycan, decorin, chondroadherin 32 , fibronectin 229 and basement membrane Collagen type IV2 . It also binds to alp1 and u2 1 integrins and thus maybe involved in cell signaling231. Thus, roles of collagen VI include structural roles such as anchoring the basement membrane to the connective tissue, and serving as scaffolds for formation of fibrillar collagen networks; and signaling roles, such as mediating the interaction of cells with the ECM.

Therefore, it could be involved in roles ranging from wound healing to tissue

232 development and architecture 2. Thus, the positive synergism displayed between injury and TNF-a for COL6A3 possibly represents an attempt to form collagen microfibrils to repair damaged tissue.

Histone H4 replacement-like protein also shows considerable synergism

(27.03%). This protein resembles histone H4, which is one of the basic units of chromatin architecture in the nucleosome. DNA wraps itself around the nucleosome composed of histones. Because of this close association with DNA, histones are involved in DNA transcription, replication, DNA damage and its repair, recombination and overall chromosomal stability 233. Histones are basic proteins with a globular domain and a charged NH 2- terminus (histone "tails") protruding from the nucleosome. These tails are highly involved in modulation of chromatin architecture, either by serving as targets for chromatin remodeling factors such as Swi/Snf and NURF; or by means of diverse post translational modifications such as acetylation and phosphorylation (for review2 3 4).

Synthesis of histones is closely associated with DNA replication during S-phase of the cell cycle, and hence histone gene expression can be used as a marker for proliferation 235 ,236. Therefore, the synergistic up regulation by TNF-a and injury of

Histone H4 replacement-like protein possibly represents increased cell proliferation due to repair of damaged tissue.

SPARC and IGFBP7 are the proteins which show reduced expression because of negative feedback between TNF-a and injury. SPARC is a glycoprotein present in the

ECM which mediates cell-matrix interactions but does not serve structural roles. It is a

Ca** binding protein which acts as a counter-adhesive protein, a modulator of growth factor activity and a cell cycle inhibitor. It is expressed in high levels in tissues undergoing remodeling, whether naturally, due to wound healing or due to disease (for reviewm2 1). SPARC is stimulated by several members of the TGF- super family including TGF-1, BMP-2 and activin, and inhibited by bFGF, IL-1, TNF-ax, dexamethasone and LPS 238-240 .

SPARC has been shown to induce production of MMPs 1, 3 and 9241; and also to activate MMP-2242 It also binds to several collagens including collagen type I, III, IV and V243. SPARC synthesis is markedly enhanced in arthritic joints (OA or RA), and in such joints has been hypothesized to induce MMP production in the surface zone, and modulate chondrocyte proliferation, matrix synthesis and mineralization in the middle and deep zones 240. A proteomic study by Wu et al 205 found SPARC to be increased in

OA cartilage compared to normal. In this context, it is highly surprising that we observe negative feedback between TNF-a and injury, resulting in down regulation of SPARC.

We would expect up regulation, both in view of the fact that SPARC elevation has been reported in OA cartilage, and that IL-1 and TNF-a alone reduce SPARC mRNA levels.

A possible reason could be high levels of apoptosis with injury, leading to a less number of SPARC producing cells. Increased transport of TNF-a due to increased permeability caused by injury and subsequent inhibition by TNF-a could be another reason for the negative feedback. However, it is unclear whether increased transport may suffice to explain extremely high negative feedback, and further studies need to be carried out to understand better the reason behind this behavior of SPARC.

IGFBP7 is the other protein which shows down regulation due to negative feedback between TNF-a and injury. This protein is known to be expressed in OA

articular cartilage. The gene coding for IGFBP-7 is present in chromosome 4q which has been associated with hip OA susceptibility by linkage mapping, but further studies

indicate that polymorphisms in IGFBP7 gene may not increase susceptibility to hip

OA 244. As IGFBP-7 only weakly inhibits IGF, this significant down regulation is

probably due to an as yet unknown function of IGFBP-7.

In our study, we have thus identified six proteins on which TNF-a and injury act

synergistically. A more complete way to analyze interactions and feedbacks between

Cytokine and injury would be to perform multiple mass spectrometry experiments using

the 8-plex iTRAQ experimental design, and analyze the data using a two way Anova. However, this study makes a start in trying to understand where the synergism between injury and cytokine in GAG loss comes from.

4.6 Partial Least Square Regression (PLSR) Analysis

4.6.1 Motivation behind the PLSR analysis and Methodology

Our next aim is to connect what happens inside the cell to variables which can be

observed outside. By performing this study, we hope to find out proteins which are most

relevant in changing some specific measurable phenotypes of cartilage. We thus have two

matrices, X composed of log-transformed protein ratios under different treatment

conditions, and Y composed of phenotypes under the same treatment conditions. We

want to find out how the underlying change in protein ratios manifests as observable

phenotypes, and hence want to find a relationship between Y and X. Since the number of

protein ratios is much higher than the number of phenotypes, we have a problem of multi-

collinearity (one or more protein ratios are highly correlated and hence, the matrix X

contains superfluous information). Ordinary multiple regression can hence not be used.

The commonly used Principal component regression (PCA) eliminates the problem of

multi-collinearity by choosing directions of maximum variance in X and using these to

develop a multiple regression model with Y. PLSR on the other hand chooses those

directions which maximize covariance between X and Y, projects X and Y data points on

these directions, and performs regression. Therefore, PLSR helps generate those

independent variables which contribute most to the relationship between X and Y (for

review 114). In our situation, this means that amongst all the protein ratios we have, we

can identify proteins which contribute most to observed phenotype. From the 74 proteins for which we have data for all 5 treatment conditions, CLEC3A and CD109 are not chosen because they increase error in the model. We use log transformed data for the remaining 72 proteins across 5 treatment conditions as the X matrix. The Y matrix is a

(4*5) matrix consisting of log transformed phenotype ratios with respect to control. The four phenotypes we choose are sGAG loss to the medium during the five day treatment, nitrite accumulation in the medium in five days, rate of proline incorporation (indicative if protein synthesis) on the sixth day (the day after the five day treatment ends), and rate of sulfate incorporation on the sixth day. The five treatments are the same as for the X matrix.

It is important to note that any relevant phenotype for which data across all treatments including (cytokine+injury) are available can be chosen, these four phenotypes are chosen because of availability of data for all the five treatments including the

(cytokine + injury) treatments. Also, sGAG release and proline and sulfate incorporation rates are biologically significant phenotypes, as they indicate catabolism (sGAG release) and anabolism (proline and sulfate incorporation). Choosing these phenotypes therefore helps understand which proteins contribute most to catabolic and anabolic activities.

4.6.2 Some Observations from the PLSR plots

Figures 8a-b show that four factors (principal components) are able to describe

100% of the variation in the X matrix (protein ratios) and Y matrix (phenotypes). Two

principal components describe 88% variation in the training data set and 70% of Y

variation in the validation data set. This difference may be due to the presence of outliers

in the protein data matrix. However, for obtaining trends about how phenotypes and proteins relate to each other, projection on the first two principal components are sufficient. Figure 8c shows the projection of different treatment conditions on the first two principal components. We see that the cytokines IL-1p and TNF-a are close to each other, the two cytokine+injury treatments are close to each other, and the injury treatment is separate from the other two.

Figure 8d shows the projection of proteins and phenotypes on the first two principal components. Proteins and phenotypes present close together in the same quadrant are highly correlated to each other, and proteins and phenotypes present in opposite quadrants and far from each other are inversely correlated. We immediately see that proline and sulfate incorporation rates, both indicators of synthesis, are highly correlated as expected. sGAG loss to medium, an indicator of cleavage of proteoglycans, and hence catabolism, is diametrically opposite to proline and sulfate incorporation rates, again as expected. Most cytoplasmic proteins cluster together tightly in the fourth quadrant. We also observe that SPARC and IGFBP7, two proteins which we earlier observed to synergistically decrease in the presence of TNF-a and injury are clustered close to sulfate and proline incorporation rates. Therefore, SPARC and IGFBP7 both seem to be indicators of synthesis or repair, which is seen to reduce on treatment with cytokine and injury. If the only role for IGFBP7 were in binding IGF, we would expect a negative correlation of IGFBP7 with synthesis, with an increase in IGFBP7 decreasing free IGF, and hence decrease anabolism. Therefore, this positive correlation of IGFBP7 with synthesis suggests a new role for IGFBP7 in repair and merits investigation. 4.6.3 Identification of proteins which contribute most to observed phenotype

From Table 5, we see that the top five positive protein-phenotype correlations are perlecan, pleiotropin, serum amyloid A3, CILP-2 and complement factor B, all with sGAG. Since sGAG loss to the medium is an indicator of catabolism, these five proteins are either key causative agents or key markers of catabolism. Also, the top five negative correlations are pleiotropin with proline and sulfate incorporation, perlecan with proline and sulfate incorporation and cathepsin B with sGAG release to the medium.

Perlecan is a large, multi-domain ECM proteoglycan expressed in almost all basement membranes, and also in mesenchymal organs and connective tissue. It is involved in a diverse range of developmental and biological processes, including chondrogenesis, endochondral ossification, angiogenesis and wound healing. Perlecan is known to interact with and sequester several growth factors, including FGF, VEGF,

CTGF, etc. Knox and Whitelock 30 hypothesize that perlecan performs such a diverse range of functions by interacting with, and altering distributions of growth factors and morphogens in developing tissue. Perlecan also interacts with several ECM molecules including fibronectin, nidogen-1, nidogen-2, PRELP, etc and thus is involved in ECM

organization (for review 29 ). Perlecan deficient mouse embryos die at embryonic day (E)

10.5 with defective cephalic development, or die just after birth with skeletal dysplasia 245 .

Late stage OA tissue samples showed elevated perlecan mRNA and protein levels in

cartilage adjacent to OA defects246, this suggests that perlecan is either involved in OA

pathogenesis, or is synthesized in OA cartilage in an attempt to repair tissue. Our study

however, shows that perlecan is highly correlated with sGAG release and highly anti-

correlated with both proline and sulfate synthesis, showing that an increase in perlecan released to the medium is an indication of catabolism, and reduced anabolism. Therefore, it is probable that the increase in perlecan observed in late stage OA is not due to an attempt at tissue repair, but rather due to worsening catabolism.

Pleiotropin is a cytokine implicated in tumor cell growth and angiogenesis, apoptosis and mitogenesis (for review 67). It is undetectable in normal adult cartilage, but is elevated in the synovial fluid of OA patients in early stages and found rarely in late

stages247. In cultured chondrocytes, pleiotropin reduced MMP-1 and MMP-13, and

increased TIMP-1 and TIMP-2 mRNA levels, indicating a role in repair 248. However, a

study by Tare et al 249 found that over expression of pleiotropin in transgenic mice led to

increased type I collagen synthesis (associated with osteoblasts), and ossification within

articular cartilage. Our result shows high correlation of pleiotropin with sGAG loss, and

high anti-correlation with proline and sulfate incorporation, and suggests a catabolic role

for pleiotropin, contradicting the possibility of a role for pleiotropin in repair. However,

since cartilage catabolism is accompanied by endochondral ossification in OA, the

association of pleiotropin with catabolism might actually imply the role of pleiotropin in

ossification.

Complement factor B and Serum Amyloid A3 are both involved in the innate

immune response. Complement factor B has been discussed earlier, and is implicated in

cartilage cell death and endochondral bone formation20 1 . Serum Amyloid A3 (SAA3) is a

member of the Serum amyloid A family, apolipoproteins associated with HDL. The

SAA3 gene is not expressed in humans 2s; however, LPS stimulated macrophages secrete

SAA3 in a murine model, suggesting a role for SAA3 in local inflammation apart from a

general acute-phase response . The high association of these innate immunity proteins with sGAG release suggests that catabolism is accompanied by inflammation and innate immune response.

CILP-2 has been discussed previously earlier in the context of TNF-a and injury acting synergistically to increase CILP-2 levels. This protein, whose function is as yet unknown, also appears to be a marker of catabolism.

It is, however, surprising that cathepsin-B, a cysteine protease which cleaves collagens252 and aggrecan 2 2 and hence plays a part in catabolism, shows such a strong negative correlation with sGAG release. One possible reason is that in our (injury + cytokine) model, cathepsin is not the primary collagenase/aggrecanase and does not contribute to sGAG release, therefore, the anti-correlation of sGAG release with cathepsin B is an artifact.

4.7 Limitations of this study

To identify the proteins on which injury and cytokine act synergistically, we combined data from two 4-plex iTRAQ experiments to obtain values for all the treatments (IL-1, TNF-a, injury, IL-1p and injury and TNF-ax and injury with respect to control). Because there exists a variation of only -10.5% between the two experiments, which is less than that typically seen between biological replicates, the data from both experiments are combined as such without correcting for any experimental bias.

Additionally, when combining data between the two 4-plex experiments, to ensure that there are no false positives identified, we first shortlist those proteins which are identified by both iTRAQ experiments, and which have all ratios across both experiments with an

FDR<0. 1. This gives us a list of only 74 proteins, and in this final list we check for proteins on which cytokine and injury exhibit synergism, ending with only 6 proteins on which TNF-a and injury show positive or negative synergism, and none for IL-1IP and injury. This methodology is exceedingly stringent, and may miss out several proteins on which cytokine and injury actually exhibit synergism, but are not counted because one or more of their FDR>O. 1. In other words, we may end up with several "false negatives".

An 8-plex iTRAQ experiment can circumvent both these problems, as we could label samples from all the treatment conditions simultaneously. This would firstly eliminate the need to combine data from two 4-plex experiments, and thus ensure no inter experimental variation. Secondly, conducting an 8-plex experiment would ensure lesser false negatives, and ensure more than just the 6 proteins we obtained on which cytokine and injury exhibited synergism.

When we performed the PLSR analysis, we considered only the first two latent vectors, to enable easy interpretation of how proteins and phenotypes relate to each other.

The first two latent vectors of our PLSR model, however, capture only -88% variation in

the training set, and 70% variation in the validation set. The addition of more latent

vectors would capture more information, but may complicate the interpretation of the

PLSR plots. CONCLUSION

In conclusion, we have used 4-plex iTRAQ labeling followed by 2D-LC/MS/MS to study the effect of the combination of cytokine and injury on cartilage explants. A global analysis of the proteins released revealed a high correlation between (IL-13 +

injury) and (TNF-a + injury) treatment conditions. A k-means cluster performed on the

protein ratios revealed three distinct clusters, cluster 1 containing proteins highly down

regulated, cluster 2 mildly up regulated and cluster 3 highly up regulated with (cytokine +

injury). Most cytoplasmic proteins are present cluster 3 and are highly elevated with

(cytokine + injury), possibly due to mechanical lysis of cells due to injury. ECM proteins

are present in either cluster 1 or cluster 2. Looking at specific protein groups, C-terminal

collagen propeptides of some fibrillar collagen chains are down regulated with (cytokine

+ injury), indicating reduced synthesis of new collagens, and a decrease in the anabolic

ability of cartilage. ECM molecules such as COMP and matrilin-3 were down regulated,

and members of the SLRP family including chondroadherin, PRELP, biglycan and

lumican were highly up regulated with (TNF-a + injury). The elevation of lumican and

biglycan, known to be expressed in early stages of collagen fibril development possibly

indicates signs of repair. Proteases MMP-2, MMP-3 and MMP-9 levels don't change

much with (cytokine + injury), but MMP-13 is highly down regulated and ADAMTS-1

highly elevated with TNF-a + injury. This suggests that ADAMTS-1 may be responsible

for the synergistic sGAG release in the presence of cytokine + injury. An increase in

TGF-62 and activin, accompanied by a decrease in LTBP1 suggests attempts to repair

cartilage. CTGF, known to be induced by TGF-D is highly down regulated with TNF-a +

injury, a behavior similar to what happens in a sheep model of OA at the mRNA level, although it is unclear why. Proenkephalin, AIMP1 and annexins A2 and A5 show high elevation with TNF-a + injury and/or IL-1 + injury. The up regulation of proenkephalin possibly helps in repair and in apoptosis caused by injury, the elevation of annexins indicates terminal differentiation and onset of apoptosis and AIMP1 may play a yet undetermined role in this situation.

Next, we combined the results from two iTRAQ experiments to obtain data from injury alone, cytokine alone and cytokine + injury to find out proteins on which injury and cytokine acted synergistically. From data combined from both experiments, we find a very high correlation between (IL-1p + injury) and (TNF-a + injury) (-95.7%), much higher than that between injury and (cytokine + injury), indicating that the domination of

injury over cytokine is not the reason for the high correlation between (IL-13 + injury)

and (TNF-a + injury). Also, the correlation between the two (injury + cytokine)

conditions is higher than the correlation between the two cytokine conditions (-90.3%),

indicating that while both cytokines act through similar pathways in chondrocytes, the

addition of injury further adds new common pathways. From the 74 proteins identified in

common between both the experiments and quantified well across ratios, we find that

TNF-a and injury exert positive synergism on CILP-2, COL6A3, Histone H4

replacement-like and aggrecan and negative synergism on SPARC and IGFBP7. While

increased transport of TNF-a caused due to mechanical damage might cause a slight

increase in aggrecan cleavage resulting in the small synergism shown for aggrecan,

increase in COL6A3 and Histone H4 replacement-like proteins are much higher and may

result from cellular pathways seeking to repair damaged cartilage.

100 A PLSR analysis performed between phenotypes and protein ratios removes superfluous information and generates interesting associations between protein ratios and phenotypes. Perlecan and pleiotropin are both highly correlated with sGAG release and highly anti-correlated with proline and sulfate incorporation rates, indicating that both these proteins are markers of catabolism. Complement factor B and serum amyloid A3, proteins associated with innate immunity are both highly correlated with sGAG release, indicating that local inflammation plays a role in catabolism. SPARC and IGFBP7 are correlated with proline and sulfate incorporation, suggesting that the negative synergism between TNF-a and injury for these proteins indicates a reduction in the ability of cartilage to repair itself. High correlation between CILP-2 and sGAG release suggests that this protein can also serve as a marker for catabolism. PLSR can thus help find unique protein-phenotype associations, which may help discover unknown functions of proteins.

Thus, our study indicates that our in vitro model of injury + cytokine affects

cartilage differently from either injury alone or cytokine alone, and causes both

catabolism and attempts to repair damaged cartilage. This model thus mimics OA

cartilage and may serve as a useful model system to study OA under controlled

conditions in vitro.

Future Work

While bovine cartilage serves as a useful in vitro model system to mimic OA like

conditions using a combination of injury and cytokines, using human cartilage explants in

our in vitro model will help avoid bovine-human differences in response to treatment, and

101 will be closer to human in vivo conditions. Also, as mentioned before, an 8-plex iTRAQ experimental system would yield more proteins on which injury and cytokine exert synergism, and could provide a better picture of metabolic pathways in the chondrocytes which are affected by injury and cytokine.

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123 APPENDIX

Appendix A.1 and Appendix A.2: Profile of proteins released to media from the first and second biological replicates respectively. Only proteins identified by greater than 2 peptides with confidence > 90% and which are well quantified across all ratios are shown here. Protein ratios are normalized using the internal standard chondroitinase ABC. Profile obtained using iTRAQ 2D-LC/MS/MS with ProteinPilotTM Software 2.0.1 (ABI). Matlab scripts are used to calculate the weighted means and standard deviations. For more detailed information on how this list was obtained, refer to Chapter 2

Column 1: GI Number of Protein Column 2: Name of Protein Column 3: Percent of proteins covered by identified peptides Column 4: Geometric mean of (TNF-a/ IL- 1p) Column 5: Standard deviation of (TNF-a/ IL- 1f) in log space Column 6: p value for the null hypothesis that (TNF-a/ IL-1p) is unity. Obtained by Wilcoxon Sign rank test or paired t test. Column 7: Geometric mean of (IL- 1f +inj/ IL- 1p) Column 8: Standard deviation of (IL-1P +inj / IL-1 ) in log space Column 9: p value for the null hypothesis that (IL-iI1 +inj / IL- 1f) is unity. Obtained by Wilcoxon Sign rank test or paired t test. Column 10: Geometric mean of (TNF-a +inj/ IL- 13) Column 11: Standard deviation of (TNF-a +inj / IL- IfP) in log space Column 12: p value for the null hypothesis that TNF-x +inj / IL- 1P) is unity. Obtained by Wilcoxon Sign rank test or paired t test. Column 13: Number of peptides used in calculating (TNF-a/ IL-1 3) ratios Column 14: Number of peptides used in calculating (IL- 1 +inj / IL- 1p) ratios Column 15: Number of peptides used in calculating (TNF-a +inj / IL- 1P) ratios

124 6 115:std 115:11 pvalue 115:mean 116 std 116:11 pvalue 116 mean117: std 117 11 pvalue 117: Nopepll5hopepll Nopepll7 Accession Name %Cov mean 9 34.79845 1.005643 0.421889 0.5458818 0.961879 0.315976 0.0001377 1.253084 0.844274 3.39E-15 346 348 370 to heparansulfate proteoglycan 2 (pedecan)[Bos taurus] gil11988897 PREDICTED:similar 0.496027 3.61E-102 1.047484 0.294038 1.60E-05 0.473026 0.639835 1.87E-103 628 682 857 giJ76610126 PREDICTED:similar to fibronectin1 isoform4 preproproteinisoform 12 [Bostaurus] 49.32261 0.435739 4 4 44 25 3.87E-24 0.654007 0.542659 0 0.461606 0.860192 1.25E-114 717 719 719 gi|16 1 collagen,type 11,alpha 1 isoform 1 [Bostaurus] 69.60323 0.826267 0.712588 0.542485 0.260546 0.0065573 0.894213 0.218261 0.2160647 1.252597 0.216957 0.05788702 4 4 4 gi|119900891 PREDICTED:hypothetical protein LOC540664 [Bos taurus] 42.53996 0.494509 6.39E-19 1.192888 0.385784 1.10E-06 1.222695 0.763684 4.86E-05 114 120 123 gi|119890645 PREDICTED:similar to alpha3 type VI collagenisoform 5 precursorisoform 1, partial[Bos taurus] 53.2872 0.65229 0.815516 0.520715 3.35E-33 1.005309 0.288304 0.7150054 1.347252 0.921421 3.49E-14 347 357 368 gi]41386685 thrombospondin1 [Bostaurus] 50.8547 3.023079 0.45549 3.63E-117 0.867879 0.434132 5.71E-22 1.198459 1.138088 0.00159624 707 688 705 gir30794280 albumin[Bos taurus] 85.99671 56.51322 1 0.358784 0.0385808 1 0.323575 0.6563822 1 0.342961 0.64236811 238 244 250 gi|34098396 ChondroitinABC endolyase1 precursor(Chondroitin ABC lyase 1)(Chondroitnase ABC) (Chondroitin sulfate endolyase) (Chondrotin ABC elminase) 32.16904 1.34625 0.355638 0 1.002979 0.413166 0.9146439 1.274493 0.578736 2.86E-06 80 81 83 gi|1 19913814 PREDICTEDsimilar to melanoma-associatedchondroitin sulfate proteoglycan 4 [Bostaurus] 0.852151 0.343306 1.93E-05 1.067136 0.297717 0.021252 1.754255 0.357203 0 87 69 79 gi|119901095 PREDICTED:similar to collagen,type XII, alpha 1 isoform1 [Bostaurus] 23.52753 0.649362 0.44438 1.60E-50 1.015564 0.284613 0.5096392 1.224062 0.499061 5.72E-28 362 371 378 gil119887130 PREDICTED:similar to Collagen,type VI, alpha1 isoform1 [Bos taurus] 53.74878 0.472729 0 0.922274 0.3414 2.97E-21 0.813244 0.913111 5.48E-15 717 728 731 gi]27806761 aggrecan1 [Bostaurus] 18.99441 0.784089 2.90E-05 2.974593 0.72733 2.24E-18 3.541618 0.842243 2.95E-19 83 103 112 gi]115495027heat shock70kDa protein 5 [Bos taurus] 62.13741 1.247723 0.528367 0.486381 0.0339878 0.864302 0.345823 9.03E-10 0.875004 0.656286 0.53356804 188 190 182 gij1 19889734PREDICTED: similar to Collagenalpha-1(XI) chain isoform 1 [Bostaurus] 42.26747 0.919981 0.410477 0.210661 0.924994 0.360826 0.0007069 1.422424 0.787659 3.27E-08 103 107 114 gi|156120777nidogen 2 [Bostaurus] 37.70115 0.96593 0459791 4.74E-09 3.405984 0.487717 0 4.166193 0.508689 0 75 81 82 gill10347570vimentin [Bostaurus} 72.31759 1.432939 0.61657 0.6375414 3.582926 0.769483 0 4.295576 0.831835 0 76 83 83 gi|119892686PREDICTED: similar to P63 protein[Bos taurus] 69.43973 0.999488 0.432873 1.74E-10 1.20587 0.286836 1.99E-07 1.41886 0.289956 0 78 79 84 gil119890597 PREDICTED: similar to alpha3 type VI collagen,partial [Bos taurus] 39.15441 0.689371 1.77E-28 1.108789 0.321063 1.06E-11 1.849601 0.658567 1.94E-44 356 368 382 gill14051163collagen. type XI, alpha2 [Bos taurus] 45.50691 0.637727 0.635712 0.0588728 1.798648 0.379839 1.27E-13 2.725651 0.388416 0 31 32 32 gil119914559 PREDICTED: similar to filamin B, beta(actin bindingprotein 278) isoform 7, partial[Bos taurus] 27.42947 1.109722 0.423167 7 4 1 2 1.69E-05 2.590192 0.482542 0 3.48788 0.529756 0 47 49 49 giJ740 8 lamin A/C [Bostaurus] 74.12587 1.221441 0.41052 0.0009554 2.917892 0.651177 0 3.617987 0.673506 0 33 39 39 gil148238040actnin, alpha4 [Bos taurus] 52.68935 1.304351 0.603734 2.95E-11 0.563283 0.33502 0 1.076385 0.635898 0.0006317 110 105 120 gi|95147674 complementfactor B [Bostaurus] 52.69383 0.83096 0.356832 7.52E-18 0.939958 0.336537 0.0009198 1.228831 9.27E-21 134 134 132 gi|119894520PREDICTED: similar to cartilageoligomeric matrix protein isoform 1 [Bostaurus] 63.62434 0.782051 0.332165 0.458171 9 1.69058 0.440162 2.55E-10 0.554469 0.379337 9.89E-13 0.318933 0.72373 8.84E-13 30 29 29 gir9902896 complementcomponent 3 [Bostaurus] 34.31668 0.003473 4.180471 0.535 0 4.920994 0.643657 0 84 74 73 gi]148230374protein disulfide isomerase-associated 3 {Bostaurus] 61.18812 1.161431 0.486953 4.95E-12 0.98958 0.329008 0.187172 1.061875 0.290694 0.00554071 88 88 91 gill15497814nucleobindin 1 [Bostaurus] 58.86076 1.344723 0.34105 0.0004671 1.543071 0.529033 4.44E-16 2.050639 0.922916 7.71 E-09 77 78 80 gi|116003881collagen, type Ill, alpha1 [Bos taurus] 42.63301 0.848729 0.569953 0.952236 3.754362 0.721279 2.14E-07 4.804789 0.687947 1.53E-08 8 14 14 gi|119919153PREDICTED: similar to AHNAKnucleoprotein [Bos taurus] 45.49967 1.006412 0.402019 7.44E-14 0.849487 0.354394 0.0036338 0.316493 0.800289 526E-06 28 29 28 gi|77404252 collagen,type alpha 1 [Bos taurus] 46.27478 0.491396 0.389364 1, 0.849852 0.480011 5.72E-08 0.753928 0.355448 0 1.395893 0.59142 6.42E-11 137 138 151 gil119888668 PREDICTED:similar to Cartilage matrixprotein precursor (Matrilin-1) [Bos taurus 68.56618 0.305244 1.66E-12 1.46885 0.463886 4.94E-07 1.61193 0.51147 1.18E-08 86 67 70 gi]164452943gelsolin a [Bos taurus] 45.3265 0.797719 0.455201 0.0019861 3.698287 0.533763 0 4.315778 0.486888 0 31 35 36 heatshock 9OkDprotein 1, alpha [Bostaurus] 42.97408 1.212443 gi]60592792 0.0008235 0.944119 0.324471 0.0694573 0.899322 0.591975 0.52273553 43 43 46 gij76621011 PREDICTED:similar to Cartilageintermediate layer protein2 precursor(CILP-2) [Bos taurus] 32.36515 0.858448 0.339405 0.371326 0.0200043 3.291955 0.423177 0 3.828104 0.430247 0 60 60 60 gil27806501 prolyl4-hydroxylase, beta subunit[Bos taurus] 62.94118 1.151039 0.0003509 1.976554 0.297421 4.060-14 2.493254 0.284578 2.22E-16 22 22 23 gil114050715 moesin[Bos taurus] 49.74003 1.180499 0.28306 1.68E-14 0.76161 0.247638 0 0571277 1.007268 1.31 E-09 78 80 81 gil153791660 extracellularmatrix protein 1 [Bostaurus] 43.13346 0.413906 0.377329 0.53044 0.0396956 3.152702 0.775665 2.49E-14 4.033919 0.6537D6 0 28 35 36 gi]114326226protein disulfide isomerase-associated 4 [Bos taurus] 60.34214 1.162803 0.406137 7.79E-07 1.176319 0.267303 5.62E-05 1.182175 0.58193 011694565 31 31 31 gi]76607158 PREDICTED:similar to Melanotransferrinprecursor (Melanoma-associated antigen p97) ]CD228antgen) [Bootaurus] 42.21922 1.369667 0.446222 0.0183304 1.744497 0.37099 1.850-09 3.435781 0.606333 0 46 52 61 gi|156120795 quiescinQ6 sutfhydryloxidase 1 [Bos taurus] 46.56085 0.895843 0.6173109 2.152968 0.440105 0 3.638051 0.545126 2.618-08 38 40 41 gi|27807289 annexinA2 [Bos taurus] 67.55162 0.971116 0.528762 7 807 7 0.0007673 0.746375 0.334907 2.25E-08 0.542329 0.732D67 5.690-12 74 70 71 gi2 44 matrixmetalloproteinase 2 [Bos taurus] 52.6475 1.116443 0.415104 0.260787 0.703519 0 0.864888 0.255386 321E-13 0.801977 0.314875 4.440-18 93 97 95 gi|119906908 PREDICTED:similar to matrix metalloproteinase-3[Bos taurus] 38.36478 63 4 3 0.551502 0.430747 3.47E-19 0.057004 0.4602 4.960-19 1.171390 0.824810 8.990-05 123 122 125 gi|76 01 PREDICTED:similar to matrilin-3[Bos taurus] 56.79013 0.421989 8.99E-141 0.82509 0.28308 1.89E-145 0.59098 0.300093 8.8-143 851 805 087 gi|122692297 chitinase3-like 1 [Bos taurus] 56.26599 0.406649 0.012083 3.746138 0.742322 7.118-15 3.737121 0.626015 1.820-14 28 31 33 gi]77735551 phosphoglyceratekinase 1 [Bos taurus] 63.30935 1.224115 0.55838 0.2546114 0.995253 0.453871 0.8281887 1.218634 0.545084 0.2898089 37 30 39 gil119921375 PREDICTED:similar to RP11-525G3.1,partial [Bos taurus] 29.9373 1.048322 0.352778 0.329199 0.0165747 0.722218 0.334845 2.26E-05 0.818708 0.532575 0.03801338 17 17 17 gil119901238 PREDICTED:similar to laminin alpha4 [Bostaurus] 27.8481 0.862922 0.960974 0.532531 0.4711225 2.95797 0.564104 0 4819707 0.576677 0 44 52 53 gi]87196501 enolase1 [Bos taurus] 65.43779 1.22407 0.40748 0.0015581 4.12454 0.572235 2440-15 5039212 0.65512 2.448-15 25 20 25 gil27807263 heat shockprotein 90kDa beta,member 1 [Bos taurus] 39.42786 0.385121 0.0008322 3.683334 0.46267 8.300-56 4.192343 0.455185 0 28 28 26 gi|1 15497900 eukaryotictranslation elongationfactor 2 [Bos taurus] 37.41259 1.220731 1.520-08 0369366 0.697547 4.640-10 0.372628 1.428103 8.200-05 24 24 24 gi|156718112latent transforming growthfactor beta bindingprotein 1 (Bos taurus] 26.23318 0.632534 0.381765 0.1327652 0.624724 0.328717 0 0.754675 0.510241 2.020-07 45 45 45 gi|115497210complement component 1, s subcomponent[Bos taurus] 32.08633 1.043111 0.262576 3 0.269485 2.200-07 0.873592 0.30648 0.005107 1.209664 0.452606 000295748 25 29 30 gil15652 168 matrilin2 [Bostaurus] 37.47391 0.766229 7 0.472504 0.1588925 2479094 0.453791 2.63E-10 2.841205 0.465213 1.020-11 19 19 20 gil11991915 PREDICTED:hypothetical protein[Bos taurus] 34.19483 1.117488 0.0529203 0.873563 0.298941 0.0008016 0851808 1.105543 0.26924046 26 30 29 gi]61845535 PREDICTED:similar to emilin[Bos taurus] 31.42857 0.898998 0.440236 0.0144912 1.014537 0.279986 0.822161 1026874 0.520569 0.74987118 30 30 30 gil164450489AE bindingprotein 1 [Bostaurus) 25.04274 0.889162 0.35892 1.648-09 0.945667 0.351833 0.2509707 1.694337 1039992 2.620-18 124 128 130 gil27805853 cartilagelinking protein 1 [Bos taurus] 49.15254 0.855114 0.327271 0.0001394 2.974565 0.867531 7.270-07 3229098 0.926168 3.73E-14 37 39 40 gil119902010 PREDICTED:similar to LOC512571protein [Bos taurus] 56.15672 1.129493 0.474311 0.428035 5.390-07 0.141562 0.895019 0 0.13D643 1130115 6.66E-16 33 33 33 gi|94966763 haptoglobin[Bos taurus] 63.34165 0.381782 0.353315 3.64E-08 0774442 0.289031 7.25E-10 0494301 1,396514 2338-06 54 54 54 gi|62988316 follistatin-like1 [Bos taurus] 63.84365 1.240039 0.448786 0.7556605 2.567655 0.852448 00009766 3.417283 0.649426 3.928-06 9 11 11 gi]76253709 heat shock70kDa protein8 [Bos taurus] 49.07692 1.034633 0.490754 2.990-12 0.993797 0.449141 0.399628 1.588508 0.800243 0570-07 8 71 72 gil147906412 hypotheticalprotein LOC615490[Bos taurus] 42.47191 0.546603 00002931 0.679186 0.4041 2.77E-05 0689328 0.904192 0.00193099 17 17 17 gi|27805991 latenttransforming growth factor betabinding protein 2 [Bos taurus] 21.03923 0.340557 0.618178 0.572417 00063074 2431227 0.692558 5.210-56 3199324 0.707839 2670-07 14 15 15 gi1 19918578 PREDICTEDsimilar to vinculinisoform meta-VCL isoform 1 [Bos taurus] 36.33157 1.412648 0 7 1 0.502278 0.81098941031804 0349224 0.80436181.634551 0518398 000035915 28 26 26 gi|278 6 8 vitrin[Bos taurus] 33.74233 0.984274 0.574226 0.0864763 3.337236 0.779516 1.508-10 4.325407 0.915289 7.150-11 24 24 24 gi]41386780 UDP-glucose pyrophosphorylase2 [Bos taurus] 50.3937 1.109126 0.5629179 0.940327 0.321806 0.2134019 0.887358 0.529919 0.19849831 18 18 17 gi]119903231PREDICTED: similar to lysyl oxidase-like3 protein [Bostaurus] 35.23936 0.885171 0.377162 0.711714 0.245211 5030-05 079728 0.921209 0.07190333 29 29 30 gil119903133 PREDICTED:hypothetical protein [Bos taurus] 43.21678 0.876185 0.450525 0.1025633 1188-05 0.900423 0.353866 0.0014157 1160271 0.532095 0.00256143 57 55 61 gi27806477 hydroxylaseprecursor [Bos taurus] 33.47107 1.155461 0.325785 lysyl 0.521609 4.238-05 2.493635 0.608438 1.20E-08 3.112225 0.706996 0 35 43 49 gi|61888856 triosephosphate isomerase1 [Bos taurus] 77.51004 1.478887 0.62236 0.0012565 0.892258 0.21622 0.0014804 0.424701 0.653629 7.368-08 21 21 20 gil27806257 collagen, type alpha2 [Bos taurus] 49.4868 0.702918 I, 0.0001454 0.834522 0.324703 0.0006167 0.971045 0.821046 0.42906726 23 23 23 gi|154707890 lysyloxidase-like 2 [Bos taurus] 29.84496 0.811225 0.316853 0.366306 0.0968463 3.274319 0.4985 364E-08 4192086 0.533889 8.81 0-09 13 13 13 gi]76682880 PREDICTED:smilar to nucleolin-relatedprotein NRP isoform1 [Bostaurus] 48.33333 1.13597 0.421419 0.0436801 3690009 0.480869 1.950-09 4.293174 0.420101 3.128-12 13 14 16 gill15498012 glycogen phosphorylase,liver [Bos taurus) 43.36075 1.201128 9 2 0.545296 1.990-05 1.436954 0.245008 8.398-14 1.584799 0.237539 0 34 34 34 gi|1154670 collagen, typeVI, alpha2 [Bostaurus] 41.00327 0.724871 0.398402 02568198 4.098743 0.506528 0 4.17203 0.53276 0 32 33 33 gir120474983annexin AS[Bos taurus] 69.47041 0.945781 0.6647152 4.020523 0775478 1.000-12 4.007973 0790645 1468-12 26 26 28 gil114052136 FK506 bindingprotein 9 [Bs taurus] 28.04878 1.023669 0.381426 37 2 3 0.300144 03502808 2.113383 0.407388 2.590-08 2.550005 0.399806 2.31 8-10 15 16 17 gil155 20 procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 [Bos taurus] 37.40157 1.053964 99 3 18.23834 1.261767 0.382879 00044553 1.967561 0.4281 4.498-07 2936799 0.473249 4.418-09 15 15 15 gil11991 5 PREDICTED:similar to filamin [Bostaurus] 47746667 1.140169 0.450024 0.0056727 3.54527 0.593232 0 4228356 0.848994 0 46 54 54 9i)75832054 actin, beta [Bostaurus] gi|27806469 peptidylprolylisomerase B [Bos taurus] 77.40384 0.966386 0.3585 03520491 1.441514 0390379 4560-08 1.719177 0423647 2.22E-16 E-05 gij61828365 PREDICTED:similar to Tropomyosin 3 isoform1 [Bos taurus] 78.62903 1.216501 0.259127 0.0281599 3321663 0.353157 1.33E-05 3.62064 0.443649 3.21 1.62E-12 gil27806723 calreticulin[Bos taurus] 36.45084 1.33925 0.388971 1.50E-05 3.942023 0671703 1780-14 3.62686 0771161 00014648 0764914 0.254156 00001114 1.27309 0.37747 0.00602867 gi|119887291PREDICTED: similar to peroxidasin homolog [Bos taurus) 27.04115 0.821375 0.287995 gill 19895885 PREDICTED:similar to EGF-blkerepeats and discoidinI-like domains 3 [Bos taurus] 36.77419 0.865865 0.172443 0.0003491 0.859876 0.224315 00001221 0.928577 0.17773 0.03530605 gi|27807437 matrixmetalloproteinase 9 [Bos taurus] 37.2191 0.971629 0.373629 0.7172178 0.785828 0307669 8.210-05 1147188 0.631454 0.18830764 gi|114051908FK506 bindingprotein 10, 65 kDa [Bos taurus] 33.27616 1.15534 0.39642 0.046568 4.541796 0872852 2.480-09 4595993 0879052 3.OOE-10 gil38566696 serum amyloidA 3 [Bos taurus] 79.38932 0.511158 0.325391 00001318 0.658872 0.296719 4.570-06 0.835857 022354 8.44E-05 gij77736401 thrombospondin4 [Bos taurus] 28.72008 1.482349 0.771648 0.0160671 0920643 0226488 0.0535164 047494 0922694 0.00077837 gil27805949 inhibinbeta A [Bos taurus] 47.05882 1.108032 0.432033 0.1934376 3.759034 1190221 6.270-06 6055416 1.259887 0.00029305 gil27807207 plasminogen activator inhibitortype 1. member 2 [Bos taurus] 52.14106 0.58862 0.400078 9.840-06 1.051048 0312012 0.3067599 1.561361 0.35485 7.36E-10 0480175 1.23E-05 gil27807367 tyrosine 3-monooxygenase/tryptophan5-monooxygenase activation protein,zeta polypeptide [Bos taurus] 61.63265 1.814499 0.540997 6.97E-08 3.974334 0515406 4.440-16 4638462 5.30E-05 gil155371895sema domain, immunoglobulindomain (1g),short basic domain,secreted. (semaphorin) 3C [Bostaurus] 36.08522 0.626223 0.552839 7.390-05 1.833769 0469972 00001064 2.611883 0591251 gi|27806697 chondroadherin [Bostaurus] 51.24654 0.578176 0.522216 0 1.113816 0373196 0.0002071 206508 0494707 6.65E-20 gi|27806147 secreted protein, acidic,cysteine-rich [Bos taurus] 48.02631 1.268557 0.360931 4340-14 0.477958 0414947 0 0 179124 1.014901 3.73E-16 gi|119888620PREDICTED: similar to alpha 1 type XVIcollagen [Bostaurus] 21.38872 1.133376 0.690707 0.3307672 0.796239 0337696 00016794 0754229 0614367 0.02244257 gi|41386719 milkfat globule-EGFfactor 8 protein [Bostaurus] 37.00234 0.791304 0.273901 0.0001075 0.619551 0354467 8.520-08 0.628954 0422447 1.87E-06 gi|76253900 GDP dissociation inhibitor2 [Bos taurus] 52.58427 1.455453 0.665949 0008821 2.596066 0.420476 1.02E-05 2.653669 0575952 8. 12E-05 gil27806351 ezrin [Bos taurus] 46.64372 1.091578 0.252855 00988308 2241708 0.281875 1.980-09 2642202 0.270054 3.02E-10 (LDH-M)[Bos taurus] 57.22892 1.178704 0.390814 00287841 2.637623 049076 9960-10 3.071962 0.59957 6.87E-10 gil1199200809 PREDICTED: similar to L-lactate dehydrogenase A chain (LDH-A)(LDH muscle subunit) gil14822350 osteoprotegerin [Bos taurus] 44.27861 0.635828 0.924922 00159708 1.665717 0.451871 2.200-05 4.118681 1045629 1.15E-06 gil110350683 biglycan[Bos taurus) 59.62059 1.09486 0.399052 00115285 2.228327 0.503143 3650-14 1.816831 0.487552 4.16E-13 gi|156120479aldolase A [Bos taurus) 45.87912 1.713039 0.466647 6.290-05 2.469162 0060426 1.700-06 3072349 0.599473 4.76E-07 gi|134085671histone cluster 1, Hic [Bos taurus] 64.78873 0.97495 0.404706 0.8458203 1.650374 0481195 0.0294921 8610595 0783553 0.00089462 gil156121245insulin-like growth factor binding protein 7 [Bos taurus] 57.80142 1.094136 0.426337 0.1213981 0.538502 0559197 2.720-08 0.397764 1046779 3.22E-07 gil27806317 annexin A8[Bos taurus] 68.19572 1.438976 0.344794 00018496 2.485153 0432846 1.8005 4.042242 0555698 2.86E-07 gil115495019 procollagen-proline.2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase),alpha polypeptide I [Bos taurus] 48.50187 0.882886 0.367268 00679135 2.495708 0.831659 00002931 2.80955 0794449 8.57E-07 gi|99028973 transcobalamin |1[Bos taurus] 40.74074 0.555233 0.459658 0002449 0.89322 0.31755 0.1206319 2.086042 0497411 3.87E-07 gil116004023 phosphoglucomutase 1 [Bos taurus] 53.20285 1.19411 0.353038 00482391 3.098721 0.547299 6.110-06 3.580732 0574067 3.24E-06 gil77404273 glyceraldehyde-3-phosphatedehydrogenase [Bos taurus] 51.65165 1.349932 0.412958 5.060-05 469257 0.709112 4220-14 5369332 0.734137 1.31 E-14 gi]40254806 heat shock 70kDa protein 1A[Bos taurus] 41.65367 1.218518 0.660476 02067183 3.891821 0.221504 5150-11 4.338255 0.207845 1.22E-11 gil164420731 transaldolase 1 [Bostaurus] 53.41246 1.647513 0.78044 0.0085615 4.19667 0.48062 00002441 4465234 0.648436 4.82E-08 0 gi|27806907 clusterin[Bos taurus] 32.57403 1.370788 0.365556 0 0.532673 0.499506 0 0.603443 0.479738 2.780-08 53571 0.784822 8.79E-10 gill14051756 heterogeneous nuclear ribonucleoproteinA2/B1 [Bostaurus) 58.94428 1.269216 0.423061 0.0054606 4.265354 0899902 gi]51491841 transketolase [Bos taurus] 33.22632 1.001777 042484 06601456 2.824623 0490068 1110-06 3972913 0.60178 1.44E-08 gi|77404217 phosphogtycerate mutase 1 (brain) [Bostaurus] 53.1496 1.420449 0.457248 1.680-05 2.984226 0.47117 3930-14 3.274677 0388514 8.30E-06 0.01025391 gi|119901059 PREDICTED:similar to COL9A1protein, partial [Bos taurus] 27.92608 0.624194 0.419346 8.106-05 0.064839 0.225205 0.0215454 0.752822 0.528793 0.74156451 gil30794358 versican [Bos taurus] 10.85478 1.341758 0.270482 0.0081088 0.71699 0.204008 1.8006 1.023065 0.351439 gil168804008 tubulin,alpha, ubiquitous[Bos taurus] 38.13747 1.128293 0.426549 0.125152 3.777497 0.530255 3.47E-10 5.599331 0.713884 1.06E-11 0.39952894 gil27806941 serine (or cysteine) proteinase inhibitor,clade A (alpha-1 antiproteinase, antitrypsin),member 1 [Bos taurus] 38.94231 0.626197 0.408455 1.03E-06 1.087044 0.370509 0.1216249 0.957381 0.350759 0.77381 0.00058879 gi]73853762 annexin I (Bos taurus] 57.80347 0.688388 0.559084 0.801079 1.263143 0.685786 0.0385797 1.638153 0.00040001 gil119331178 SPARC related modularcalcium binding 1 [Bostaurus] 33.41014 0.729588 0.323378 4.740-06 0.731745 0.242091 6.800-08 0.616842 0729459 4.30E-08 gi|41386727 protein kinase C substrate 80K-H[Bos taurus] 21.95122 1.283851 0.412294 0.0045111 3.884186 0.617272 6.300-10 3596102 0.706315 1.831E-10 gil 19903961 PREDICTED:similar to Protein disulfideisomerase associated 6 isoform7 [Bos taurus] 40.67796 1.533899 1.239035 0.0981442 3.564787 0911481 3.030-08 4.920584 1.044945 gil27806739 apolipoproteinE[Bostaurus] 56.64557 1.054537 0.307699 0.3506565 0628845 0.339228 3080-08 0.648509 0.475921 1.18E-05 gi]77736275 calumenin [Bos taurus] 45.07937 0.883407 0.436466 00494385 2.731974 0.342826 7.710-10 3.035601 0310606 6.01 E-11 1.71E-06 gill 19900887 PREDICTED:similar to collagen XXVIIproalpha 1 chain precursor; preproprotein[Bos taurus] 25.86558 0.755179 0.415235 0.00621 0888638 0.36141 08164648 2.123001 0.409484 gi|l 19908661 PREDICTED:similar to Laminin gamma-i chain precursor (LamininB2 chain) [Bostaurus] 28.74016 1.08494 0.340788 1 0.827303 0.389165 0.0384872 0.742105 1166543 1 gil41386707 vascular celladhesion molecule 1 [Bostaurus] 27.0636 1.583077 0.40522 0.0002906 0.532135 0528182 0.0001742 0.882593 0341505 0.11070289 gil1 19912531 PREDICTED:similar to mannose receptor, C type 2 [Bos taurus] 17.45602 1.25875 0.477965 0.0562236 1.172813 0.242125 00180407 1.253979 0.746504 0.1999653 gil157074106 lamin B1 [Bos taurus] 43.00341 1.07196 0.454305 0.8505681 1.065838 0.308361 00021871 3.308644 0.312812 0.00024721 gi]27806489 proenkephalin [Bostaurus] 64.25856 0.952292 0.418135 1 7.706637 1.083169 4330-07 12.92838 1.420231 6.07E-06 gi|27805823 dermatan sulfate proteoglycan3 [Bos taurus] 26.47975 0.869887 0.219256 0.0008093 1.87483 0.286394 1.560-09 1.079035 0.830543 0.17895611 6.78E-07 gil149773543 steroid-sensitive protein 1 [Bos taurus] 29.24528 0.940334 0.471483 0.5422245 1.337722 0.359504 0.0035578 4.364235 0730059 gi|119900517PREDICTED: similar to KIAA11027protein [Bos taurus] 21.95986 0.828445 0.994186 0.496875 2.306418 0.605184 0.0008231 3.343639 0.889053 2.23E-05 gi|1 15496928 EGF-containingfibulin-like extracellular matrixprotein 2 [Bostaurus] 37.47178 0.8642 0.253817 0.0109751 0.84039 0295555 0.008025 0.639841 1.00329 0.03911204 gi|27806623 fibromodulin[Bos taurus] 35.2 0.961092 0.294838 0.006021 1.173788 0304319 4.840-14 1.285537 0509777 9.00E-13 gi|148230364heterogeneous nuclear nbonucleoprotein D [Bos taurus] 41.17647 1.313532 0.470427 0.0984023 4581735 0.403819 2070-07 5790203 0.396794 5.72E-08 gi|l 15497628 reticulocalbin2, EF-hand calcium bindingdomain [Bostaurus] 30.91483 1.01621 0.59814 08954841 3.288107 0.808578 1.050-06 4.260653 0.758722 4.14E-08 gil119908681PREDICTED: similar to superficialzone protein [Bos taurus] 24.15186 0.752636 0.778006 0.129217 1.654838 0303415 3790-05 2.538553 0.63436 8.91E-05 gil156121327galectin 3 [Bos taurus] 38.86792 1.084874 0.500628 0.1772784 25512 064266 1.330-15 3928305 0.670743 0 gil66792902 vanin 1 [Bostaurus] 26.66667 0.388952 0.930631 00001055 1.220544 0.252873 0003363 0881697 0.433523 0.05794988 gill 14053121 reticulocalbin3, EF-hand calcium bindingdomain [Bos taurus] 34.14634 1.847044 0.718336 0.0013333 2.905404 0.856876 2290-06 4.103241 0.641637 0.00012207 gij76253701 decorin (Bostaurus] 49.72222 0.730415 0.440608 4.230-06 1.108696 0413871 00897178 0.694329 0.522041 4.45E-05 gil115496400 dihydropyrimidinase-bike2 [Bos taurus] 32.16783 1.377001 0.288088 00027255 3.100286 0.478821 2820-05 3.50074 0.456305 1.01E-05 gil27806853 lumican [Bostaurus] 35.67252 0.972895 0.301185 0.4589595 1338501 0257497 6490-10 2.23113 0309532 8.75E-07 0 gir76670918 PREDICTED:similar to histone H4 [Bos taurus} 83.49515 0.82703 0.405577 1.880-09 1.37621 025284 9610-12 2707767 0451848 gi|115496892non-metastatic cells 2, protein (NM23B)expressed in [Bostaurus] 61.18421 1.105545 0.210209 00468387 3.00549 0607591 2100-06 0408232 0.943364 7.28E-08 gi|114051505 serpin peptdase inhibitor,clade H (heat shock protein 47), member 1, (collagen bindingprotein 1(Boo taurus] 36,1244 0.848087 0.362412 0.0351232 1481406 0432926 0.0007492 2208003 0.34101 1.23E-08 gi|114052731 tubulin, beta polypeptide [Bos taurus] 36.93694 1.032283 0.352411 0.7898694 3.855163 0380602 2.720-06 4.388894 0533753 9.47E-06 gi|119903031 PREDICTEDhypothetical protein[Bos taurus] 25.41322 1.712802 0.342161 2520-12 3.081876 0.488811 0 3.107097 0.490403 0 gi|119913640 PREDICTED:similar to KIAA0051isoform 1 [Bos taurus] 20.07481 0.958993 0.426455 06774568 2.536098 0.811613 00002813 3.587219 0.567402 3.54E-06 gi]78369684 platelet-denvedgrowth factor receptor-likeprotein [Bos taurus] 35.2 0.84085 0.295958 0.0089347 0747438 0.240368 3.220-05 1154384 028933 0.02406251 gi|78369242 alpha-actinn [Bos taurus] 38.90134 1.389165 0.49615 0.0307261 2.214585 0481167 0.0002806 2.756921 0533143 0.0001105 gi]129277510superoxide dismutase 3, exracellular [Bostaurus] 37.3444 0.909759 0.26464 00382466 0.687629 0.32273 7010-07 0.544909 0.462822 1.58E-07 giJ68299807 eukaryotictranslation elongation factor 1 alpha 1 [Bostaurus] 29.87013 1.047109 0.340916 00670238 277265 0.850019 354E-08 3.486767 0.884485 1.84E1-09 0.00059758 gi|115497328 procollagenC-endopeptidase enhancer 2 [Bos taurus] 36.29808 0.689359 0.234566 2.890-06 0922919 0292597 0.1495701 1.7825 0.651812 gi|115495641 aldo-keto reductase family1, memberAl (aldehyde reductase) [Bos taurus] 49.53846 1.22208 0.379178 0.0395591 3.068088 0579483 3380-06 3.802311 056824 5.36E-07 gil76635416 PREDICTED:similar to coat protein delta-cop isotorm 1 [Bos taurus] 29.7456 1.08162 0.421572 0.5838214 3.853607 0483844 0.0001858 4.440202 0538426 0.00018981 gi]76613946 PREDICTED:similar to Histone H1.5[Bos taurus} 57.52212 0.819695 0.450096 0.0325495 1149782 059554 0.2488871 4845611 0.88057 2.64E-07 gi|31341666 mannosidase, alpha, class 2B, member 1 [Bos taurus] 23.72372 0.886321 0.446803 0.222994 1.14973 0304272 00627517 1343001 07043 0.06036566 23.12704 1.378899 0.582837 0.0605531 2.492031 0.596562 0.0001733 2746881 0582638 gi|119912215 PREDICTED:hypothetical proteinisoform 2 [Bostaurus] 6.89E-05 gi|62751849 Parkinsondisease (autosomalrecessive, early onset) 7 [Bostaurus] 7089947 1.612388 0.31221 2.578-05 3.98492 0400179 2.81E-09 3.820856 0.679266 2.75E-08 9i[139949116slit-like 2 [Boslooms] 28.52897 1.338105 0429092 00002926 0.904302 0.27225 0.0308789 1.227725 0.591427 0.13297131 28.18429 1243113 0.239059 0.0227445 2.67794 0.8840080.0013486 3.156577 0.779729 0.00134692 gi[1561205Dl4 05 26 53 heat shock70kD protein binding protein [Bos taurus] gill1 procollagenC-endopeptidase enhancer[Bos taurus] 41.24188 0.747768 039671 0.0010757 0.907298 0240483 0.0277299 0.975545 0.645648 0.8278468 gi27807349 complementcomponent 1 inhibitor[Bos taurus] 2284957 1.934870408844 3.788-10 028844 0.8422580.00023321.154815 0.379542 0.01799357 gi|27807167peroxiredoxin 6 [Bos taurus] 58.25 1.574858 0.471361 00005459 3.539818 0.728845 1.10E-064.112762 0.687028 4.97E-09 gi|77735541valosin-containing protein [Bos taurus] 3325082 1155745 1.015324 0.578125 1.982845 0.741708 0.015625 3.081692 0.629709 0.015625 gil148238309 aldolaseC, fructose-bisphosphate [Bostaurus[ 4835185 1.292778 0.681553 0.193125 3.157838 0.8997380.0002856 3.731659 1.033639 0.015625 gil27807007 insulin-likegrowth factor bindingprotein 3 [Bostaurus[ 371134 0.788581 0.778124 0.0288555 1761299 0.338075 1.65E-06 1.708408 1.5065580.08921194 gi75812940 phosphabdylethanolaminebinding protein [Bos taurus] 6258885 1.289117 030763 0.00282 3.01298 0.489092 3.13E-09 3.746912 0.524693 1.19E-09 gil27806671 cathepsinB [Bostaurus] 31.34329 0.943841 0205782 0.0327489 1.389304 0.2582490.0081208 0.504166 0.829374 0.0330881 gi|119920777PREDICTED similar to collagen,type IX, alpha2 [Bostaurus] 2981104 0.54183 0.958949 0.00289950.822455 0.2880620.0019401 0.609309 0.603253 0.00108429 gi|119901068PREDICTED: similar to Collagen,type IX,alpha 1, partial[Bos taurus] 54.0148 0.482305 0,458428 0.00032640.758333 0.1933130.0005563 0.499832 0.555497 0.00140861 gi27805999 matrixmetalloproteinase 13 [Bostaurus] 2590234 0449574 0.439799 0004883 0.398849 0.248278 1.14E-090.58289 0.213158 6.72E-08 gil27806637 fibrillin1 [Bostaurus] 1605712 1158511 0.333925 0.31250850119 0.1402440.0080701 0691041 1.667035 0.46885429 gil27806617 GDP dissociationinhibitor 1 [Bostaurus] 3534878 1.814872 0.9995 0.0098531 3508770 0.5482110.0005312 3.634862 0.593508 2.34E1-09 gil27808640 peptidoglycanrecognition protein 1 [Bostaurus] 51.05263 045803 0.326152 1.70E-080.808319 0.257445 0.003467 0.400714 0.724958 0.00027455 gi|157427782 insulin-likegrowth factor binding protein 5 [Bostaurus] 50.92251 1181072 0354524 00280809 0.906282 0.48406 0.9013177 2.080596 1.098557 0.00157819 gi|119895711PREDICTED: similar to cytokine-likeprotein ZCYTO7 isoform 1 [Bostaurus] 2748781 0507187 0.877454 3.35E-06 1.17427 0.37824 0.0084102 1.9352331.077054 0.00245713 gi|78369298stromal cell derivedfactor 4 [Bostaurus] 42.25352 0778141 0.33739 0012125 0.85096 0.1598280.0034501 1.083191 0.272478 0.24000671 gi|77735539procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide || [Bostaurus] 24.22998 077955 0452039 005827 2.820207 0.308505 4.25E-062.514681 0.302407 4.98E-06 gi|27806197tyrosine 3-monooxygenase/typtophan 5-monooxygenase activation protein, epsilon polypeptide[Bos taurus] 72.94118 1.358303 0.443855 00002031 3.00073 0.552971 3.00E-11 3.319728 0.62026 1.44E-11 gi|119919662PREDICTED: similar to LOC539445protein [Bos taurus} 189 0.585452 0780314 0.0452885 4.897555 0.831237 9.61E-05 5.847062 0.859128 2.02E-05 gi|27805983leukocyte cell-derived chemotaxin 2 [Bostaurus 642341 0.409485 0331874 7]74E-08 0.990859 0.3119310.6551496 1.049618 1.145164 0.70533302 gi|1 16003813 amyloidbeta A4 protein[Bos taurus] 29.06475 1.538547 0.230885 00014379 0.840781 0.298271 0.086367 1.247608 0.218664 0.01595411 gi|129270171gremlin-1 [Bos taurus] 51.83044 0793555 0.87352 0141028 1.001231 0.3376480.8968756 1.140038 1.154018 0.01479114 gil148228060 stem cell growthfactor [Bos taurus] 3240741 0848954 0.267988 00194377 1.002715 0.27112 0.9519144 1.23396 0.252748 0.00390625 gi|119915902 PREDICTED:similar to Thioredoxindomain-containing proei5precursorThioredoin-keproteinpEndoplasmic p465 re(dculuoprotein Epp446iso)otm1 [Bar 3947389 1.258969 0.559088 0.05354583.870127 0.817854 4.28E-13 4.664796 0.711726 4.55E-14 gi|27806401 secretedphosphoprotein 1 [Bos taurus] 37.78979 0.903294 0.221566 0.0022755 1.915212 0.235771 4.44E-16 1.401041 0.245955 2.70E-10 gi|76613223 PREDICTED:similar tochitinase [Bos taurus] 39.01809 030445 0.493303 00004883 0.209851 0.404558 7.54E-10 0.07019 0.829902 5.78E-09 gi|77404209 eukaryotictranslation initiation factor 4A2 [Bostaurus] 3438803 1.233772 0388759 0.21812943.109795 0.252134 0.0010031 3.010434 0.426075 0.00498072 gil119917871PREDICTED: similar to serin proteasewith IGF-bindingmotif [Bos taurus] 2020849 0.771777 0.243891 8.73E-050.081847 0.202598 0.224032 0.981734 0.433694 0.29577637 gil164448628carboxypeptidase X [Bostaurus] 19.88301 0.788564 0268784 0.001439 1.008874 0.249295 0.9681949 1.339499 0.394484 0.19335938 gil113205970glial fibrillaryacidic protein[Bos taurus] 48.83178 1.027088 0483528 0.8502459 2.292389 0.6884970.0082859 3.098982 0.936698 0.03125 gi178045555 acidic (leucine-rich)nuclear phosphoprotein 32 family,member B [Bostaurus) 28438 1.143311 0510882 0.3222514 3344078 0.476514 1.78E-05 4.841424 0.63541 1.97E-05 gi|119895049 PREDICTED: similar to LaminB2 [Bostaurus] 32.78148 1.343139 0.667012 0.1147527 1.874172 0.482114 0.00117 2.522016 0.602373 0.00041745 gi]27806829osteoglycin [Bos taurus] 34.78281 0.858085 0.220385 0.00211511.021554 0.284641 0.7655521 1.061728 0.3270.34082302 gi]78045491fascin homolog1, actin-bundlingprotein [Bos taurus] 23.32857 1.334458 0.4889 0.01083013.119513 0.442908 0.0024413.158849 0.428223 8.73E-09 gil119908208 PREDICTED: similar to Calpain2, (m/11)large subunit isoform 3 [Bostaurus] 23.83014 0.777731 0.805924 0.25078992.057831 0.459223 0.00270782.089055 0.255707 0.00015682 gi[62751777cofilin 1 (non-muscle)[Bos taurus] 59.03814 1.249729 0.371271 0.08799523.522417 0.42509 2.95E-05 4.929116 0.442864 9.21E-06 gi|78042524 thioredoxindomain containing 4 (endoplasmic reticulum)[Bos taurus] 2508207 0.995942 0.314542 0.833009 2.889886 0.573365 0.03125 2.574683 0.61755 0.00110559 gil27806449 dystroglycanI [Bostaurus] 18.32402 1114704 0.428948 0.12392920.989739 0.21681 0.6524901 1.604039 0.364926 0.00193099 gi]94966781 eukaryotictranslation elongation factor 1 gamma[Bos taurus] 26.59091 1.182179 0847073 0.45095113.044482 0.592818 0.0011996 3.21599 0.638340.00131691 gi|164420791transforming growthfactor, beta2 [Bostaurus] 30.43478 0742424 0.259859 00022421 1.87391 g.261033 9.46E-05 2.987197 0.773021 0.00067956 gil 14053333fibrinogen-ike 2 [Bostaurus] 3850784 1.17009 0309784 0.04671230.427778 0.195793 9.33E-09 0.444388 0.340456 1.78E-06 gi[95006989 ribonuclease, RNaseA family,4 [Bostaurus] 57.1428 0804356 0.385366 2.98E-09 0.917848 0.249674 0.0003852 0.639449 0.405761 1.40E-07 gi]27806081peroxiredoxin 1 [Bostaurus] 47.7389 1.145655 0.251919 0.11600143.559536 0.494199 0.08025234.558172 0.460837 8.24E-05 gi|27806561lactate dehydrogenase B [Bostaurus] 47.30539 1.217427 0.422007 0.09978333.073825 0.570874 0.000102 3.625548 0.586296 4.38E-05 gi|77735425proteasome alpha 7 subunit[Bos taurus] 4508452 1.3595 0.250214 0.00751124.311514 0.535948 4.73E-05 5.37498 0.447148 7.15E-06 gi|27806679colony stimulating factor 1 (macrophage) [Bostaurus] 21.94115 1.5128 0.285552 0.015625 0.008248 0.535002 0.0208893 1.487397 0.40853 0.00297115 gi|119917542 PREDICTED: similar to tolloid-like2 proteinisoform 2 [Bostaurus] 18.33333 0.558517 0.413338 0.00183580.932274 0.44272 0.1525279 1.677954 0.435032 0.00392804 gi|115497078splicing factor, arginine/serine-rich1 (splicing factor 2, alternatesplicing factor) [Bos taurus] 54.83871 1.197937 0.333368 0.03353993.03228 0.520847 1.40E-06 3.276995 0.605293 2.84E-06 gi|165905571leucine aminopeptdase 3 [Bostaurus] 29.28709 1.483983 0514998 0.04256542.252881 0.725022 0.0111017 2.875392 0.698814 0.00308652 gill4051129 retinoicacid receptor responder (tazarotene induced)2 [Bos taurus] 5123457 0668587 0341849 2.548-09 0.705825 0.385304 2.47E-07 0.775408 1.0322070.08804999 gil157073966 reticulocalbin1, EF-hand calciumbinding domain [Bos taurus] 33.83686 110571 0.39971800947473 3.231942 0.338122 1.31E-08 3.505302 0.382501 2.27E1-08 gi]27806541 matrixmetalloproteinase 1 [Bostaurus] 31.5565 0.272697 0491877 1.30E-050.887994 0.145805 8.60E-06 0.954822 0.11586 0.7421875 gir41386760 CD14anbgen[Bostaurus] 3646113 0.952161 019569 0.25332950.378479 0.350338 1.01E-07 0.619293 0.556359 0.00207345 gill 19915070PREDICTED: similar to CUB andEGF containing protein [Bos taurus] 14.50151 0 78g134 0409551 0.0356442 0.90833 0.309445 0.4955312 1.431923 0.783978 0.08278948 gil27807377 glutamic-oxaloacetictransaminase 2. mitochondrial(aspartate aminotransferase2) [Bostaurus] 36.74419 1.406094 0270802 00067711 3.928337 0.298463 1.67E-05 4.352382 0.349685 2.49E-05 gi|78369510 ribonuclease/angiogenininhibitor 1 [Bostaurus] 2939597 1.501005 0325888 0.00704174.131356 0.247004 0.03125 3.17242 0.74366 0.00274529 gi|118601868 heat shock 90kDa protein 1, beta [Bostaurus] 3991713 1.179283 0338089 0.1518089 3.748732 0.368913 5.46E-05 4.628471 0.337131 1.70E-05 gi|27806825pepbdylglycine alpha-amidating monooxygenase [Bos taurus] 17.90123 066916 0.256285 0.00261280.921522 0.1398 0.0751198 1.025040.244148 0.73498679 gi|82697375 histoneH2B-like [Bos taurus) 73.015g7 0701006 0.441253 00220058 1.387118 0.449981 0.0345741 3.246232 0.402594 3.08E-05 gi|1154971743'-phosphoadenosine 5'-phosphosultate synthase 2 [Bostaurus] 299197 1.099204 0395566 0.3926138 3408799 0.393327 4.44E-06 4.956252 0.403776 8.39E-07 gi|119901198 PREDICTED:similar to Man9-mannosidase[Bos taurus] 27.6176 0669272 0337197 00039794 1.227779 042194 0.122205 1.555083 0.420027 0.0070192 giJ28603774 RhoGDP dissociationinhibitor (GDI) alpha [Bos taurus] 46583 1693909 0506794 0.015072 3518611 0682599 0.0013047 4.0184230.633009 0.00056677 giJ71037405 heat shock27kDa protein 1 [Bostaurus] 50 0.99641 0.516659 0.975981 4.504843 0.777647 0.000323 5.119911 0.722672 0.00013365 giJ27806751alpha-2-HS-glycoprotein [Bos taurus] 25.90529 0280017 0351435 4939-08 0.44843 0.269395 2.37E-07 0.211971 0.516017 2.45E-07 gi|77735769complement component 1. r subcomponent[Bos taurus] 2994924 0559876 0.399 0.0093022 0.608092 0477975 0.0267146 0.404853 0.967008 0.03929655 gil 19894807PREDICTED: interleukin enhancer binding factor 3 isoform2 [Bostaurus] 2095075 1.456753 0582043 0.1058396 2103822 0.172287 0.0001595 2.924806 0.509752 0.0625 gi[94966765glucose phosphate isomerase [Bos taurus] 29.08438 1.019204 0.584551 09159895 2.748777 0.93774 0.0082061 3.179529 0.941867 0.00742862 gi[115496067nucleobindn 2 [Bostaurus] 2843373 0.977995 0519511 0.8450852 1.283333 0.328989 0.0082484 1.505560.279417 9.05E-05 gi]27805977keratin 10 [Bostaurus] 23.19392 0794292 0.417656 0.0875888 0.918927 0.357075 0.368511 D.926095 0.310298 0.38139633 gil119901449PREDICTED similar to PDNP1[Bos taurus] 20.31873 1122001 0.570995 0.3842895 2.127114 1.0820980.0116401 2.076167 1.308587 0.03135567 gi[77736203malate dehydrogenase 1, NAD(soluble) [Bos taurus] 30.23952 1620919 0254319 00001127 4.000058 0.345863 8.00E-074.334892 0.225396 2.75E-08 gil41386798poly(A) binding protein, cytoplasmic 1 [Bostaurus] 29.24528 1.241022 0.516059 0.1875 2.920083 0.559438 0.001099 4.082206 0.496975 0.00017076 gil27806881epididymal secretory protein E1 [Bos taurus] 3087249 0.454079 0.389 0002295 1.695337 0.153522 0.000394 1.488269 0.099131 0.00020578 gil27806415scinderin [Bos taurus] 2293708 1.300330.289782 00434031 2.595421 0.60842 0.007284 3.324911 0.661368 0.00423199 gi|76610312 PREDICTED:similar to neuropilin2 isoform8 [Bostaurus] 2915767 1.3179350334412 00120733 0.788258 0.331260.0126559 0.965049 0.22483 0.53905575 gil157744500tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypephde [Boo taurus] 51 01215 1.195710 0515928 0.327959 2888529 0.3380740.0005647 3.56837 0.347956 0.00029579 gir27806673 cathepsinL2 [Bostaurus] 3502994 1190907 0253439 0.1028135 1.025708 0.10081 0.6850116 1.033617 0.762904 0.625 gi]30794292 lactotransarrin[Boo tauras4 2387006 0.69982 0.444285 0.0310855 0.538608 0.9787910.0738598 0.571186 1.170383 0.15162747 0.448173 00201767 0.914237 0365116 02366615 0617444 0571456 0.10564933 gi|76620378 PREDICTED:similar to cytokine-likeprotein C17 [Bos taurus] 52.55474 0.772804 gi|125991942 Sec23 homologA [Bostaurus] 16.92708 1.40908 0.550793 0.1706566 4321113 0.756614 0.011496 4706377 0.624164 0.01229541 23.28482 0.195757 0.44865 0.000302 0.406175 034643 0.0002543 0.329361 0,643969 0.00172116 gi|845798534 lipopolysaccharidebinding protein [Bos taurus] gi|11990114 PREDICTED: similar to malate dehydrogenase decarboxylase(NADP+) [Bos taurus] 20.62147 1.467496 0.340693 00106179 4.224115 0.462441 1.671-07 4.233192 0493512 6.82E-08 7.49E-13 gill14051157 complement component 1, q subcomponent, 8 chain [Bos taurus] 31.17409 1.103934 0.2474 00117622 0.631504 0.3034 0.0002543 1.784666 0.272691 gi115497294 canopy 2 homolog [Bos taurus] 57.14286 1.031638 0.370242 0.7466377 3.754973 0393293 5.045-07 3777664 0.363764 0.00390625 gi|76651183 PREDICTED:similar to histone H3 [Bostaurus] 55.14706 0.842391 0.226061 00045065 1.355866 0233258 0.0001135 2.695744 0069296 4.41E-06 0.00020993 gi|27807523 ribosomal protein P2 [Bos taurus] 57.3913 1.883075 0.8785 00516846 4409362 0.981136 0.0012072 6.355423 0687346 gi|156121049 dimethylargininedimethylaminohydrolase 1 [Bos taurus] 55.08772 1.236429 0.555719 0.4416299 2.976156 0.624865 0049293 2773325 0.600857 0.05132816 0.241434 0.57150453 gi|75832065 TIMP metallopeptidase inhibitor2 [Bos taurus] 40.90909 0.867925 0.293951 0.0170916 1.479176 1.09336 0.0412596 1.025353 gi]61888850 adenylate kinase 1 [Bostaurus] 38.14433 1.136216 0.430822 0.3033108 3023313 0565108 0.000306 4901766 0.59492 5. 18E-05 0.00186412 gi|28875793 thrombospondin2 [Bos taurus] 13.24786 1.005457 0.336821 0.9553344 1109125 0210213 0.1260952 1.450622 0313212 gi|119923009PREDICTED similarto put. HMG-17protein [Bostaurus] 42.22222 0.646482 0.829652 0.0304121 2144563 0499891 1.110-05 4168457 0659649 3.26E-07 0377662 gi|148232266fibulin 1 [Bos taurus] 15.58074 0.887858 0.247168 0625 0605337 0.326422 00923653 0.664477 0.28146037 0.20613 0.46117897 gi|119890932PREDICTED: similar to Lamininbeta-1 chain precursor (LamininB1 chain) [Bos taurus] 15.35245 1.354883 0.234493 00060169 1148729 0.449674 0.3464434 1.046109 1 gi|119896006PREDICTED: similar to mannosidase, alpha, class 2A, member 1 [Bos taurus] 16.59389 1.243177 0.281497 0.4375 0993192 0.234221 0.6643037 1.267403 0479661 0.58778549 gl155372035 nidogen 1 [Bostaurus] 16.6801 1.231962 0.36954 0.1034256 0877971 0.561423 0.4293699 1.070923 0.456856 0300522 0743839 0.00229874 gi]62460514 angropoetin-ike 7 [Bostaurus] 20.93023 0.968973 0.470794 0.6187797 0.373047 0497327 0000686 0597922 4.75E-05 gil164414726thymosin beta 4 [Bos taurus] 81.81818 1.30961 0.482918 01060539 6.545266 0.647427 0.000154 9.062092 1250631 1.242364 0.296875 gi|27806095 protein S, alpha [Bos taurus 18.66667 1.17304 0.332141 0296675 1.146996 1368479 0.296675 gi|45429977 staphylococcal nuclease and tudor domain containing 1 [Bos taurus] 28.24176 0.986197 0.378524 0.6972496 3.137771 0.444296 00002761 4365947 0.341663 2.20E-05 gi|27807209 alpha-2-plasmininhibitor [Bos taurus] 18.90244 0.942 0.386282 0.6624672 1.042147 0.242856 0763677 3.120632 0.202312 0.00050626 gill 16004039 peptidylprolylisomerase C (cyclophilinC) [Bos taurus] 44.33962 0.746961 0.398982 444E-05 0.90611 0322438 0.0274032 1.201796 0.446562 0.00667512 gi|119914274PREDICTED: hypothetical proteinisoform 1 [Bos taurus] 20.91429 1.114657 0.268242 03297717 3.595856 0387989 0.0056739 3.135764 0308311 0.00174715 gil27806553 microtubule-associatedprotein 4 [Bostaurus] 25.74627 0.784726 0.556233 02964631 1.941072 0453118 0024877 2.856925 047348 0.00772229 gi45430019 ribosomal proteinL12 [Bostaurus] 43.0303 1.051228 0.153254 0.3578261 3.399142 0.621972 00625 4.543992 0804589 0.00372183 gil119919151 PREDICTED:similar to AHNAKnucleoprotein isoform1 [Bostaurus] 36.92494 1.238706 0.309334 0.0900441 2.705998 0.997999 00059426 3.486668 0.71653 0.00164087 gi|119919898 PREDICTED:hypothetical protein[Bos taurus] 24.85714 0.696563 0.354593 0.0002441 0873866 0.394239 00651016 1113116 0.402894 0.15939404 gi|119908402 PREDICTED:similar to Calsyntenin-1,partial [Bos taurus] 17.28135 0.971916 0.307732 07515049 0.937539 0.153447 0.1634666 1306979 0.201769 0.00539029 0.00183715 gl157785567 calnexin [Bostaurus] 19.56155 0.981782 0.4997D2 09194103 3.107429 0.510515 00078239 3.498451 0343663 giJ78369456 nucleophosmin(nucleolar phosphoproteinB23, numatrin) [Bos taurus] 33.67347 1.151309 0.476669 0.4582779 2.610443 0.613626 0.0154491 3.539745 0.916422 0.02854164 0.03157637 gil119331192 H2Ahistone family,member X [Bos taurus] 62.23776 0.721476 0.046847 0.0032687 1.331159 0389176 0.2144017 3.195369 0.528406 0579555 9.74E-06 gil27807469 peroxiredoxin2 [Bostaurus] 46.23116 1.335529 0.376182 0.0165782 4.071044 0.517156 112E-05 4.946965 0.6916 0.00195313 gi[77736471 SH3 domain bindingglutamic acid-rich proteinlike 3 [Bos taurus] 53.76344 1.523003 0.241733 0.0005666 3.9735 0.602612 0.0009766 4.226144 0.00976563 gir76611607 PREDICTED:similar to C1QC proteinisoform 1 [Bos taurus] 21.69118 1.196257 0.499483 0.1373665 0.713353 0360547 0.0018497 1715993 0.691086 gill18151330 tyrosine 3-monooxygenaseltryptophan5-monooxygenase activation protein, theta polypepsue 005 taurus] 53.46939 1.666969 0.181633 00001768 4.070146 0340142 2.885-05 4.270728 0.235607 3.79E-06 0.61365111 gi|119892706 PREDICTED:similar to chondroitin4-O-sulfotransferase [Bos taurus] 33.28197 1.328366 0.280622 0.0160601 0.750151 0.456758 0.1076629 1.084646 0.033707 1.61E-05 gil60592767 ribosomal protein, large, PO[Bos taurus] 38.67925 2.339525 1.705012 0494 4967163 0752382 00006669 5.276421 06488 5.02E-06 gi|62751593 proflin 1 [Bos taurus] 47.85714 0.984943 0.229807 0.7732803 2.827048 0.262665 2.065-07 3961251 0.95482 gi|78042564 CNDPdipeptidase 2 [Bos taurus] 28 0.759355 0.657534 0.3126366 1393516 0.063048 00613242 2.115963 0.493526 0.02201728 gi27806625 frizzled-relatedprotein [Bos taurus] 27.69231 0.611032 0.397021 270E-07 0.947817 0.181766 0.0071035 1.868062 0.433897 2.04E-14 gil119901416 PREDICTED:similar to Syncrip protein [Bostaurus] 30.4078 0.978475 0.537585 0.8965113 245728 0.593261 0.0083154 2.944606 0.662037 0.00694147 gil29135295 aspartate aminotransferase I [Bos taurus] 29.05569 1.4351 0.81286 0.5316989 4.332814 0.399891 0.0649655 4.030831 0.157902 0.03530835 gil110626119 insulin-likegrowth factor bindingprotein 6 {Bostaurus] 39.28692 0.515588 0.843656 0.0021144 0.6635 .30365 0.0002922 0.787142 0.499883 0.06738705 0.772411 0.17339178 gi|119907521 PREDICTED:similar to NuMAprotein [Bos taurus] 24.79962 1.310653 0.767904 0.4724615 1.570161 0.931907 0.3025977 1.900436 0.12274193 gi|155371853 thrombospondinrepeat containing1 [Bos taurus] 16.07477 0.609054 1.186849 0.246469 0.692773 0.161206 0.0026575 0.600518 0.842992 gi}114052384 heterogeneous nuclear ribonucleoproteinA1 [Bos taurus 32.5 1.401466 0.278818 0.0398761 2.349795 0.462218 0.0101637 3.707999 0.375063 0.00207823 5.67E-08 gi|115495555 endoplasmic reticulumprotein 29 [Bos taurus] 40.31008 1.209341 0.469429 0.2132029 5887609 0.59221 5635-08 6.924352 0.649171 0.009058 gij27806279 caldesmon 1 [Bos taurus] 27.66355 1.521052 0.861412 0.3484215 3.234318 0.655 0,0466966 5.043265 0771672 giJ78042498 phosphoglycerate dehydrogenase [Bos taurus] 29.26829 1.091927 0.23322 0.293771 1.915942 0.338410 00049064 2.520743 0.607506 0.00418842 gi|27806447 prosaporsin[Bos taurus] 15.42857 0.780148 0.148658 0.0169044 0.994096 0,250669 0.8962366 0597473 0.756674 0.14392217 gil27805981 ribosomal protein SA [Bostaurus] 30.50847 2.017567 1.029917 0.1442065 6706982 0.714377 00002276 7.159304 0659366 0.00013219 0.05113466 gil155372057 cysteine-rich secretory protein LCCLdomain containing 1 [Bos taurus] 23.2 0.718178 0.660928 0.1807677 1.217623 0.210773 0.3434273 1.521064 0.491197 gil125630701 collagen triple helix repeat containing1 [Bos taurus] 46.50206 0.939134 0.253669 0.3765814 0.866708 0.194334 00261033 1.124384 0.360969 0.26137904 gill18151356 angiogenin, ribonuclease. RNase A family,5 [Bos taurus] 42.56757 0.561035 0.269208 5.04E-05 0.904662 0.266371 0.3125 0.770496 0.740227 0.7421875 gil28461187 myosin, lightchain 6, alkali,smooth muscle and non-muscle[Bos taurus] 44.37086 1.189206 0.334906 0.0966763 2.372D42 0.481176 0.0076125 2.789196 0.525725 0.0078125 0.00050396 gir27805805 fatty acid bindingprotein 5 [Bos taurus] 53.33334 1.143116 0.588056 0.5057353 2.664728 0.465376 0.0007116 3.373152 0.539626 0.00476912 gi56119114 purine nucleoside phosphorylase [Bos taurus] 22.83737 1.077707 0.16977 0.386629 2.49569 0.480878 0.0420877 328739 0.453214 gi|61097917 cystatin E/M [Bos taurus] 41.61074 1.588616 0.617223 0.0730954 2.46493 0470962 0.0035763 1023269 0645059 0.91396118 E-05 gi|1 19888578 PREDICTED:hypothetical protein [Bos taurus] 33,33333 1.305207 0.579915 00531219 5.156013 1.36457 4.395-05 5291402 1.331999 2.91 gi|56710317 keratin 5 (epidermolysisbullosa simplex, Dowing-Meara/Kobner/Weber-Cockaynetypes] [Bostaurus] 28.1198 0.568797 0.044792 0.0247297 0755315 0.62039 05163629 0796984 0.22566 0.28874226 gil119919488 PREDICTED:similar to cathepsin D isoform 1 [Bos taurus] 25.97087 1.343171 0.029007 0.0015513 2.009387 0265239 00227652 1591036 043976 0.11860294 gi]62751650poly(rC) binding protein 1 [Bos taurus] 33.98876 1.676168 1786418 0.5455095 3122778 0.520259 00021674 4.541486 0514743 0.00014232 gi|62460494hemoglobin, gamma [Bostaurus] 70.34483 2.79713 0.550535 00125265 1.199678 0402669 02966016 3962602 0.365669 0.00195396 gi|116004251 granulin [Bos taurus] 16.18399 0.937838 0.30186 0.5792656 1.237269 021475 0.0693237 0351941 1.002473 0.05740508 0.00165727 gi|27806813 pleiotrophir [Bos taurus] 42.2619 0.334023 0.63773 0.0002441 1.375822 0.39724 0001486 1.622514 0.624073 0.09747653 gi|30466254 gelsolin-likecapping protein[Bos taurus] 19.19771 1.580628 0.619966 02067068 2.304159 0.90919 0.150557 2794152 0.666304 gil28461287 hepatoma-derived growthfactor (high-mobilitygroup protein 1-like)[Bos taurus] 51.88285 1.156699 0.776058 0.627433 274657 0.75906 00130126 3205741 0.596991 0.00329443 gil78369458 proteasome (prosome, macropain) subunit, alpha type. 1 [Bos taurus] 25.47528 1.348022 0.391818 0.015625 3.217466 0590741 0.0002686 3.529642 0.839054 0.00121962 7 0.1087656 gil2780714 C-type lectin domain family3 member A [Bos taurus] 38.07107 D.487568 0.137961 0.0006384 0.713065 0.236462 0.0251242 0728636 0.403697 0.05549302 gil27806139 secreted frizzled-relatedprotein 1 [Bos taurus] 35.71429 0.600276 0.353855 4.14E-05 1.010426 0.232691 0936477 1.375163 0.703576 0.20064388 gi|115497736 offactomedin-like3 [Bos taurus] 19.21182 0.461024 0.437169 0.0046268 115529 0.332775 02520667 076319 0.569631 0.00093924 gi|134085635 glyoxalase I {Bos taurus] 35.86957 1.343524 0.194285 0.03125 3.196009 0.427377 0.0002126 4.158412 0.723263 gi|119919689 PREDICTED:similar to RNAbinding motifprotein, X-linkedisoform 5 [Bos taurus 48.59335 0.957059 0.484712 0.6080712 3.306448 0.533767 00073567 5.9913 063796 0.00393705 17.04835 1.130043 0.300774 0.3276844 1.40175 0.407355 00565616 2.059305 0453447 0.00679523 gil1198927757 PREDICTED:similar to myosin, heavy polypeptide 9, non-muscle [Bos taurus] gi]6275184 ribosomal proteinLlOa [Bos taurus) 37.78802 1.390803 0.337679 0.067115 5065662 0.510943 00005196 5632550 0.559054 0.000056759 gi|164518978 diazepam bindinginhibitor [Bos taurus] 72.41379 1.865069 0.402858 910E-06 4.57462 0415176 144E-09 5756974 0.420637 3.51E-10 gi|77735725 cathepsin H [Bos taurus] 29.55224 1.142346 0.205271 0.1595653 1.684237 0.456006 00477095 2.935988 0.451667 0.00628833 gil119926480 PREDICTED:similar to Meteorin,glial cell differentiationregulator-like [Bos taurus] 25.24272 0.59365 0.376463 00018407 0.64177 0328059 00629004 0692403 043771 0.01847104 gil119907290 PREDICTED:hypothetical protein[Bos taurus} 20.61856 1.765132 0.057721 0.0016523 0322906 0.194399 00041666 0.476651 1.69626 0.38921172 22.22222 1.183397 0.220437 0.1976026 2.247146 1.107326 0.0793661 3.249727 0.986715 0.01837422 gi]77735597 FK506-bindingprotein 4 [Bos taurus] 14.94565 1.197811 0.733036 0.6022613 2.296675 0.279972 0017423 2.512035 376722 gi|157074098 leprecan 1 [Bos taurus] 0.02575885 38 7.18E-07 10 8 10 gi]8885 16 superoxidedismutase 2, mitochondrial[Bos taurus] 2747748 1.235636 0.493475 00829534 0.734209 0.227854 00007784 1.999054 0261175 6 6 6 gill15432055tyrosine 3-monooxygenaseltryptophan5-monooxygenase activation protein, beta polypeptide[Bos taurus] 55.69106 1.422587 0.077752 174E-05 3.501318 0192254 2.94E-06 3.858543 0211649 319E-06 3.1876850.565521 000271062 5 5 5 gil157785569coatomer protein complex, subunit alpha [Bos taurus] 129085 1.3169660.397573 00897317 2610025 0.47391300029117 4 5 6 7 gil1199050DPREDICTEDsimilar togrowth-anest-specioc protein (809 taurus) 7765276 0721107 0376862 0.04859110.697037 0.093336 0.03125 0.548286 0.769348 0.02458663 0.47596106 9 9 8 gil122692559oncostatn Mreceptor [Bos taurus] 31.17284 1.280483 0.106645 0.0001005 0.6362 0270181 8.02E-05 1.042406 0.240358 0.0625 5 5 5 gi76613071 PREDICTEDsimilar to class mu glutathione S-transferase isoform 1 [Bostaurus] 5183486 1.338978 0.684998 0.125 3909715 0.427273 00005115 4.55274 0.491575 002123441 3 4 4 gi]83715992coatomer protein complex, subunit gamma 1 [Bos taurus] 18.7643 1.383876 0.198783 00552539 2835368 0.600945 00156205 3.367195 0.788878 000018219 3 4 4 gi|27806103polypyrimidine tract bindingprotein 1 [Bos taurus] 18.39736 1.071288 0.177561 043833034928856 0.45951800021435 7.5342880.258851 023030411 4 4 4 gi|95147666periostin, osteoblast specific factor [Bostaurus] 21.822800667162 0.5532701248816 0941027 00817 0.12681170.741324 0.575318 0.613798001001715 4 4 4 gi|77736071heterogeneous nuclear rbonucleoprotein K [Bos taurus] 3189655 0892417 0420267 08583256 2468258 0.65711700291513 3.461617 0.740719000166614 5 5 5 gi|119914936PREDICTED similar to histoneHix [Bos taurus] 4252336 0.9021830.701122 08305065 1.3043080.498831 01688872 5.633982 1.5496260.505138 000198304 11 11 11 gil6246D582 complementcomponent 1, q subcomponent, A chain [Bos taurus] 40.573771213977 0.2851660008303 0734811 0.25533700001522 2.5496730.608262 006165172 1 3 3 gil119902257PREDICTED: similar to peptidyl-Procis trans isomeraseisoform 1[Bos taurus] 57.31707-1.00065 1 -1 2222372 0.781190.1267682 0.1203970.00293502 6 6 6 gil27806743alpha-1-microglobulin/bikunin [Bos taurus] 33.230641.014587 0.191366 08085857 0.9703770.158274 04552212 1.202043 42 17 17 gil1990 80PREDICTED: similar to collagentype 5 alpha1 {Bostaurus] 22.426470.831915 0.302113 0180258 0927065 0.21652100354010 0.83402074081 0.98111659 17 3216-07 14 14 14 gill15494984 apolipoprotein D [Bostaurus] 34.391530.354994 0.444943 1 19E-081,046545 0.348715 0.5554434 0.452896 0.449429 1 -1 1 2 1 gi|77735583 S-adenosylhomocysteine hydrolase[Bos taurus] 37.037D4 -1.00065 1 -1 2728891 0.533151 0.1631087 -1 0.01803671 2 3 3 gill 14051445protein phosphatase 2A, regulatorysubunit B [Bos taurus] 19.1356 0.808148 0.109661 03252149 2717641 0.488088 0.0365571 4.41776 0.505432 0.0145299 3 3 3 gi|114052264 phosphoglucomutase 3 (Bos taurus] 2121771 1.213355 0270846 02173425 2.652676 0.096038 0.001358 2.75446 0308063 2 2 2 high-mobilitygroup box1 {Bostaurus] 29.76744 0934584 0016017 0.0726401 3357907 0.782882 0.1962704 5.707625 0.760044 0.13755545 gi41386729 5 5 5 gil119904118PREDICTED similar to ChainA, NeutrophilGelatnase-Associated tipocalinp6 IsA NovelBacteriostacAgentThat nereresith Siderophore-Mediated IronL 219182 0o31c243 00001014 0387581 0363147 0.0010616 0.18633 0.793478 000174783 0.5846554 3 3 3 gil119919315 PREDICTED:similar to latenttransforming growthfactor beta bindingprotein 3 [Bostaurus] 17.73504 0.754775 0406388 02250735 1112067 0.066451 0.0642077 0.708965 1.331028 3 3 3 gil119920682PREDICTED: similar to adlican[Bos taurus] 15.83567 1.099632 0.772836 0789192 1131677 0.328484 04663631 1.191522 0.470687 045043484 004812702 3 3 3 gi|164452935SWAP-70 protein [Bos taurus] 20.51282 1.125470.509696 0622627 3064104 0.674581 0.054061 3.790778 0.758144 00659195 2 2 2 gil155372051hypothetcal protein LOC535277 [Bos taurus) 52.8168 1.476054 0685859 0.45391873102647 0.331506 0.0912452 3.180255 0.245301 6.08E-07 10 10 10 gil27806085peroxiredoxin 4 {Bostaurus] 4708029 0.886062 0.381269 01790651 1.576858 0.295458 5.216-05 2.110987 0.276311 00193342 3 3 3 gi]27806087probneatgirine-nchandleuome-nchrepaatprotem]Bostaurus 19.98504 1.028341 0.225889 0.791198 1766713 0.430917 0.082431 2.247018 0.285457 0.01141375 2 2 3 gil156523068ubiquitin-activating enzyme E1 [Bostaurus] 14.27221 1835553 0.961293 0.4203646 5.673488 0.316458 0.057302 8.848527 0.510027 4 4 4 gil76631783PREDICTED similar to chondrorho6-sulohansterase ]Bos taurus] 29.64509 0.989903 0230848 0.901174 0.870173 0.15354 0.0717274 1.118471 0.131443 0.0914539 4 4 4 cell growthregulator with EF-handdomain 1 [Bos taurus] 30.15267 1.374686 0564551 0.2030251 1.244621 0.3458 0.1742345 1.469283 0.338737 0.04651187 gil114051730 4 4 4 gil119884990PREDICTED similar to ceruloplasminisotorm 1]Bos taurus] 14.24528 0.510658 0.346303 0.0112042 0.773488 0.184435 0.0209447 0.459270 0.556732 002741702 9.27E-05 3 6 6 gi|156120555glyoxalase domain containing 4 {Bostaurus] 34.56376 1.361714 0.523666 0.2793152 4272346 0.430063 7.496-05 4.233143 0.449098 0.08149262 3 3 3 gij114326274 crystallin, zeta (quinone reductase) [Bos taurus] 34.84848 1.415376 0.253623 0.0760381 3465257 0.09736 0.0818 3.899744 0.13153 8 8 8 connective tissue growthfactor [Bostaurus] 20.3434 0.557302 0.359124 0.000204 0.996824 0.101397 0.7113753 0.134078 0.741618 1.10-05 gil27806677 2 3 3 proteasome(prosome, macropain)subunit, alpha type, 6 {Bostaurus] 40.2439 0.99902 0.37092 0.99438833.892598 0.253461 0.00507584.470833 0.273579 0.00530201 gill1405313549 7 7 7 gil278068 melanomainhibitory activity [Bos taurus] 39.23077 0.93442 0413171 0.55020780.74613 0.394438 0.0275114 1.870968 0.317091 844188343 4 4 4 gil114052350WD repeatdomain 1 [Bostaurus] 24.91748 1.328011 0.317688 0.08196092.835062 0.8448 0.0144696 3.231304 0.589377 0.01099513 6 3 4 3 1 inhibitorof metalloproteinase1 [Bostaurus] 32.36719 1.30685 0.251035 01004857 0.847312 0.272364 0.567031 0.540126 1.701831 0.46123532 gil278061 tissue 7 7 7 gil119905546 PREDICTED:similar to Cu/Zn superoxide dismutase [Bostaurus] 36.17021 1.751774 0.020838 0.00632993.824468 0.549542 6.88-05 3.578872 0.383382 0.019625 2 2 2 gi]94966811 alpha-iacid glycoprotein (Bos taurus] 37.12871 0.640945 0.68266 0.68488920.238141 0.21781 0.04784 0.180854 8.18886 0.03443773 0.120 5 5 5 gil156523084matrin 3 [Bostaurus] 23.81017 1.160861 0.498817 0.37806072.281601 0.88777 0.125 3.118349 0.901589 8.04815696 3 2 3 gi]990289635' nucleotidase,ecto [Bos taurus] 17.073170.964298 0.151043 0.6027264 1.523897 0.150462 8.1120271 1.975657 8.391831 3.194459 1.677478 0.39216793 2 2 2 gi]27807517settee (or osisteine] proteinase inhibitor, dade 8 (ocatbumin], member9][Bos taurus] 33.06878 1.568132 1.134077 0.56727582.791528 1.382881 0.3732823 1.016-05 2 5 9 gil62460568 eukatyobotranslaton elongaton factor 1 beta 2 [Sostautus] 27.11111 1.042784 0.568523 0.90645353.033238 0.42681 0.0010695 4.09589 0.615496 0.0046598 2 3 3 gil147902079glycyl-tRNA synthetase {Bos taurus] 21.92102 1.411195 0.087404 0.07892224.581574 0.30509 0.00842674.982354 0.274885 00078125 6 8 8 gi77735461 canopy4 homolog[Bos taurus] 24.68619 1.163013 0494403 0.3315886 3.65057 065318 8.766-05 4.676185 0.764743 0.00610483 4 5 5 gi29135329 glutathioneS-transferase pi {Bostaurus] 4285714 1.26077 0.144679 00182182 2.979707 0.11706 7.976-06 3.837103 0.287837 00913343 3 3 3 gi27805995 mannosidase,beta A, lysosomal [Bostaurus] 14.90338392585 0239537 00103341 0.768706 0.076163 0.0129617 0.490066 0.483761 009888402 4 4 4 gil114052595 galectin 3 bindingprotein [Bos taurus] 16.21622 1163616 0312811 0.258239 0855052 0.088827 0.01318830.725326 0.38382 0.08096119 5 5 5 gij77404434 hypoxanthinephosphonbosyltransferase 1 [Bos taurus] 20.6422 1.736061 0.481642 00224143 3252737 0.4512 0.00110554.128777 0.525803 7 4 4 4 gil11991101 PREDICTED:hypothetical proteinisoform 6 [Bostaurus] 2056834 0.879581 0.624866 0.5835043 1.916642 0333636 0.01451052.200812 0.459368 0.01577774 2 3 3 gi[76642911 PREDICTED:similar to IL-10-induciblechemokine [Bos taurus] 99 0.875858 0123182 0.27094020.800304 0299641 0.1833167 0.829947 1.195581 0.43601883 2 2 2 PREDICTED:similar to spectrin,beta, non-erythrocytic isoform1 [Bos taurus] 14.47313 0.713101 059002 0.4498234 1.833073 0.596717 0.2888106 2.515698 0.597666 0.1807333 gil119903493 1 2 2 2 gil156121073ARP2 actin-related protein 2 homolog (Bos taurus] 23.09645 1.001828 0.697862 0.98818462.304177 0.029813 0.011229 2.067647 0.874D45 0.27182784 1 3 3 gil77735683S100 calciumbinding protein A2 [Bostaurus] 36.08247 -1.00065 1 -1 2.466222 0.120242 0.0028888 2.563626 0.158206 0.06438019 2 2 3 calcyphosine[Bos taurus] 28.54233 1.388068 0421027 03516481 3.522336 0.11175 0.0278185 4.282173 0.328754 0.00812933 gill15497428 5 5 5 gi|27807481 ubiquitin-likeprotein SMT3B [Bos taurus] 38.94737 1.348473 0.36111 00560946 3.350866 0.477124 0.625 4.313778 0.401973 00625 8.61e61t80-326646 0.05383599 2 2 2 gi|1 16003917ar upstreamelement-binding Protein Sostaurus] 19.44012 2.11329 0269365 011127844.654771 0.367708 0.0745735 3 3 3 gi77736560 stathminl/oncoprotein 18 [Bostaurus] 48.99029 1.092151 0325027 05704541 0995114 0342775 6.4828973 1496251 0.214272 004242202 77 8 0.01348308 2 3 3 gi|11549 2 aconitase1, soluble [Bostaurus] 12.26097 1.240157 0.17276 02382912 3423462 0.386158 0.0155636 4.481167 0.439657 4 4 4 gi]77736371actin relatedprotein 203 complex subunit 2 [Bos taurus 27.66667 1.018386 0.197026 0.8136051 2258753 0622534 0.0331859 296798 0.588514 0.01288204 4 4 4 gi[29135293farnesyl diphosphate synthase [Bos taurus] 1898017 1.203639 0435333 03082513 3.539508 0.239562 00096248 3.779224 0.267713 0.00073658 3 3 3 gi]51036244 eukaryotictranslation initiation factor 5A[Bos taurus] 24.02597 1453199 1.286417 0.5439189 2.954515 0170368 0.003964 3.754468 0.132889 0.00161227 3 4 4 gi]84000347 interleukinenhancer binding factor 2 (Bostaurus] 27.4359 1.828959 0.671820 01530618 2537814 0.533873 0.0154008 3.62311 0.465778 005411404 2 2 2 gil157073982von Willebrandfactor A domaincontaining 1 Bos taurus] 1684915 1.099628 0.131237 03810268 0807840 0.102784 0.1945011 1.090683 0.050353 017938155 0.240058 0.0158773 3 3 3 gil114051892UDP-N-acteylglucosamine pyrophosphorylase 1 [Bos taurus] 20.114941.226566 0282574 02136395 2.4475330.459173 0.401649 2.107073 4 4 giJ77735453alcohol dehydrogenuse 5 class 111) chi polppepride[Sos taurus] 14.70588 1.048268 0.260757 0.6424549 2.946919 0.36427 0.0035499 3.528374 0.332388 0.00163491 4 1788229 0.273644014437637 2 2 2 gi]76609798PREDICTED: similar to GalNAc-T5[Bos taurus] 143617 1.02627 0.2010308401129 1303342 0.4151650.4245001 5 5 5 gil77735537ephrin-Al [Bostaurus] 20.97061 113559 0.365597 0.327316 0.839321 0362003 0.1809834 1.18891 0.33226 0.1682474 4 4 4 gil114052973 glypican 1 [Bostaurus] 254025 1.210355 0.77746 053101 0.797344 0.122 0.011785 0.986551 0.435525 093417054 14 14 14 gi[76691395PREDICTED: similar to mammaryserum amyloidA3.2 [Bos taurus] 76.33588 0416971 0.352713 2.02E-08 0408124 0.374995 8.01-09 096142 0.234995 038254998 3 3 3 giJ77735939proliferating cell nuclearantigen [Bos taurus] 33.33333 0.761819 0420592 02466368 1.739169 0431616 0.0869073 2.034908 0.234062 00694241 17 17 17 gi|27807275matrix Gla protein[Bos taurus] 4466019 1.287522 0396059 00016206 0.754217 063986 0.0183724 1.280808 0662351 0.04100272 7 7 7 gil27806703CD44 antigen [Bos taurus] 17.75956 1100475 0.406985 0406822 040386 0.32497 3.886-05 0.68014 0.502869 0.02643126 7 0.00252709 5 5 5 gil15727990parathymosin[Bos taurus] 28.43137 1744462 0.642378 0.0492541 3952378 0628303 0.0021777 4150371 0.681311 9 2 3 2 gil15612105biotinidase[Bos taurus) 8 0.814699 0455306 05260767 1.140688 0236245 6.3163806 1.005905 0.151722 0.94970084 3 3 3 gi[91680535proteasome activator subunit1 [Bostaurus] 35.34136 1.091096 0.459176 0.6840743 2735559 0.288460 0.0130874 4546808 0.418258 0.01200284 4 4 4 gi|75812956GTP bindingprotein Rabla [Bos taurus] 28.01106 0667297 0.163042 0.0853354 4.239819 0.35D6830.0012976 5.530162 0.377976 0.00097051 2 2 2 gil164420709retinol binding protein 4. plasma[Bos taurus] 40.29851 0593539 0.486594 D.2726927 0637269 0.156723 0.2517599 0300645 0.06204 0.0183279 000088134 6 7 7 gill 39948301branched chain aminotransferase 1, cytosulic 85 taurus] 17.61696 1.334924 0.373477 0.04141872335966 0576351 0.00141222.805883 0.64513 2 2 2 gil164420725dipeptidyl peptidase Ill [Bostaurus] 20.13699 1.801793 0048435 0.02560064.015375 0217609 0.048973 4672917 0.390478 0.07862271 6.926-06 6 6 6 gi[76638716PREDICTED: similar to ubpiguihonisotorm 12 [Boo taurus] 94.63760 1.204583 0.27885 0.06551012.015621 0.246148 0.0001837 2.392524 0.159873 44.76744 0.552975 0.604769 00662065 1244118 0.095675 00077462 27926 0300125 0.00220895 gil122692375 hypotheticalprotein LOC783871[Bos taurus] 0.526258 0.3153689 2.232197 0.526929 0.0634759 3044657 059854 0.04329931 gi|114052248 plastin 3 [Bos taurus] 25.03912 1.323663 0.548921 0083639 1.350388 0.033151 00001309 0.654959 035796 0.2957879 PREDICTED:carilage-derived morphogenetic protein 2 [Bos taurus 18.51064 0.567102 gi|119906762 2.0671330.989501 0.3774703 1.87352 0.796051 D035399461 A [Bos taurus] 16.44613 -1.00065 1 -1 gil115495441 hexosaminidase 087366 1.447548 gi119920091PREDICTED similar to ventroptin[Bos taurus] 23.39956 0.937338 0.048121 0.0769204 068945 0.5151650.2079753 0.96743644 gil62177164tumor protein, translationally-controlled1 [Bostaurus] 35.46512 1.158834 0.443541 0.62196 2,340446 0.412916 0.1498302 3.121972 0.566442 0.15279323 gi]62751339 proteasome (prosome, macropain) subunit, beta type, 2 [Bostaurus] 29.35323 1.330207 0.274933 0.1225159 3.127191 0.379439 00006479 4.246609 0.660994 0.00237663 1.341008 0.807716 03724710 3.140066 0.564662 00101359 3.704626 0525602 0.00555204 gi78369310 stress-nduced-phosphoprotein 1 (Hsp70/Hsp9O-organizingprotein) [Bos taurus] 32.22836 0.1750179 0.975966 0.08125 05616646 0979913 0205985 0.87372895 gi|27806555 legumain [Bostaurus] 24.01848 0.768093 0.152944 3.248512 1.009726 0.0440782 450022 0.875604 0.0157647 g|87080799 small induciblecytokine subfamily E. member 1 [Bos taurus] 37.57764 0.978151 0.51457 0.9427495 2.029195 1041695 0.2346279 1919908 1320611 0.34246752 gil77735431 ornithineaminotransferase [Bos taurus] 15.03417 1.395334 0.98313 04870478 gill 15497922 hypotheticalprotein LOC505636[Bos taurus] 28.51711 -1.00065 1 -1 1.581124 0400711 0.2610647 226249 0236818 0.08990153 gi]157279995dipeptdyl-peptdase 7 [Bos taurus] 23.97541 0.857851 0.549094 06691034 1304726 0.197209 0.2269135 1.089601 0.096418 0.31989787 gil155372073 cullin-associatedand neddylation-dissociated1 [Bos taurus] 18.86179 0.695073 0.378225 02896027 2.907305 0.49621 01440313 3759088 0843245 0.1926009 0.730217 0224571 02104108 0.684151 0102243 0.24606708 similar to Carboxypeptdase X 2 (M14family) [Bos taurus] 18.60158 0.797696 0.095673 01288634 gil119917901 PREDICTED: 054895 0.09948294 gil27806887 fusion (involvedin t(1216) in malignant liposarcoma) [Bostaurus] 32.61719 1.181835 0.481151 0.606781 4238665 0.550486 01180872 5.516432 0.00035965 gil27806783thioredoxin [Bos taurus] 42.85714 1.444023 0.440707 0.054952 3509945 0.475087 00010569 419445 0.412321 1493062 0.605143 gi|155372333 lectin, mannose-binding2 [Bos taurus] 23.11978 1.153124 0.410591 0.4790391 127617 0.343735 0226436 0.23972742 1254963 0.811092 0.55656123 gi|84370199 6-phosphogiuconolactonase [Bos taurus] 30.23256 0.576743 0.018861 0.0106797 1.205925 0040074 0.0688709 4 0516322 0.0625 gi|783696 5 SH3 domain binding glutamicacid-rich proteinlike [Bos taurus] 47.36842 1.577395 0.086695 0059265 3.14094 0.4042 00038984 4378125 0.04702865 gi|27805945 inositol(myo)-1(or4)-monophosphatase 1 [Bostaurus] 27.43682 1.400499 0.331543 0.1288063 2.957581 0692891 00602492 3363121 0.681528 0.00794 giJ77735429 proteasome alpha 3 subunit [Bos taurus] 38.82353 0.932204 0.22374 06348077 2.281576 0313706 0.1182173 2.446468 0.00276909 gi|77735823 ribosomal protein S20 [Bos taurus) 31.09244 0.790159 0.17284 0.0290852 1.737038 0.268753 00099104 3.188097 0.532714 0.0080911 25.87719 0.927323 0.676895 0.8051482 2611393 0.755215 0.0874872 3160965 0.680789 0.05172991 gil1 140513477 69 29 cytidinemonophosphate (UMP-CMP)kinase 1, cytosolic[Bos taurus) gil66 0 spectrin, alpha, non-erythrocytic1 (alpha-fodrin)[Bos taurus] 16.58576 1.184876 0.388282 0537734 2947246 0.371875 01068734 3840887 0.388681 0.08957329 19.76744 0.884934 1.269861 0.798205 0.41275 0.65961 0.0776335 0.487832 0.680651 027704591 gi|1 140522709 chordin-like2 [Bos taurus] gi|12672282 BCL2-associated athanogene 3 [Bostaurus] 27.0855 2.802923 1.289169 03502997 3.458467 0048328 0.0122142 4.271478 0.13829 0.02969692 0.236823 gi|114051291tissue specifictransplantation antigen P35B [Bos taurus] 23.98754 1.654949 0.535261 01433048 4.137355 0278018 0.0061372 4.214638 0.00431224 gi]78042510 haloacid dehalogenase-likehydrolase domain containing2 Bos taurus] 24.71042 0.94649 0.757689 0.9053114 3.266997 0.791617 00033087 4.125668 0317018 0.00013437 gil149642873hyaluronan and proteoglycanlink protein3 {Bostaurus] 41.38889 1.158435 0.537488 0.4291358 1.01502 0.217778 0.625 1.241183 0.2373 0.04251385 38.37209 0.609115 0.452781 0.1114128 0.931329 050876 0.8898663 1754287 0.451165 0.03690212 gil11993014897 PREDICTED: similar to RIKENcDNA 1600002004 gene, partal [Bostaurus] gil278064 plasminogen activator inhibitor-1[Bos taurus] 16.41791 2.200635 0.29896 0.004716 4.486546 0380705 0.0014754 8.368697 0.330522 0.00034211 gil115497482 zinc binding alcoholdehydrogenase, domain containing1 [Bostaurus] 13.10541 -1.00065 1 -1 1.893992 0.327402 0.1582271 2.316179 0.902675 0.07474413 gil119879173PREDICTED: similar to putativeMAPK activating proteinisoform 2 [Bos taurus 15.89649 1.426563 0.799612 0.5317789 3.512832 0.665082 0.0123022 5.059416 0.032401 0.00310552 gill 15497442 5-aminoimidazole-4-carboxamideribonucleotide formyltransferase/IMPcyclohydrolase [Bos taurus] 13.85135 0.945259 0.440325 0.8357586 3.088379 0.800938 0.2143968 3.857588 0.686000 0.15539029 gil134085825carboxypeptdase Z[Bos taurus] 18.47826 0.559446 0.166333 0.0128532 0.856328 0.130455 0.0905337 1.188414 0.289823 0.26980953 0.07874667 gi|75832090 isocitrate dehydrogenase 1 (NADP+),soluble [Bos taurus] 24.63768 1.045861 0.344877 0.8372108 2.802093 0.576889 0.1841776 2.929273 0.27283 0.715079 gi27807129 thioredoxinreductase 1 [Bos taurus] 19.23848 1.282764 0.3450D6 0.3805389 2.327132 0.981346 0.3316377 25314 0.22974867 00547898 2.6914 1.083964 0.15213374 gil119908206 PREDICTED:hypothetcal protein[Bos taurus] 17.80303 1.214952 0.224236 0.1830811 2.43674 0.887958 gil114052667 RAD23 homolog B [Bos taurus] 19.36274 -1.00065 1 -1 2757201 0.625255 0.1879261 3.188905 0.658176 0.17267951 0.02318819 gil149642987S100 calcium bindingprotein Al1 (calgizzarin)[Bos taurus] 29.12621 1.030253 0.247403 0.8497925 2.568362 0.112691 0.0374885 4.252827 0.107625 89 0.00704158 gi|284611 lectin, galactoside-binding,soluble, 1 (galectin 1) [Bos taurus] 30.37037 1.224775 0.175201 0.0447682 220260 0.339462 0.8069073 2.735369 0.439069 0493875 0.1588096 3.749839 0.737501 0.16996106 gi27807075 S100 calcium-bindingprotein A4 [Bos taurus] 42.57426 0.879406 0.024022 0.0878722 2.602672 0.3733367 2.407823 1.479507 0.43922794 gi27806067 plasminogen activator, urokinase receptor [Bos taurus] 16.36364 1.121041 0.414642 0.6754922 1.833391 0.813034 0.329782 1.259486 0.08457753 gil27806725 cathelicidinantimicrobial peptide [Bos taurus] 17.89474 0.1597 0.784296 0.0066379 1.138599 0.187596 0.1580967 0.146586 0.00572903 gi|27806273 chemokine (C-C motif)ligand 2D [Bostaurus] 19.79167 0.194847 0.555875 0.0179878 0.419872 0.220179 0.0100069 0.482231 0.07135406 gi|l14052322 alpha-N-acetylgalactosaminidase[Bos taurus] 12.89538 1.139529 0.595913 0.8404207 1.51588 0.332239 0.0911832 1.883756 0.367761 0.05477894 gil114050905 SERPINE1mRNA bindingprotein 1 [Bos taurus] 27.27273 -1.00065 1 -1 3.400362 0.220987 0.0584873 5.344381 0.284973 1.831562 0.088643 0.00521001 gi]76669880 PREDICTED:similar to LRTS841 [Bos taurus] 22.9765 0.448547 0.945133 0.167938 0.759383 0.335878 0.1712008 gi|119915943 PREDICTED:similar to serine (or cysteine) proteinase inhibitor,clade B (ovalbumin),member 1 isoform 1 [Bos taurus] 24.41176 0.866515 0.14408 0.2903878 3.970274 0.119787 0.0272171 4866283 0.500872 0.0979879 0.347958 0.528402 1.937879 0.32838 0.1533487 1.906841 0.396522 0.1861921 gi119912825 PREDICTED:solute carrier family9 (sodium/hydrogenexchanger), member 3 regulator1 {Bostaurus] 29.61957 1.16955 gi76608584 PREDICTED:similar to KIAA0653protein isoform 1 (Bostaurus] 12.87425 1.184635 0.264365 0.0080988 0.252437 0.804793 2.606-05 0.641921 082809 0.05737305 gi|27807445 peroxiredoxin5 precursor [Bostaurus) 36.07306 1.113993 0.326673 0.4880402 4.136891 0171283 0.0023432 5.802393 0.11972 0.00074166 0.063241 gi|155371881 alanyl-tRNAsynthetase [Bostaurus] 18.69835 1.087238 0.469583 0.7796875 3232799 0.34756 00922806 3.627258 0.01531197 0.149403 gi|1 15496898 Hi histone family,member D [Bos taurus] 28.86598 0.849143 0.420366 0.5716697 2.006775 0033187 0.0150018 9.050744 0.02115459 0439552 gil119924945 PREDICTED similarto agrin [Bos taurus] 16.48301 0.866545 0.317657 05250638 0823302 1.139062 07801894 1.01548 0.95468536 0.031078 gil119922764 PREDICTED:similar to MSTP006[Bos taurus] 25.0715 1.305384 0.165716 0.1887662 3.244213 0.394372 0.1041913 3.761606 0.00731903 gi61888866tropomyosin 1 alpha chain [Bos taurus] 50.35211 0.833011 0.034991 0.0593724 1.718769 0.723139 0.3723729 1.984483 0.477072 0.2093176 0.12674482182267 1.0053760.19034862 giJ60592784 hexokinase 1 [Bos taurus] 11.88659 0.685052 0.923112 0.55616252.195976 0.321184 0.520491 0.42900195 gi[76649536 PREDICTED:similar to fibulin2 precursor, [Bostaurus] 9.444905 1.109473 0.049989 0.1483473 1.321788 0.510396 04727018 1.376373 gi27806923brain ribonuclease [Bos taurus] 41.31736 0.723807 0.332298 0.0029219 0.96977 0.130105 02522524 0.273223 0656186 2.68E-05 gi|119909757 PREDICTED:similar to E-selectin ligand-1 [Bostaurus] 16.37131 1.093871 0.410077 0.7344326 2.581782 0169757 0.0560792 1.506366 1.445697 0.66626145 0.51129 0.138435 gi158262747glutamine-fhuctose-6-phosphate transaminase 1 [Bos taurus] 24.81645 1.231305 0.084642 0.1256687 2535182 0.377752 01259132 3.108689 4334730 0.218424 0.00353319 gi|27807193 platelet-activatingfactor acetylhydrolase, isoform lb, beta subunit 30kDa [Bostaurus] 24.45415 1.027925 0.91947 09620578 2.814638 011982 00021668 27.17283 -1.00065 1 -1 2.035217 0816025 0.3268293 2365465 0.387398 0.13818516 gi|1199071327 3 PREDICTED:similar to Hyoul protein [Bos taurus] gi|8269 34 cystatin B (stefin B) [Bos taurus] 71.42857 1.478201 0.182256 0.0332131 2501006 0.120338 00030223 2.85596 0.131669 0.00251171 0.2601742447641 0.257507 gi|116004245 retinoblastoma bindingprotein 4 [Bos taurus] 17.41176 -1.00065 1 -1 1.9875140.601259 0.08917497 3 3 0.505891 gil14823 12 dermatan 4 sulfotransferase 1 [Bostaurus] 17.81915 0.740922 0.011548 001198860.728226 0.348724 03094067 1246645 0.38991166 gil119915491 PREDICTED:similar to Complement C4-Aprecursor (Acidiccomplement C4) [Bos taurus] 17.96785 0.166893 0.97678 0.1662811 1082212 0.420537 0743752 0.316188 2.318201 0.4957697 0.09976674 gi78042520 eukaryotictranslation initiationfactor 4B [Bos taurus] 25.40984 -1.00065 1 -1 3.503268 0512061 0.1264097 2.816826 0333674 gil119894607 PREDICTED:similar to peptidoglycanrecognition proteinL [Bostaurus] 20.13536 4.792911 0.487863 0.0004903 0395206 0.772723 00175063 1.139641 0.698255 0.57900678 gi|157427940activated RNApolymerase 11transcription cofactor 4 [Bos taurus 41.73228 1.13341 0.21623 0.4487548 4.359615 0183893 0.0025105 5755411 0.17363 0.00156915 0.01695884 gi|77735427 protein phosphatase 1, regulatory subunit 7 [Bos taurus] 25.83333 -1.00065 1 -1 3.783091 0.000171 4.036-05 5.134357 0.088936 gij77735717 proteasome (prosome, macropain) subunit, alpha type, 4 [Bos taurus] 27.20307 1.218217 0.382513 0.3274525 3.009222 0.61398 0046819 3.822741 0.658926 0.03526476 0.88726404 gil29135303 glutaminyl-peptidecyclotransferase (glutaminylcyclase) [Bostaurus] 27.42382 -1.00065 1 -1 0.955946 0524784 0.8743152 0939491 0.711602 0.14534993 gil117935057 dihydroxyacetonekinase 2 [Bos taurus] 23.18339 1.770718 0.856066 0.4035772 2710145 0.889998 02540701 2.181308 0.368757 0.1471004 gi|122692601 hypotheticalprotein LOC526913[Bos taurus] 33.91813 -1.00065 1 -1 -1.00642 1 -1 5440118 0.813041 gil157151716 annexin A6(Bos taurus] 22.28826 0.875483 0.2842 0.5159396 1.580233 0.112174 0077197 1.94607 0.114224 0.05340423 gil27807521 prostaglandinH2 D-isomerase [Bos taurus] 35.6021 2.053996 0.214395 0.0139374 0.468867 0.252016 0.0169907 0277285 1.052004 0.09295571 gi|114051526 coactosin-like1 [Bos taurus] 30.28169 1.478547 0.347001 0.2615507 1.981741 0.060627 02472422 3.391487 0498548 0.12571595 taurus] 23.96857 -1.00065 1 -1 -1.00642 1 -1 2.645573 0.610104 0.23100851 gi|76615127 PREDICTED:similar to Dihydrolipoamidedehydrogenase isoform2 [Bos 12.93532 1.28624 0.582208 0.5406334 2.614265 0.289968 0.0940907 2701291 0.075577 gi|76671278 PREDICTEDsmiar to vesicle aminetransport protein 1 isoform 1 [Bos taurus] 0.02371975 1.8926480.828082 023114 2775658 0.9621350.11767174 gij76652407 PREDICTEDsimilar to ChainA, CrystalStructure Of HumanRangapl-Ubc9 isoform 2 [Bostaurus] 27.8481 0.838683 0.297707 076523 31.84713 0.299926 0.348412 0.0896313 1.559483 0.354058 0.240169 6.89452 0.560858 0.09003547 gi27806129 ribosomal proteinL24 [Bos taurus] 0.4071360.0330273 3.087672 0.318510.01254228 gil122692537 damage-specific DNAbinding protein 1, 127kDa [Bos taurus] 12.45614 1.110984 0.044019 0.027213 2.412275 73 7 0.095755 0.05912638 gill991 8 PREDICTEDsimilar to FAM20Cprotein [Bos taurus} 24.71783 0.76106 0.07298 0.0827319 0.747602 0043851 0.045987 0.604188 0.444676 0.43302788 gi]119901084PREDICTED: similar to CD109 [Bos taurus] 36.22449 1.019964 0.426654 0.680915 0.94923 0409485 0.6351143 1.113193 2786537 0.968469 0.00180243 gi|41386699 heat shock 70kDa protein2 [Bos taurus] 34.54834 0.843417 0.785543 0.5746141 1769436 0913078 0.078125 0.1217369 4361099 0846208 0.17475981 gi|157074012 EF-hand domain family,member D2[Bos taurus] 24.38017 1.793432 0.112593 00600325 2865644 041333 0.0002585 3.301008 0318316 0.00026735 gi|122692293 chlorideintracellular channel 4 [Bos taurus] 27.27273 1.17274 0.328963 01946439 2.710524 0261827 0.0073721 8.410554 0.399028 0.00557626 gi]77735589 splicing factor, argininelserine-rich2 [Bos taurus] 33.48416 1.270996 0.145926 00547751 7.86308 0443527 1175707 0.2887952 3.836271 1819622 0.3728619 g]155372143 arginine-rich,mutated in early stage tumors [Bos taurus] 18.43575 0.848986 1.101397 0.9118423 3.28157 0.27815 0.496713 0.11969159 gil27805833 chemokine (C-X-Cmotif) ligand 5 [Bostaurus] 23.21429 0.179465 0.263135 00476897 0670318 0245752 0.1034332 0602405 0.356736 00089668 1431962 1.303577 1 gill18151144 tenascin C [Bos taurus] 50.91638 D.770316 0.294486 00256867 0.103929 2.95617 0.266369 0.07687783 gil119914306PREDICTED similar to 1-phosphatidylinositol-4,5-bisphosphatephosphodiesterase delta 1 (Phosphoinositidephospholipase C] ]PLC-deha-l ]Phospholpase 15.621 1.035261 0.323216 08653991 2.394548 0.291299 02821737 053295 2.306675 0.67666383 gi|77735633 cysteine-rich,angiogenic inducer, 61 [Bos taurus] 10.20942 0.883789 0.51206 0.7074747 1745372 0533266 0.2614632 1122839 0.125486 0.07607451 gi|158937293fibroblast growth factor receptor 1 [Bos taurus] 12.56098 1.753452 0.47468 0.0421556 0559447 0521762 0.6378362 0.331646 0.726595 0.0629296 gi|41386687 leukemia inhibitoryfactor (cholinergicdifferentiation factor) [Bos taurus] 17.82178 -1.00065 1 -1 0.963503 0190164 2.573371 0.426845 0.03114173 gil115495701 pentraxin 3 [Bos taurus] 29.58115 1.670089 0.348366 00667453 1.193437 0490979 04771401 0.39146 0.02730964 gill 15497506 LIMand cysteine-rich domains 1 [Bos taurus] 27.27273 1.321589 0.146172 0.1604673 2.747591 0.409624 00206214 2530716 5.402224 0.035946 0.00664913 gi|27806365 UDP-glucose dehydrogenase [Bostaurus] 12.75304 1.648265 0.581918 0.3305285 4.526757 016537 0.0342841 0240222 00700653 1.789695 0.171036 0.01362673 superfamily,member 6b [Bostaurus] 30.06757 0.677278 0.157293 0025035 1.419267 gi|155372327tumor necrosis factor receptor 5113621 0.525556 0.00293428 gi|169658384non-metastatic cells 1, protein(NM23A) expressed in [Bos taurus] 51.97368 1.599096 0.496178 01421363 3221894 0487525 00066466 0.06457963 gi|119888815PREDICTED: similar to GALEprotein isoform 2 [Bos taurus] 14.39394 1.003638 0.243785 0.9764014 367117 1123597 0.1024996 4.28586 0.971872 02172251 2.065455 0.464456 0.06451703 calcium-bindingprotein isoform 2 [Bostaurus] 13.68876 0.873921 0.473286 05486248 1459335 0521968 gi|76607797 PREDICTED similar to cis Golgi-localized 5.293795 0.185178 0.03090209 gi|62752D12 ribosomal protein L18[Bos taurus] 45.74468 -1.00065 1 -1 4234364 0.124859 00270982 1.915535 0.39931 0.18618914 gi|147902328thymopoietin [Bos taurus] 24.86911 1.629695 0.244807 01535627 1171980 0.032739 0.0668263 0.355249 00046603 4.140374 0.414895 0.00220445 sensitive element bindingprotein 1 [Bostaurus] 28.7037 0.913776 0.424419 0.580643 257372 gil27807361 nuclease 4.291759 0.545594 0.02173644 gil119895504 PREDICTED:similar to heterogeneous ribonucleoproteinAO [Bos taurus] 18.72131 1.612215 0.242052 0.0388583 2692241 0.504924 0.0396778 4.3053 0.029314 6.46E-05 gir77736367 poly(rC)-bindingprotein 2 [Bos taurus] 23.20917 -1.00065 1 -1 2838597 0370689 0.0228783 4.459772 0.743368 0.15217439 gi]77736353 dimethylargininedimethylaminohydrolase 2 [Bostaurus] 23.85965 -1.00065 1 -1 -1.00642 1 -1 1.678967 0.156796 0.09394594 gi|114051796 glucosidase, beta, acid (includes glucosylceramidase)[Bos taurus] 15.67164 0.764494 0.202429 02246044 1.254877 0.384624 04501166 3.148392 0.620932 0.16512648 gi62751960 nbosomal proteinS26 [Bos taurus] 42.6087 0.485585 0.151816 00652849 1.730674 0.56823 0.3025819 1.088801 0.253514 0.48996056 gil119879493 PREDICTED:similar to limbicsystem-associated membrane protein6c- [Bos taurus] 16.17977 1.159068 0.19806 0.204879 0798589 011301 0.0361716 14.54193 1.193093 0.00747859 gil145279649tumor necrosis factor alpha [Bos taurus] 6.837607 14.20984 1.293349 0.0096325 0911562 0.678383 0.8157827 4.959856 0.14599 0.02842712 gil119938155 PREDICTED:similar to AHNAKnucleoprotein, partial {Bostaurus) 66.79587 0.849069 0.201162 0.3441443 3.886245 0.072836 0.0168176 7 77 3 7 0.0030011 4.463149 0.121149 0.00104883 gil 540 eukaryotictranslation initiationfactor 4A isoform 1 [Bos taurus] 40.64039 1.16605 0.353513 0.3926128 4233965 0.197121 gil28461231 ATPase, He transporting, lysosomal accessory protein1 [Bostaurus) 16.88034 0.960254 0.077019 0.3130112 1.005032 0.197032 0987851 1.422343 0.092712 0.01090967 7 3.240050 1.513455 0.35835129 gil119895 32 PREDICTED:similar to synaptopodin [Bos taurus] 12.58503 -1.00065 1 -1 2.424683 1.728491 0.4877888 114 05 42 3 2.089131 0.997176 0.37288398 esterase Li [Bos taurus] 13.88889 0.962528 0.00119 00690604 1.689114 0.859838 0.3554163 gi| 1 ubiquitincarboxyl-terminal 0.20072435 gir76633778 PREDICTED:similar to syndecan 4 [Bos taurus] 17.08543 0.683179 0.248639 0.0617444 0.418593 0.797848 0.1108239 0.821749 0.63134 0.33214533 gl1 54707892 Clq and tumor necrosis factor related protein 5 [Bos taurus] 21.39918 0.847331 0.101863 0.1856581 1.392054 0.068518 0.0658721 0.832781 0.214649 0.0273438 0.929361 0.662288 0.677246D9 gil153791208 immunoglobulinsuperfamily containingleucine-rich repeat [Bos taurus] 11.91589 0.83523 0.581162 0.2152827 0.591791 0.597903 gil119890624 PREDICTED:similar to insulin responsive sequence DNA bindingprotein- [Bos taurus] 10.12048 0.901494 0.025889 0.0770335 0.820672 0.319856 0.4170883 0.784367 0.215142 0.26075991 0.347844 0.310227 0.09103958 gil27806675 cystatin C [Bos taurus] 35.81081 0.311661 0.505464 0.1332276 040507 0.032231 00110491 0.998028 2.136357 0.9987981 gil164259354 reticulon 4 isoform 2 [Bos taurus] 9.49868 0.777559 1.6576 0.8083492 1.871928 1.281245 0.5037745 0.347009 0.11458395 gil115497820 parvalbumin[Bos taurus) 36.36364 1.014075 0.188671 0.9088892 4.286481 0.434321 00928578 2.549938 0.06945593 gir84000233 hypotheticalprotein LOC517857[Bos taurus] 23.75 1.171824 0.508378 0.6403982 3306598 0281712 0.073567 4.85265 0.352999 3.034521 0.605806 0.01319904 gi|27806297 biliverdinreductase B (flavinreductase (NADPH))[Bos taurus] 18.93204 1.564585 0.63012 01331828 2.125048 0.58417 0.0344776 2.284475 1.615261 0.48644602 gi|77736489 high-mobilitygroup nucleosome bindingdomain 1 [Bos taurus] 32.67327 D.444938 0.555178 0.2062116 1.943734 1161423 0.4539377 -1 1 -1 gil1 15497074 esterase D/formylglutathionehydrolase [Bos taurus] 12.76596 -1.00065 1 -1 4369147 0504538 0.1362306 1.36881 0.39305904 gil77735391 regakine 1 precursor [Bos taurus] 18.47826 0.451793 0.098061 0.0384317 0667916 0.53123 0.3602041 0.388718 0.00441075 gil62460528 nascent polypeptide-associatedcomplex alpha subunit [Bos taurus] 13.02326 1.830435 0.724095 0.3382421 4.504433 0.378398 0.0783849 5.934570 0.025173 0.45322823 gill 15497050 isopentenyl-diphosphate delta isomerase [Bos taurus] 27.1777 0.877947 0.778639 0.7897649 1.459049 0.325729 0.256461 2.013007 1.231721 4.633014 0.04837 0.06984299 gil77735825 cathepsin K [Bos taurus] 35.62874 0.934242 0.047902 0.2097279 2.224044 0.048229 0.0189731 3.477498 0.235773 0.05885961 gi77735687 proteasome (prosome, macropain) subunit, beta type, 6 [Bos taurus] 32.21757 1.333942 0.047645 0.0515986 3050473 032389 0.0905222 1.225383 0056163 0.00187667 gi|27806823 osteomodulin[Bos taurus] 14.45498 0.774023 0.249999 00593557 1.07641 0153721 02961305 0.263572 0.05644745 gij82697405 LSM3homolog, U6 small nuclear RNAassociated [Bos taurus] 31.37255 1.468764 0.902448 05450427 3.680527 0607336 0.1436591 4.276493 0.1617409305859 1548858 0.5022670.32627669 gil115497398neuron derived neurotrophic factor [Bos taurus] 39.05326 0.670412 0.278612 020918070997721 gil119879765 PREDICTED:similar to eukaryotic translationinitiation factor 4 gamma, 1 [Bos taurus] 18.13773 1.564344 0.75853 04416059 5483087 0106958 0.01968 7244449 0.08588 0.01349983 0.798797 0.13406917 gi|82697365 ribosomal protein S19 [Bos taurus] 35.17241 -1.00065 1 -1 1.692251 0.653953 03518414 6243338 7 0.32563774 gil15 954420 angiopoietin-like2 [Bostaurus] 16.22718 1.028046 0.593377 0.9198322 1139332 0241889 0.3285328 1.277148 0.47329 gir84000197 FK506 bindingprotein 3, 25kDa [Bos taurus] 24.55357 0.812919 0.853254 0.7084508 1.916825 0.544387 0.2499603 2.67173 0.989421 0.29183193 gi|41386683 beta-2-microglobulin[Bos taurus] 39.83051 1.416328 0.47085 0.0759663 083683 0.320825 0.1371149 2.344783 0.035110 0.00680338 0.358809 0.00813073 giI62751407 fast skeletal myosin chain 2 [Bos taurus] 19.18605 1.240037 0.337093 02527990 3.986864 0.165871 00023171 4.063859 light 0.471435 0.08121488 gill 19919943 PREDICTED:similar to methyl-CpG-bindingprotein 2 isoform 1 [Bos taurus] 25.71943 -1.00065 1 -1 -1.00642 1 -1 8006162 0.148516 0.04781956 gi]77736355 RAN,member RAS oncogene family[Bos taurus] 22.68519 0.739306 0.430781 0307744 2.028151 0204308 0.0903641 2.630704 1.23592804900739 2.4549420.030363 0.17932944 gi|149642675 ribosomal proteinS17 [Bos taurus] 32.59259 0.542396 0.748698 0.3436696 1.800149 0619099 03311537 1.5652250837969 0.47235345 gi|45430009 fumarylacetoacetate hydrolase domain containing2A [Bostaurus] 26.11465 1.274767 0.170997 02121555 1.709406 0.242594 003125 028535 0.6809720.00127969 gi|154152039 scrapie responsive protein 1 [Bos taurus] 45.91837 0.738699 0.312672 001862760421795 01358267 2750132 0.031820.00981346 gi|119907227 PREDICTED:hypothetical proteinLOC535533 isoform 2 [Bos taurus] 13.22141 -1.00065 1 1 2202432 0.346186 4.1789970.122533 5.76E-05 gi|84000371 H2Ahistone family,member V [Bos taurus] 48.4375 0.760566 0458467 0182695 1.6091690324879 00251315 3993368 0.422779 giJ27807173 S100 calcium bindingprotein A10 [Bos taurus] 38.14433 D.839503 0.497589 026758712623931 0360347 9788-09 8.99E-10 0.08998154 gill 19927320 PREDICTEDsimilar to lamininB2, partial [Bos taurus] 13.90729 1.083504 0.254515 0.6386092 0965657 0045925 0.317017 1.357422 0.088716 2.488608 0.160546 0.05480918 gil7662D955 PREDICTED:hypothetical protein [Bostaurus] 59.52381 0.694827 0.419084 0.3265113 1135705 0147721 0.3436365 50 0.15935018 giJ77736063 dynactin2 (p ) [Bos taurus] 16.62531 -1.00065 1 -1 2.792355 0.054156 0.0165552 3.203302 0.807278 gi|149642591 0100 calcium bindingprotein Al [Bostaurus] 17.02128 0.991839 0.070916 0.6413485 3.244039 0.087176 00232301 4.796569 0.248085 0.04927254 0.114788 0.00183849 giJ78369246 acireductone dioxygenase 1 [Bos taurus] 27.93296 1.213911 0.401982 03527036 255191 0.258428 00121312 2.915009 0.775939 0.14892666 gi]77735889 mesoderm development candidate 2 [Bostaurus] 28.87931 1.077581 0.23601 0.6374274 3.852189 0.986506 0.2201164 4.933039 32.09876 -1.00065 1 -1 1.82684 0.790179 0.170527 4.057833 0.901211 0.19448039 gill 14052282 nuclear casein kinase and cyclin-dependent kinase substrate 1 [Bos taurus] 14.250611.524025 0.197044 0.1436533 0.892664 0.3424905050319 1.3738410.052724 0.05168312 gil1 19888616 PREDICTED:similar to beta 1,3-N-acetylglucosaminyltransferase7 [Bos taurus] 19.028341.273419 0.057796 0.0744743.178216 0.414290.1112683 4.085951 0460966 gil115497034ubiquitin specific peptidase 14 [Bos taurus] 0.10132238 18.63116-1.00065 1 -1 -10642 1 1 4.9249030.174657 0.03414982 gil27806277 calpain, small subunit 1 [Bos taurus] 15.223081.353102 1.00327 0.4493391.529699 0.415258 0.0620404 1025928 0563429 0.90400419 gi|27806265 leukocytecell derived chemotaxin 1 precursor [Bostaurus] 0.804016 1.044613 2.026587 1.22306 0.4508602 2.810786 1.083154 0.30210375 protein(Bos taurus] 28.4 0.7429104 gi|119909677PREDICTED: similar to MGC84382 0.5340143 2.758603 0.059322 0.01823637 giI122692323ysozyme [Bostaurus] 55.10204 0.742951 0.688586 0.53975271.160912 0.324298 0.428584 0.0186749 3.646303 0.546461 8.86E-05 3.957525 0.598624 8.86E-05 macrophagemigration inhibitoryfactor (glycosylation-inhibitingfactor) [Bos taurus] 20.86957 1.296906 gil75812914 0.687048 0.1720918 4.493164 0.881982 0.17833888 gill 15496488SEC22 vesicle traffickingprotein homolog B [Bostaurus] 16.33466 1.291398 0.488746 0.4800242 3.392663 0.0444805 2.047706 0.037289 gill 15497346adaptor-related protein complex 2, beta 1 subunit[Bos taurus] 10.30494 1.497709 0.414254 0.2969266 1.574513 0.063904 0.01623032 1.543475 0.159946 0.1154222 1.40067 0.171665 0.15578092 9i|76629502 PREDICTED:simiartoTGOLN2 protein [Bostaurus] 16.77632 0.927459 0.279476 0.677674 0.157536 0.7457215 2.663787 0.08286 0.02637374 gi|76635454 PREDICTED:similar to OAF homolog(Drosophila) [Bos taurus] 28.36879 0.958118 0.023639 0.1659524 0.974144 0.001541 0.0006607 2.831372 0.086064 gi|134085842glucuronidase, beta [Bostaurus] 4.907975 0.704209 0.263444 0.224216 2.083319 0.02578849 Accession Name %Cov mean 115 stdl15l1pualue115:11stl1 1lluel161meanl17 stdl171lpcaluell7l4Nopepl11 Nopep 16 upept1? gi|119888979PREDICTED: similar to heparan sulfate proteoglycan 2 (perlecan)[Bos taurus] 26.34935 0.837063 0.403478 1.9DE-i5 0.95259 0.373037 00088365 0998097 0.883258 0000136975 247 258 255 1.36E-93 1.052178 0.423887 0.0007858 0484351 0.840485 2.82E-93 598 591 589 giJ766101267 PREDICTEDsimilar to fibronectin 1 isoform4 preproproteinisoform 12 [Bostaurus] 44.75021 0.463156 0.480728 gil16441442 collagen,type 11, alpha 1 isoform2 [Bostaurus] 55.00705 0.854572 0.495462 6.13E-35 0.840488 0.589894 3.08E-96 0.535849 0.830221 4.770-111 743 739 744 8 gil307942 D albumin[Bos taurus] 85.66722 2.45993 0.479478 2.04E-111 0883801 0.495928 1.49E-18 09846 1091581 0.974578433 674 650 68 gil34098396 ChondroitnABC endolyase1 precursor(Chondroitin ABC lyase1) (Chondroitinase ABC) (Chondroitin sulfate endolyase) (Chondroits ABC elroinase) 52.88932 1 0.419691 0.989830327 1 0387833 0.3194828 1 0.320736 0436577998 219 220 225 gil41386685 thrombospondin 1 [Bostaurus] 47.69231 0.73053 0.493838 1.57E.31 1.057974 0.33551 7.05E-06 0.951059 0.737226 0200718485 275 280 281 gil119890645PREDICTED similar toalpha 3 typeVI collagenisoform 5 precursorisoform 1, partial[Bos taurus] 45.04037 0.582081 0.538468 5.40E-19 1107817 0,499157 0.0086458 0.957857 0.81187 0034312499 110 110 112 gil1 19900891PREDICTED: hypothetical protein LOC540664 [Bos taurus] 32.81527 0.849801 0.104069 0194181551 1.112003 0.3771220.7060237 1.162073 0.126294 0.229409638 2 2 2 gil27806761 aggrecan1 [Bostaurus] 17.40438 0.752685 0.512405 0 0816278 0.43587 2.38E-49 0744753 0806282 552E-39 671 879 895 gi|119901095PREDICTED similar to collagen, typeXII, alpha 1 isoform 1 [Bostaurus] 22.98335 0.719242 0.381935 1.72E-13 0982038 0.345564 0.2847857 1153545 0388945 928E-06 59 64 68 gi|l 15495027heat shock7DkDa protein 5 [Bostaurus] 63.35878 1.109696 0.480298 0024460731 2823067 0.642853 0 277909 0682487 0 108 116 126 gi|119887130PREDICTED similar to Collagen, type VI,alpha 1 isoform1 [Bostaurus] 42.64849 0.622398 0.325312 721E-55 1.008858 0.301594 0.023367 0.874573 0379201 533E-17 334 343 339 gi|119913814PREDICTED similar to melanoma-associatedchondroitin sulfate proteoglycan 4 [Bostaurus] 24.45019 1.040585 0.249999 0.047922995 0.928117 0283046 0.0002174 0.927416 0425555 0076052035 74 74 75 gi|119890597PREDICTED similar to alpha 3 typeVI collagen,partial [Bos taurus] 36.76471 0.62676 0.480739 8.58E-11 1010198 0.290596 0.857575 104808 0377368 0038908652 79 81 81 gi|119919153PREDICTED: similar to AHNAK nucleoprotein [Bos taurus] 41.59497 1.039134 0.370255 06078493 3.092819 0.743213 1310-07 2.638091 0.733315 5700-07 12 17 17 gill10347570vimentin [Bostaurus] 62.23176 1.380347 0492979 1.490-10 2.542251 0886741 0 2.738 0.713441 8390-14 65 71 74 gi|114051163 collagen, type XI, alpha2 [Bostaurus] 40.14977 0.686068 0.464923 4990-29 1113795 0.347323 4410-08 1.629073 0.646425 3.730-36 307 323 333 gi|119892686PREDICTED similar to P63 protein[Bos taurus] 67.06282 0.906607 0.321133 0000194084 3.247247 0.734753 0 2.653344 0.745841 0 80 87 88 gi|119914559PREDICTED: similar to filamin B. beta(actin bindingprotein 278) isoform 7, partial[Bos taurus] 27.82132 1.194375 0.540658 0.006403559 1.848702 0.363359 7.370-14 1.74755 0497287 2430-12 37 37 36 gil148238040actinin, alpha 4 [Bostaurus] 47.63996 1.26428 0.394529 0000114481 2578089 0.812227 0 2.664101 0694165 0 40 43 46 gi[77404182lamin A/C[Bos taurus] 59.26573 1.217868 0.359493 5030-06 2.17346 0.466516 0 2.624695 0.54318 0 43 43 43 gi|l56120777nidogen 2 [Bostaurus] 32.64368 0.808751 0.343373 8150-12 0.908361 0.298581 5.870-08 0.955872 0.576384 0707942141 81 63 84 3330-15 3260966 0.579182 2560-06 28 29 29 gil605927927 heat shock90kD protein 1, alpha [Bos taurus] 47.3397 1.205972 0.474195 000537598 2.949889 0.538509 gil14823034 proteindisulfide isomerase-associated 3 [Bos taurus] 62.57426 1.0893 0.446279 0109789208 3.817216 0.748355 0 3.793069 0.691047 0 67 86 86 gil119894520 PREDICTEDsimilar to cartilageoligomeric matrix proteinisoform 1 [Bos taurus] 46.03175 0.818788 0.487005 4.580-07 0827782 0.533429 7.250-09 0.551296 1.118423 1420-10 88 86 86 gi|164452943gelsolin a [Bos taurus] 40.97311 0.695187 0.386157 1150-14 1.649112 0.580126 7.88E-07 1348877 0.557911 1770-06 58 59 59 gi|99028969complement component 3 [Bostaurus] 28.95846 1.063743 0.280662 0074060176 0.811825 0.384343 2.840-12 0.356582 0.903084 2310-10 33 33 32 gi|27806501prolyl 4-hydroxylase,beta subunit [Bos taurus] 66.47059 1.073538 0.345317 0.008549401 3.010583 0.583446 0 2.977922 06411 0 81 89 90 gi|153791660extracellular matrix protein 1 [Bostaurus] 54.54546 0.338604 0.455412 0 0815204 0.344116 1.88E-09 0.306434 0899426 1.230-11 59 60 61 gil119889734PREDICTED: similar to Collagenalpha-l(XI) chain isoform1 [Bostaurus] 31.97578 0.839092 0.38777 827E-13 0.847742 0.337052 222E-18 0.711882 0.505987 6350-18 146 146 147 gil115497814nucleobindin 1 [Bostaurus] 60.12658 1.121138 0.374925 0.000237774 1.011852 0.356226 0.5324485 0.883468 0.302727 4.810-05 78 77 77 gil119888668 PREDICTED:similar to Cartilagematrix proteinprecursor (Matrilin-1) [Bos taurus] 50.73529 0.835415 0.390752 1.870-11 0.803501 0.338838 0 0.959839 0552348 0.866595362 125 125 133 gil27807447 matrixmetalloproteinase 2 [Bos taurus] 55.06808 0.761708 0.557064 2.990-08 0739579 0.450868 6.17E-10 0.440702 0635573 7.55E-11 55 56 56 gi|87196501enolase 1 [Bostaurus] 60.36866 1.041078 0.58665 0393765163 2.791033 0.721168 0 3407778 0.805833 0 65 68 69 gil95147674 complementfactor B [Bostaurus] 43.88962 0.6658 0.400061 0 0.58171 0.479446 0 0.717888 0.478212 2.101114 98 00 105 37.10692 0.379018 0.633204 0 1.006669 0.293237 0.739739 0.80568 0.306294 1.76E-08 81 81 81 gil1199069084 3 PREDICTED:similar to matrixmetalloproteinase-3 [Bos taurus] gi|766301 PREDICTED:similar to matrilin-3[Bos taurus] 50.97002 0.56015 0.453447 0 0.588751 0.493742 0 0.831069 0.762416 0.000940778 104 105 104 gill14050715moesin [Bos taurus] 52.513 1.033309 0.317744 0.761636645 1.700946 0.484271 1.820-5 1.727707 0.454487 1.60-06 15 16 17 gi|114326226 protein disufide isomerase-associated 4 [Bostaurus] 44.32348 1.173791 0.603892 004049287 2.897887 0.799682 2.77E-12 2.8862 0.777428 1.466-13 31 33 36 gi]122692297chibnase 3-like 1 [Bos taurus] 53.70844 0.355438 0.422411 3010-117 0.5802 0.28437 1.070-119 0.322687 0.443066 2.590-118 706 729 719 gl155372023procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 [Bos taurus] 36.08924 1.008474 0.263717 0796128801 2.369590 0.441774 2.22E-14 2.565107 0.42218 4.44E-16 27 28 28 gi]27807289 annexinA2 [Bostaurus] 63.71682 0.864522 0.449534 002005348 1.620062 0.561806 1.300-06 1.719897 0.64929 3.190-07 27 27 29 gil156120795quiescin Q6 sulfhydryloxidase 1 [Bos taurus] 45.14991 0.885577 0.576208 0.00094702 166031 0.408226 0 2.953609 0.547068 2.43E-13 52 63 71 gil27805853 cartilagelinking protein 1 [Bos taurus] 50.64746 0.877402 0.365959 3.3-07 0.935443 0.383396 2.310-07 0.975970 0.305248 0.397689938 113 113 118 gi|119921375 PREDICTED: similar to RP11-525G3.1, partial [Bostaurus] 23.35423 0.810071 0.469326 0.003648842 0.819274 0.593539 0.0105995 0.902332 0.48741 0.013061608 27 28 28 gi|41386780 UDP-glucosepyrophosphorylase 2 [Bos taurus] 48.8189 1.035136 0.322768 0388958428 2.778324 0.534484 4.440-16 2.508068 0.654265 1.740-12 31 32 31 gil119901238 PREDICTED:similar to lamininalpha 4 [Bostaurus] 21.79417 1.092885 0.561388 0.304105828 0796267 0.304242 3.800-05 0.832184 0.345183 0.004018315 21 21 21 gi|115497900 eukaryotc translation elongation factor 2 [Bostaurus] 36.71329 1.176607 0.423182 0010127881 2.801989 0.578151 1.320-12 2.58226 0.607902 1.35E-11 24 26 26 giJ61888856 triosephosphate isomerase1 [Bos taurus] 72.69076 1.374135 0.51317 3.290-07 3.010825 0.60713 0 2.84055 0.664001 0 45 70 71 gil27807263 heat shockprotein 90kDa beta, member 1 [Bos taurus] 38.80597 0.947458 0.348699 0.34980025 3010549 0.575034 5.790-10 3.428719 0.729066 2.900-09 19 19 19 gil147906412 hypotheticalprotein LOC615490 [Bos taurus] 37.52809 0.610225 0.662467 5.600-10 0.877448 0.350422 6.520-05 1.077281 0.858107 0807656019 51 53 53 gir61845535 PREDICTED:similar to emilin(Bos taurus] 30.04926 0.90724 0.350471 0.008698904 0990708 0.313715 0.5207495 0.891227 0429387 0.040499183 38 38 39 gi|76607158PREDICTED similar to Melanotransferrinprecursor (Melanoma-associated antigen p97) (CD228 antgen) 905taurus] 33.55886 1.171926 0.320153 0001172306 1.171209 0.318859 0.0142498 0.980021 0.53173 0.933285925 28 26 26 gi|27806469 peptidylprolylisomerase B {Bostaurus] 75.48077 0.775224 0.405526 1100-09 1.497094 0.508777 2.23E-08 1.26326 0.381398 373E-10 63 63 63 gi]116003881collagen, type 111,alpha 1 [Bostaurus] 42.90587 0.728525 0.476838 3.680-08 1.435887 0.461725 4.93E-06 1.348145 0.735978 0.00842754 54 54 54 gil115497210complement component 1. s subcomponent[Bos taurus] 28.77698 0.825658 0.276615 2.8-07 0681547 0.260751 2.220-16 0.586669 0340813 2.220-16 39 39 39 giJ77735551 phosphoglyceratekinase 1 [Bos taurus] 65.94724 1.014866 0.457414 0.787327151 3.25846 0.813019 2.200-14 2.909108 0960754 3.190-11 31 36 35 gil27806147 secretedprotein, acidic, cysteine-rich [Bos taurus] 52.30263 1.112631 0.432879 0000437327 0.433091 0.455733 0 0.267344 0.904945 0 103 103 103 gi|110350683 biglycan[Bos taurus] 53.11653 0.90193 0.361646 3.30-05 1943041 0496503 0 1.452485 0.547575 0 102 116 114 gij75832054 actin,beta [Bostaurus] 47.46667 1.142032 0.488555 0009011311 3.081512 0.857538 0 2.87937 0.85164 0 62 67 67 gi|119919953 PREDICTEDsimilar to filamin[Bos taurus] 18.10017 1.119296 0.299691 0095124695 1617911 0.446145 0.0002908 2.246832 0578947 1970-05 12 12 12 gi|115495019 procollagen-proline,2-oxoglutarate 4-dioxy genase (proline 4-hydroxylase),alpha polypeptide I [Bostaurus] 42.50936 0.962406 0.370927 0510699563 2836113 0.724586 3920-08 3161361 0.881849 1.330-08 21 22 22 gi|27806559 lactatedehydrogenase A [Bostaurus] 61.44578 1.095854 0.396579 013030691 2.451282 0.68869 6230-09 2.131829 0.74977 6410-08 22 24 26 gi|115496702collagen, type VI,alpha 2 [Bostaurus] 48.74591 0.672606 0.454125 1.036-06 1.329379 0.349184 2850-08 1142093 0302527 0.006284216 26 20 26 gi|119902010PREDICTED: similar to LOC512571protein [Bos taurus] 44.40298 1.167611 0.372884 0.001632762 3468224 0.44042 0 2.93251 0.500255 0 32 35 37 gi|27806477lysyl hydroxylaseprecursor [Bos taurus] 48.76033 1.014552 0.303548 0650464916 0.97276 0.340762 0.0345908 1.026278 0.288379 0.201064286 40 43 42 gi|76253709heat shock 70kDa protein 8 [Bos taurus] 33.69231 0.941139 0.884305 0.206054688 1.833408 1.178599 0.0136719 1.933138 1.566201 0.088752455 11 11 11 gil119919157 PREDICTED: hypothetical protein [Bos taurus] 30.6163 1.057389 0.447895 0423419308 2.443185 0.80538 2630-09 2.379748 0.58095 1.23E-09 20 22 22 gi|77404252 collagen,type 1, alpha 1 [Bos taurus] 42.72044 0.588984 0.431925 1.700-05 0838006 0.491821 0.0245361 0.423029 0958062 0.000610352 14 14 14 gi|62988316 follistatin-like1 [Bostaurus] 49.83713 0.920442 0.311904 0006624735 0.892288 0271447 1.330-15 0.295712 1.0329 3.570-08 40 40 40 gi|77404217 phosphoglyceratemutase 1 (brain)[Bos taurus] 61.02362 1.290904 0.353771 1800-06 3140921 0.546255 0 2.441112 0599982 1.200-14 32 35 35 gi|156523168matrilin 2 [Bostaurus] 31.10647 0.622527 0.379856 1560-07 0764861 0.24757 5.710-07 0711202 0.52512 000068081 20 20 20 gi|27807367 tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide[Bos taurus] 61.22449 1.352603 0.55751 0000570644 3000337 0.625155 3.350-14 274531 0596008 1.580-14 30 30 31 gi|27806723 catreticulin[Bos taurus] 37.88969 1.191075 0.373938 0013218783 3.972464 0.640949 3020-11 3033952 0901157 3.250-07 17 20 19 gi|148238309 aldolaseC, fructose-bisphosphate [Bostaurus] 42.85714 1.040839 0.319976 1 2.175947 0.71009 00002261 2.069744 0.669801 0000177336 10 12 12 gir77404273 glyceraldehyde-3-phosphatedehydrogenase [Bos taurus] 40.84084 1.284868 0.298604 4.930-05 4767639 0457855 3.33E-14 4.94648 0.055547 4.830-13 19 19 19 gill14052136FK506 bindingprotein 9 [Bos taurus] 32.92683 0.952533 0.353901 0700480746 3.459247 0.833595 1.040-14 2866455 0.815351 2.930-13 34 36 36 gi]76621011PREDICTED: similar to Cartilageintermediate layer protein 2 precursor(CILP-2) [Bos taurus] 24.6473 0897385 0.315468 0.006546787 0.871611 0.355929 0.00078 0.904995 0.38123 0.047402976 35 36 38 gi]27806941 serine(or cysteine)proteinase inhibitor, clade A (alpha-1 antiproteinase,antitrypsin), member 1 [Bos taurus] 39.18269 0.654795 0.323534 6.920-11 0863964 0.268318 9.840-05 0.688005 0.338425 1.01E-08 29 28 28 gil94966763haptoglobin [Bos taurus] 45.63591 0.480795 0.458112 1.130-11 0188748 0.806206 7.330-15 0.155989 0.921258 7.970-14 28 26 26 gi|27806697chondroadherin [Bos taurus] 48.75346 0.627942 0.422765 0 1.134407 0.298218 288-07 1.558928 0.401822 2.090-16 99 103 102 0.19047228 1.893274 0.499106 6.26E-05 2.384881 0.700173 5.80E-05 gi|157074106lamin B1 [Bostaurus] 4027304 1125927 0.427507 D 0.409857 0.63264 1.66E-13 gi|27806907 clusterin [Bostaurus] 31.890060.994265 0459222 0.8912971140.516234 0560052 0.1909616 0.564658 0.721397 0.00012594 gi|27806257 collagen,type 1,alpha 2 [Bostaurus] 42.00880.572324 0.327987 8.68E-09 0.915573 0.473782 0.0085296810.942108 0.340611 0.2022533 0.863529 0.450099 0.088978291 1 [Bostaurus] 22.820510791002 0.481149 gi|164450489AE bindingprotein 2.148625 1.114603 0.147246 1.902193 1.325119 0.247878468 gi|61828365 PREDICTED:similar to Tropomyosin3 isoform1 [Bostaurus] 6572581 1.2039890.533307 0.387538885 0 0.24743 1.262241 5.91E-07 gi|156718112 transforming growthfactor beta bindingprotein 1 [Bostaurus] 22.34679 0477334 0.39839 2.22E-16 0.30131 0.545653 latent 0.380719 0.0001744 1.727248 0.411099 6.77E-10 gi|27807207 plasminogen activator inhibitortype 1, member 2 [Bos taurus] 34.50882 0.747163 0.406881 4.12E-05 1.330537 0.0003589 1.664574 0.30045 4.00E-15 gi|27806853 lumican [Bostaurus] 41.22807 0.957583 0.452189 0.462619285 1.44796 0.556375 0.381484 7.86E-08 gil115498012 glycogenphosphorylase, liver[Bos taurus) 3184499 0.935102 0.333769 0.341791907 3.125026 0.374674 2.44E-09 2.55356 6.45E-07 2.221409 1.340117 0.001018829 gi]82751849 Parkinson disease (autosomal recessive, early onset) 7 [Bos taurus) 5978836 1167313 0.482983 0.051651999 2.577366 0.825105 0.0032635 0.788065 0.372537 0.006361088 gil27807437 matrixmetalloproteinase 9 [Bos taurus] 3244382 0845298 0.356417 0.024928977 D.797066 0.361873 5.39E-05 2.645173 0.599273 0.00075482 gi|76253900 GDPdissociation inhibitor2 [Bostaurus] 4874157 1 008134 0404379 0.93617784 3.215671 0.434908 1.840324 0.475653 1.49E-08 gil155371895 sema domain, immunoglobulindomain (1g),short basic domain. secreted, (semaphon) 3C [Bus taurus] 3475366 0.656269 041703 1.73E-06 1.498072 0.455662 9.49E-06 1.30E-06 3.288567 0.43516 2.17E-D6 gir76682880 PREDICTEDsimilar to nucleolin-relatedprotein NRP isoform 1 [Bos taurus] 3972222 1146393 0.255354 0.0641942833.295887 0.398104 7.94E-06 0.419281 0.36339 7.84E-10 gi]38566696 serum amyloidA 3 [Bos taurus] 72.51908 0448264 0.570317 7.26E-07 0.562113 0.513318 0.5745095 0.936048 0.411462 0.523185861 gi27806781 vitrin [Bos taurus] 32.97546 0799637 0423876 0.018105078 1.069017 0.473747 0.456215 0 gil156121245 insulin-likegrowth factor bindingprotein 7 [Bos taurus] 5567376 0.774017 0.289178 6.68E-11 0.578059 0.483977 7.99E-15 0.417834 0.0019531 2.664316 0.71645 5.79E-05 gi|78369242 alpha-actinin[Bos taurus] 3733184 1.397087 0398414 0.006582678 2.490301 0.516419 0.5830078 0.789571 0.565253 0.053630741 gi|119903231 PREDICTEDsimilar to lysyl oxidase-like3 protein[Bos taurus] 29.65426 0872476 0356122 0.060376673 0.965418 0.314061 0.796064 0.000140134 gi|119903133 PREDICTEDhypothetical protein [Bostaurus] 37.06294 0.673846 0.235276 4.00E-09 0.809814 0.284731 6.34E-05 0.611837 1.025448 0.029541016 gi|77736401 thrombospondin4 [Bos taurus] 28.72008 0946067 0666918 0.668260819 0.770715 0.318171 0.0004471 0.596724 54.91713 091483 0.369929 0.174969615 2.546949 0.420705 0.0002764 1.367317 0.394717 9.48E-05 gi|120474983 annexin A5 [Bos taurus] 7.93E-08 gi27805949 inhibinbeta A [Bos taurus] 3764706 1.07804 0.333489 0.279569061 4.1447 0.734835 1.22E-07 5.027523 0.819795 4 5.21E-08 2.603798 0.660478 2.94E-08 gij4025 806 heat shock 70kDa protein 1A[Bos taurus] 3588144 1.09679 0438155 0.238874125 2.523834 0.644209 4.44E-07 0.715834 0.255707 3.06E-07 gil119895885 PREDICTEDsimilar to EGF4ikerepeats and discoidinI-like domains 3 [Bos taurus] 2645161 0.773023 0.352321 0.000231289 0.705205 0.297968 52.22552 1.238868 0.37494 0.028022175 3.935189 0.538403 1.12E-06 3.411074 0.552981 2.94E-06 gi|164420731 transaldolase 1 [Bostaurus] 4.001638 0.068226 0.015194624 gi|155371863 histone cluster 1, Hld [Bos taurus] 6063349 0.851828 0.328314 0.500616253 1.433246 0.26743 0.2306782 2.528015 0.70501 1.11E-08 gi|168804008 tubulin,alpha, ubiquitous[Bos taurus] 42.79379 0.965033 0.308589 0.498755942 2.635921 0.538609 2.05E-10 2.691735 0.926881 0.000427246 gil156120479 aldolase A [Bostaurus] 39.83516 1.456509 0.8686 0.00643178 2.571771 0.845196 0.0003052 3.846697 0.733993 2.D3E-08 gil1 14051756 heterogeneous nuclear ribonucleoproteinA2/B1 [Bos taurus] 43.10851 1.261221 0.429936 0.009074546 4.906097 0.859797 2.37E-08 6.706334 1.185234 1.79E-08 gil27806489 proenkephalin [Bostaurus] 60.8365 0.900143 0.625208 0.350254639 4.301779 1.027594 5.73E-08 0.0001398 1.948011 0.615649 4.67E-05 gil1 14053121 reticulocalbin3, EF-handcalcium bindingdomain [Bostaurus] 36.89024 0952057 0.602992 0.709051716 2.056151 0.620444 1.849762 0.45305 8.43E-07 gi]27806085 peroxiredoxin4 [Bos taurus] 56.93431 0.88498 0258455 0.019177603 1.985809 0.399244 7.34E-08 0.788375 0.197172895 gil115497328 procollagen C-endopeptidase enhancer 2 [Bostaurus] 39.06346 0.70963 0.290526 0.000212765 1.00785 0.357468 0.9441931 1.298553 gi|119912215 PREDICTED:hypothetical protein isoform2 [Bos taurus] 1933682 1.171852 0.346696 0.081894695 2.440332 0.262848 5.13E-07 2.30525 0.416675 2.15E-05 8.72E-06 5.61E-06 gi]94966781 eukaryotictranslation elongation factor 1 gamma [Bostaurus] 2888364 1.172907 0.475952 0.138293414 2.707686 0.693884 3.070175 0.768143 5.40E-062.000045 0.382546 4.36E-05 gi[27806351 ezrin [Bostaurus] 36.8881 0.965646 0.296138 0.630514667 2.090992 0.294809 0.178795263 2.561417 0.547593 0.0015722 2.768278 0.754362 0.001969849 isoform 1 [Bos taurus] 361552 1.206622 0.426321 gi|119918578PREDICTED: smilar to vinculinisoform meta-VCL 0.00692 0.633805 0.607659 0.00116154 gi|119331178SPARC related modular calcium binding 1 [Bos taurus] 29.7235 0833238 0.325341 1.75E-06 0.858276 0.295083 1.120131 0.444784 0.021602435 gi[76253701 decorin [Bostaurus] 39.72222 0.909011 0.434115 0.075654317 1.10709 0.44731 0.1091174 0.8959932 0.750986 0.408437 6.75E-05 gil114052653 procollagen C-endopeptidase enhancer [Bos taurus] 40.79823 0.727481 0.485901 8.63E-05 1.009105 0.279098 0.00369553 2.955933 0.792807 8.33E-10 2.895901 0.857683 3.25E-09 eukaryobc translation elongationfactor 1 alpha 1 [Bostaurus] 43.93939 0.903605 0.225695 gil68299807 1.B2E-05 3.373809 0.649795 3.93E-12 3.928961 0.766198 6.51E-12 gi]115496892non-metastatic cells 2, protein (NM23B)expressed in [Bos taurus] 5855263 1.066869 0.557906 70.87379 0.779528 0.307805 5.28E-14 1.333828 0.284532 0 2.038513 0.349828 2.76E-22 gi76670918 PREDICTED:similar to histone H4 [Bos taurus] 0.0002955 2.283279 0.802632 0.000532804 gil115495641aldo-keto reductase family1, memberAl (aldehyde reductase) [Bos taurus] 49.30789 1.181813 0.431983 0.125970649 2.978355 0.952032 0.5676450.073378059 gil154707890lysyl oxidase-like2 [Bos taurus] 2209302 0.801612 0.381171 0.006041276 0.900239 0.32636 0.0604612 0.81011 0.027624 1.4423270.30729 3.38E-06 gill14051505 serpin peptdase inhibitor,clade H (heat shock protein 47), member 1, (collagen bindingprotein 1) [Bos taurus] 354067 0633137 0.478665 6.D9E-05 1.3755 0.49172 0.0001156 gil114051908 FK506 bindingprotein 10,65 kDa [Bos taurus] 3859348 0.745777 0700688 0.010130054 2.053107 1.D43318 1.663922 0.977401 0.001238017 3.58E-06 gi|118601868 heat shock 90kDa protein 1, beta [Bos taurus 4323204 1.179926 0432622 0.134949764 2.829475 0.442488 8.27E-06 2.952465 0.423224 0.001825279 gi|119900887 PREDICTED:similar to collagen XXVilproalpha 1 chain precursor; preproprotein [Bostaurus] 24.28717 0711018 0.35738 0.001865616 0.994869 0.329713 0.7952449 1.379617 0.351506 0.677598 5.52E-05 3.198501 0.50656 8.15E-06 gi|94966765 glucose phosphate isomerase [Bos taurus] 26.75045 1.337168 0.39997 0.013554497 3.40858 0.252591 0.802247 0.0078125 gi|30794292 lactotransferrin[Bos taurus] 27.11864 0.372085 0.501949 8.97E-05 0.320559 0.720169 0.0003259 0.940242 0.710279484 gi|119917101 PREDICTED:hypothetical proteinisoform 6 [Bostaurus] 25.71042 1.018847 0.342963 0.83106789 1.279463 0.699551 0.1886494 1.072044 0.56859 0.000477678 gil115496400 dthydropyrimidinase-like2 [Bos taurus] 23.07692 1.099276 0.41174 0.1953125 2.509343 0.626335 0.0078125 2.315977 0.647019 7.12E-06 gil15707396 reticulocalbin1, EF-hand calcium binding domain [Bos taurus] 37.16012 0.930906 0.492291 0.419250873 2.43038 0.584041 1.35E-07 2.089754 0.649615 1.01E-08 gil27806197 tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide [Bostaurus] 06.47059 1.278383 0.321317 0.000136922 2.992986 0.542725 2.48E-10 2.74771 0.514091 0.791706 0.033521707 gi|27805991 latenttransforming growthfactor beta binding protein2 [Bos taurus] 1477331 0.540961 0606565 0.015955232 0.740266 0.418005 0.0447558 3 1.56E-05 gi|2780662 fibromodulin[Bos taurus] 28 0859221 0.363144 2.87E-10 1.139816 0384728 3.58E-07 1.062949 0.426523 gil119887291 PREDICTEDsimilar to peroxidasin homolog [Bostaurus] 175049 0770344 0.231358 0.001244457 0.957782 0.262863 0.3729791 1.10643 0.254901 0.086168407 3.63E-12 gil119915902 PREDICTEDsimilar to Thioredoxindomain-containing protein 5 precursor (Thioredoxin-likeprotein p46) (Endoplasmic reboutumprotein ERpd6) isoform 1 [Bust 3971292 0.997541 0.50088 0.981283756 3.006458 0.723948 1.20E-12 3.144248 0.837481 2.876172 0.387523 2.20E-06 gil78369310 stress4nduced-phosphoprotein 1 (Hsp70/Hsp90-organizingprotein) [Bos taurus] 290976 1.506858 0.390306 0.001864326 2.846165 0.286288 2.84E-07 0.380839 8.41E-05 gil119892775 PREDICTEDsimilar to myosin. heavy polypeptide 9. non-muscle[Bos taurus] 22.79898 1202307 0.357698 D.072756884 1.343691 0.473816 0.033177 1.873962 4.182297 0.430176 7.47E-05 gi|41386727 protein kinase C substrate 80K-H[Bos taurus] 19.88743 1.219497 0.313394 0.074649923 3.827347 0.373161 5.64E-05 1.595401 0.43057 0.000324779 gi|1 19908681 PREDICTED:similar to superficialzone protein[Bos taurus] 23.88721 0.632282 0.948914 0.004192246 2.049133 0.405612 2.42E-05 7.87E-06 1.198242 0.263457 0.001529606 gi27805823 dermatan sulfate proteoglycan3 [Bostaurus] 24.29907 0.778807 0270498 0.000254386 1.51588 0.304836 1.79E-060.720128 0.197288 1.50E-07 gi|119888620 PREDICTED:similar to alpha 1 type XVIcollagen [Bos taurus] 23.00062 0.761568 0.540382 0.019970875 0.757169 0.223901 0.1907425 0.864673 0.63068 0.196108186 gil139949116 slit-like2 [Bos taurus] 2734027 1052909 0.806197 0.679115689 0.946403 0.281463 0.86595 0.935589 0.002545598 gi 27805983 leukocytecell-derived chemotaxin 2 [Bos taurus] 5761589 0.478727 0.264371 1.73E-06 0.937929 0.310296 0.0517578 2.288128 0.49468 7.88E-05 gil27806317 annexin A6 [Bos taurus] 48.62385 1-333883 0.500675 0.050782434 1.393843 0.413415 0.0164323 0.0001465 4.23411 0.759813 0.001953125 gil148223509 osteoprotegerin [Bostaurus] 3706468 0765069 033217t 0.005260277 1.871569 0.445891 0.821616 0.430317 0.005111049 gi|119917871 PREDICTED:similar to serin protease with IGF-bindingmotif [Bos taurus] 2507375 0.676009 0208364 2.25E-10 0.896397 0.249134 0.0019443 E-07 gi|119903961 PREDICTED similarto Protein disulfideisomerase associated 6 isoform 7 [Bos taurus] 3395932 0.964435 0657748 0.752539626 2.195263 0.728227 2.96E-06 2.236703 0.799112 4.11 0.242164 0.447340792 gi|31341666 mannosidase, alpha, class 2B, member 1 [Bos taurus] 2102102 082622 0.327152 0.049479839 1.230651 0.269044 0.0219369 0.940036 0.000128137 gil115496067 nucleobindn 2 [Bos taurus] 41.20482 0916578 0.272702 0.095089985 1.38818 0.367229 0.0001809 1.255011 0.272421 0.017885709 gi|30794358 versican [8os taurus] 12.18574 1.108672 0436888 0.333419449 0.743294 0.488374 0.0248765 0.793328 0.316939 0.128364 0.274317 9.50E-10 gi76613223 PREDICTED:similar to chitiase [Bos taurus} 3255814 0.396545 0195772 3.44E-08 0.215502 0.273773 8.26E-09 0.0002268 2.24359 0.507328 0.000111094 gi77736275 calumenin [Bos taurus] 39.36508 0.969705 0.682093 0.853675631 3.123114 0.769672 1.124706 gil28461273 glutathione S-transferase M1 [Bos taurus] 55.9633 1.308305 0.483219 0.105825187 3.714805 0.914706 0.0016373 2.9947 0.009286548 2.64342 0.6928230.000656927 gil116004023 phosphoglucomutase 1 [Bos taurus] 24.91103 1.026151 0.396244 0.792354392 2.236643 0.464371 0.0002214 0.0001169 0.944823 0.003718017 gil115497628 reticulocalbin2, EF-hand calcium bindingdomain [Bostaurus] 29653 1.162354 0.492613 0.285804312 3.610974 0.673524 2.647829 gi]27807349 complement component 1 inhibitor[Bos taurus] 22.86325 1132734 0.364022 0.017929343 0364105 0.705031 1.94E-10 0.58983 0.326639 1.56E-11 8. gi]27806625 ftizled-retated protein Bus taurus] 2.84615 0.627622 0.260391 1.81E-10 1.008216 0.255657 0.8784018 1.566374 0.435435 13E-07 0.300595249 2.622966 0.510622 0.0012207 1.77584 0.790103 0.046451449 gi|78369510 ribonuclease/angiogenin inhibitor1 [Bos taurus] 29.38597 1.126192 0.324758 01010121323005314 0.519199 5.93E-05 2.537452 0.70156 0.000908615 gill 18151330 tyrosine3-monooxygenaseltryptophan 5-monooxygenase activation protein,theta polypeptide[Bos taurus] 55.91837 1.199329 0.39451 7.496-06 0.640309 0364921 1.05E-05 0.539079 0.500076 4.52E-06 gi[41386719 milkfat globule-EGFfactor 8 protein [Bostaurus] 33.48946 D.79112 0.202064 0011395109 0.944692 0.50241 0.4270805 0.791509 0.5186360.027304536 gi[119919898PREDICTED: hypothetical protein [Bos taurus] 26.28571 0.775134 0.46563 0 6 0.000392235 4.069971 0942095 9.61E-07 3.66979 0.501802 0.00390625 gi|278 561 lactate dehydrogenase B [Bos taurus] 47.60479 1.508136 0.342936 0.156403106 3.70924 0.5117 1.72E-05 3.00167 0.674195 0.000267971 gil156121049dimethylarginine dimethylaminohydrolase 1 [Bostaurus] 39.29825 1.237758 0.3963 0.4396537942.902697 0.571167 1.97E-05 2.843166 0.533737 8.15E-06 gil115497174 3-phosphoadenosine 5'-phosphosulfate synthase 2 [Bos taurus] 31.21951 0.949886 0.251807 0.7200533491.944109 0.311161 0.0003285 2.039338 0.733335 0009154083 gi|119895049PREDICTED: similar to Lamin 92 [Bostaurus] 35.92715 1.020326 0.212387 3.559594 0.440376 0.03125 3.951069 0.654932 0.00067055 gi76635416 PREDICTED:similar to coat protein delta-cop isoform 1 [Bos taurus] 27.0057 1.193124 0.247001 0.091502996 7 0.701524 0.257354 443E-05 0.628839 0.397688 0.001953125 gil12927 510 superoxide dismutase 3, extracellular [Bos taurus] 41.49378 0.926996 0.387711 0.376073329 2.779999 0.444469 2.98E-11 0.861641 0.406777 0.051614392 gil27806401 secreted phosphoprotein1 [Bostaurus] 45.68345 0.720812 0.458792 0.000291367 1.622905 0.402909 0.0196995 3.790859 0.157842 7.53E-07 gi|149773543steroid-sensitive protein 1 [Bostaurus] 20.96436 0.605496 0.607317 0056229617 070331 0.456362 0.0032712 1.744261 0.580574 0.0000840259 gi|27806679 colony stimulatingfactor 1 (macrophage) [Bos taurus] 27.07581 2.070581 0.802553 0001457649 4.020656 0.641951 3.20E-08 3.845949 0.670613 1.93E-08 gi|51491841 transketolase [Bostaurus] 27.60835 1.028374 0.531252 0.920956016 3797069 0.730459 5.136-05 3.948679 0.723811 3.84E-05 gi|119913642 PREDICTED:similar to KIAA0051[Bos taurus] 22.83019 1.436444 0.503329 0.033195974 0903793 0363619 0.3132383 0.86015 0.154319 0.026026895 gi]119908661PREDICTED: similar to Laminin gamma-i chain precursor (Laminin62 chain) [Bos taurus] 21.33858 0.926587 0.285185 0392433702 1.250975 0444424 0.1217071 0.734348 0.350268 0.109375 gi]78369298 stromal cell derived factor 4 [Bos taurus] 32.67606 1.012411 0.517378 0.92627127 94 0.401752 6.14E-11 2.189436 0.481435 4.73E-09 gi|75812 0 phosphatidylethanolaminebinding protein [Bostaurus] 49.73262 1.099546 0.353673 0116220906 2.426716 27 3 2.469264 0.572251 0.0003883 2.399148 0.432184 6.69E-05 gill1405 1 tubulin,beta polypeptide[Bos taurus] 22.2973 1.038783 0.49663 0.759217249 0.704996 0.346392 0.0010251 0.47D103 0.39876 1.39E-05 gi|27806739 apolipoproteinE [Bostaurus] 45.88608 0.733549 0.252604 0.000341695 7 64 0792692 0.299915 0.0046462 0.71025 0.459951 0.014955195 gi|2 808 D peptidoglycanrecognition protein1 [Bos taurus] 46.31579 0.639251 0.33389 0.000409542 4067222 0.399759 1.80E-09 5.140501 0.459027 1.67E-09 gil27807167 peroxiredoxin6 [Bos taurus] 48.66071 1.323234 0.296632 000390625 0.396909 0629676 0.0010998 0.340626 0.850908 0.003100862 gil114053333 fibrinogen-like2 [Bos taurus] 44.89796 0.749614 0.318248 0.013672157 1.529371 0213163 0.0010414 2.604213 0.216527 1.82E-05 gi|82697375 histone H2B-like[Bos taurus] 78.57143 0.778357 0.309967 0.0359793 3.6169 0.629535 6.51E-14 3.302432 0.463762 2.22E-16 gi|119903031 PREDICTED:hypothetical protein [Bos taurus] 19.6281 1.617291 0.439488 3.916-09 4 6 2.991697 0.520536 0.0008649 2.56434 0.543379 0001575162 gil119901 1 PREDICTED:similar to Syncrip protein [Bos taurus] 31.11702 0.824457 0.443684 0299776229 0670677 5.30E-05 2.496215 0.555573 8.87E-05 gi]71037405 heat shock 27kDa protein 1 [Bostaurus] 41.66667 0.930182 0.428426 0.517012213 3426523 0.975401 0.270109 0.5734683 0.826073 0.343311 0.056168721 gi|1 16003813 amyloidbeta A4protein [Bos taurus) 15.68345 1.404063 0.335795 0.002337023 0.397699 0.4115302 0.95028 0.407553 0.026398981 gi|119901059 PREDICTED:similar to COL9A1protein, partial [Bos taurus] 19.71253 0.699285 0.338444 9.736-05 1.053999 1.132333 0.506994 0.3249347 0.683994 0.580632 0.013026282 giJ76611607 PREDICTED:similar to C1QC proteinisoform 1 [Bos taurus] 25.36765 0.734554 0.67762 0.054991937 3.996-05 1.974962 1141225 0.0140974 0.79563 0.2939 0.000226808 gi[75832065 TIMP metatlopeptidase inhibitor2 [Bos taurus] 33.18182 0.65191 0.497802 0.997929406 0.999099 0.355969 0.9104525 1.009918 0.288851 0.826414819 gi|119896006 PREDICTED:similar to mannosidase, alpha, class 2A,member 1 [Bos taurus] 18.42795 0.999529 0.28011 2.312701 0.562955 0.0672586 2.274411 0.987044 0.092233717 gi|148230364heterogeneous nuclear ribonucleoproteinD [Bos taurus] 35.62092 1.082545 0.371005 0.643733331 0.599967 0.423997 0.0071728 0.643632 0.3127 0.004795902 keratin 10 [Bostaurus] 23.95437 0.714804 0.141639 0.000401162 gi27805977 0.097204 1.21E-05 0.422755 0.718362 0.0625 protein S, alpha [Bos taurus] 22.66667 0.691925 0.320239 0.125 0.496097 gi]27806095 1.644241 0.656075 1.497581 0.6134 0.125 gil119900517PREDICTED: similar to KtAAl027protein [Bos taurus) 22.2747 0.848161 0.076552 0033191021 0.1236341 1.756-09 1.392702 0.34499 2.52E-05 0.552479 0.504984 5.84E-10 gil66792902 vanin 1 [Bostaurus] 17.64706 0.405888 0.843808 0.973799 0.077667 0.0034577 1.0253 0.426367 0.782321414 gil119917901 PREDICTED:similar to CarboxypeptidaseX 2 (M14family) [Bos taurus] 26.51715 0.686724 0.290136 0.014026424 2.345791 0229969 4.91E-05 2.025963 0.324223 0.000538952 gil125991942 Sec23 homolog A [Bos taurus] 1809896 1.12328 0.277486 0.245595267 3 1.966-05 0.424693 0.313313 2.11E-06 0.314787 0.298409 1.79E-07 gi|8457985 binding protein [Bostaurus] 23.07692 0.248446 0.43585 lipopolysaccharide 3.051097 0.456294 0.0014673 3.277184 0.402737 0.000132843 gil113205970 glialfibrillary acidic protein [Bos taurus] 52.1028 1.142308 0.609279 0.517790539 0.000139795 0.947792 0.321252 0.4893589 0.904175 0.268745 0.306090759 gi[27806541 matrixmetalloproteinase 1 [Bos taurus] 26.01279 0.421773 0.29099 0.842916695 1.023016 0.299 0.9042234 0.975588 0.302229 0.882598401 gi[78369684 platelet-derivedgrowth factor receptor-likeprotein [Bos taurus] 36.8 0.980503 0.400557 0.391170505 2.369377 0.703193 0.0946462 2.645973 0.754333 0.013462004 gi]27807517 serine (or cysteine) proteinase inhibitor,clade B (ovalbumin),member 6 [Bos taurus] 31.21693 1.448994 0.534954 0.045796595 0.906221 0.29523 0.066161 0.473723 0.520026 0.010437137 gil115496928 EGF-containingfibuhn-ike extracellular matrixprotein 2 [Bos taurus] 31.37698 0.776944 0.283558 0.75013 0.403742 2.33E-05 0.505201 0.481114 3.88E-05 gi]95006989 ribonuclease, RNase A family,4 [Bos taurus] 59.86394 0.470833 0.438507 4.976-11 1.339209 0.502969 0.0078125 0.989947 0.48982 0974569727 gi|28875793 thrombospondin2 [Bos taurus] 13.84615 0.843256 0.307429 0.059936619 0.425033 0.4221623 1.059235 0.482058 0.454602142 gi|27806449 dystroglycan 1 [Bos taurus] 14.07821 0.874871 0.407648 01159614 0947697 3.503711 0.532703 8.05E-06 2.868775 0.680483 0.000138879 gi62751777 cofilo 1 (non-muscle)[Bos taurus] 71.08434 1.299646 0.549125 0072047296 0.697934 0.0003912 3.177227 0.894847 0.002474373 giJ27807469 peroxiredoxin2 [Bos taurus) 39.19598 1.246076 0.520799 0.156919757 3.716369 9 0.617036 2.13E-05 2.942636 0.474047 2.34E-06 gi|7740420 eukaryotictranslation initiation factor 4A2 [Bos taurus] 21.86732 1.360362 0.492577 0.01996463 3.009031 9 0.000100372 0.619703 0.599036 0.00092 0.743661 0.792982 0.077022558 gil11405112 retinoicacid receptor responder (tazarotene induced) 2 [Bostaurus] 50 0.588901 0.517981 3 27 2.929179 0.742356 4.99E-11 3.182978 0.698586 1.47E-12 gil156121 galectin 3 [Bostaurus] 36.60377 1.050061 0.481141 0.473661304 1.57945 0.397929 6.18E-06 1.605253 0.868891 0.004185803 gi27806813 pleiotrophin[Bos taurus] 38.69048 0.458214 0.616786 1236-06 1.377917 0.137072 0.0019626 0.799599 0.164351 0.014688243 gi|95147666 periostin, osteoblast specificfactor [Bos taurus] 23.62003 0.620313 0.577812 0.056077025 0.77993524 4.090764 0.960567 0.0037025 2.538432 1.212873 0.040063294 gi|119919662 PREDICTED:similar to LOC539445 protein [Bos taurus] 16.21271 1.032594 0.352598 2.430097 0.402511 0.015625 2.910937 0.498942 0.000167836 gi|77735541 valosin-containingprotein [Bos taurus] 28.0397 1.600285 0.979663 0015625 0.521179669 2.692939 0.68635 4.04E-06 2.839283 0.838384 5.79E-06 gi|60592767 ribosomal protein, large, PO [Bostaurus] 46.22642 0.939665 0.34456 2.619545 0.902403 0.0063833 2.659301 0.402291 0.000329245 giJ77735461 canopy 4 homolog [Bos taurus] 12.5523 0.961306 0.529227 0.971797147 0.6595186941.094649 0420726 0.2957764 1.210667 0.336312 0.005811501 gil27806829 osteoglycir [Bos taurus] 34.11371 1.043169 0.513296 0017260013 099271 0.2515140.1768685 1.310973 0.096146 0.000669569 gi(99028973 transcobalamin |1[Bos taurus] 22.91667 0.68501 0.310794 7 9 2.719342 0416479 0.0004127 2.725266 0.373542 0.000206011 9i157 8556 coatomer protein complex, subunit alpha [Bos taurus] 20.75163 1.1022230.276264 0.315996319 0973912 0.060754 0.1121356 2.588773 0.378437 4.66E-15 gij61808545 PREDICTED:similar to histone H2A isoform 1 [Bos taurus] 62.30769 0.694453 0.450515 0003090092 2.676727 0.590959 0.0060942 3.409638 0.638057 0.003296838 gi(28461287 hepatoma-derivedgrowth factor (high-mobilitygroup protein 1-tike)[Bos taurus] 42.67783 1.064069 0.347332 0.637719162 3 2 1.207639 0.0110729 2.175438 1.000295 0.003571663 gil5106 44 eukaryotictranslation initiationfactor 5A [Bos taurus] 24.02597 0.993644 1.03353 0.990709602 2.223171 0.675319797 3.090223 0959097 0.0004906 2.787343 1.069869 0.001644201 gi|75832045 HLA-Bassociated transcript 1 [Bos taurus] 28.27103 1.04593 0.454598 0.455245 0.197909 0.0011572 0.504189 0.487159 0.028514686 gi|41386707 vascular celladhesion molecule 1 [Bos taurus] 20.43302 0.896205 0.529789 0.59039496 0.058640939 0.99664 0376304 0.8056481 0.955899 0.592788 0.860490913 gil119915070 PREDICTED:similar to CUB and EGFcontaining protein [Bos taurus] 10.2719 0.702824 0.509628 0963963 0.209996 0.478133 0.970308 0.191641 0.80011816 gil134085613alpha 1 type XVIIIcollagen [Bostaurus] 19.15456 1.108286 0.310159 03426475 0.439693459 2.414472 0.569202 0.007906 1.574095 0.628333 0.074235276 gi|115497294 canopy 2 homolog [Bos taurus] 33.51648 0.901592 0.387093 0147267129 2524294 0.693279 7.98E-05 2.336087 0.851779 0.000365813 gilo14053135proteasome (prosome, macropain) subunit, alpha type, 6 [Bostaurus] 31.30081 1.161236 0.344706 0.963199927 2.214517 052779 0.0033862 2.012049 0.722936 D.0625 gil119894807 PREDICTED:interleukin enhancer bindingfactor 3 isoform2 [Bos taurus] 17.3913 1.005649 0.464716 97 4.737242 0.6969 6.58E-08 3.947945 0.629687 8.06E-08 gil164518 8 diazepam bindinginhibitor [Bos taurus] 79.31035 1.461292 0.433004 0.000649227 1193621 0.197493 0.024503 0.951472 0.337637 0.724154961 gi|73853762 annexin I [Bos taurus] 36.12717 0.760038 0.297116 004109943 0.390959 0.3313349 0.684714 0.355503 2.22E-07 gill14051157 complement component 1, q subcomponent, B chain [Bos taurus] 22.67206 0.661922 0.425196 5 576-07 1013925 9 0.02120626 2.319909 0.44932 6.38E-08 2.025029 0.491916 1.06E-06 gi|780454 1 fascin homolog 1, actin-bundlingprotein [Bos taurus] 22.10953 1.222581 0.434414 3.344199 0.549291 0.0001651 3.197614 0.676538 0.000564204 gi|78045555 acidic (leucine-rich)nuclear phosphoprotein32 family,member B[Bos taurus] 34.09962 1.171976 0.326434 0.111557495 2 0.419721 0.4342584 1.326475 0.856312 0.275477997 gi|15742778 insulin-likegrowth factor binding protein 5 [Bos taurus] 49.07749 0.954689 0.404841 0.706974927 0.917127 1.504305 0.199503 8.6E-05 1.875692 0.823987 0.109375 gi]27807007 insulin-likegrowth factor binding protein 3 [Bos taurus] 35.05155 0.746612 0.194963 0.000490913 0.954642 0.354793 0.1841908 0.59448 0.86671 0.100457964 gi|129270171 gremlin-1[Bos taurus] 63.58696 0.752901 0.895 0.45527291 0.732573 0.274444 0.0418192 0.476848 0.369024 0.010811399 gi|119926480 PREDICTED:similar to Meteorin,glial cell differentiationregulator-like [Bos taurus] 22.3301 0.404822 0.399363 0.007304042 21.92116 1.009579 0.180837 0.900093969 3.14199 0.510159 0.0077449 2.457633 0.748651 0.092871086 gi78042524 thioredoxindomain containing4 (endoplasmic reticulum)[Bos taurus] 0277661 0.000986481 gi|77736203 malatedehydrogenase 1, NAD(soluble) [Bos taurus] 31.13773 1.3796260.184951 0.015124221 3.408354 0.185321 0.0003249 3.44296 gi27806415 scinderin[Bos taurus] 23.21678 0.885651 0.5675610.447981718 2.157923 0.404807 1.35E-05 2.696082 0.643891 2.12E-05 0.03125 gi56119114 purinenucleoside phosphorylase[Bos taurus] 19.72318 1.3324070.367264 0.125 2.345824 0.527841 0.0068653 2.89181 0.609906 gi]77736471 SH3domain binding glutamic acid-rich protein like 3 (Bostaurus] 53.76344 1.0250670.347011 0.845657197 2.129681 0.617309 0.0404264 1.770043 0442193 D.031622411 gi]45429977 staphylococcal nuclease andtudor domaincontaining 1 {Bostaurus] 27.58242 0.900734 0.198308 0.321008378 1.61418 0.719711 0.1593815 1.790095 0.859 0.138907486 gil28603774 RhoGDP dissociationinhibitor (GDI) alpha [Bos taurus] 45.58823 1.392308 0.437747 0.027607744 3.050149 0.610149 0.0004622 2.868464 06141 0.000564349 gil62460494 hemoglobin,gamma [Bos taurus] 69.65517 1.44846 0.234906 0.00699086 1.621779 1.029319 0.2152592 1.633981 0842019 0.064122109 gi|77735429 proteasomealpha 3 subunit{Bos taurus] 29.80392 0.682914 0.537326 0.084294821 1.290988 0.835646 0.4044338 1.204605 0977631 0.544925809 gi|62460582 complementcomponent 1. q subcomponent,A chain [Bos taurus] 48.77049 0.593049 0.500713 0.000548737 0.906695 0.377041 0.1851973 0.413569 0656242 0.000976563 gi|156523068ubiquitin-acvating enzyme E1 [Bostaurus] 15.02836 1.245575 0.282183 0.065427349 2.665289 0.731677 0.0130567 2.8885020447800 0.001499399 gi61888850 adenylatekinase 1 (Bostaurus] 40.72165 1.0459 0.218217 0.537002948 2.966651 0.609538 0.0047413 2.913725 071626 0.008160725 gi(119914274PREDICTED hypothetical protein isoform 1 [Bostaurus] 21.82857 1.221701 0.56517 0.467953282 3.302064 0.412142 0.0189709 3.197819 0422648 0.020047615 gi|27805999 matrixmetalloproteinase 13 [Bos taurus] 23.35456 0.380074 D.254298 0.001622704 0.51614 0.273737 0.0057 0.329036 0200248 0.0000591469 gi157074098 leprecan1 [Bos taurus] 17.25543 0.996475 0.287665 0.992062477 2.37895 0.319461 5.21E-05 2.130793 029614 6.02E-05 gi1156120501heat shock 70kD protein bindingprotein [Bos taurus] 21.68022 1.354057 0.380122 0.005344143 2.752202 0.538342 0.0019531 2.730784 0.044717 0.001953125 gi115497728 aconitase 1, soluble(Bos taurus] 14.28571 1.117697 0.491219 D.502761117 3.589575 0.37128 0.0003907 3.841086 0.308882 4.16E-05 gil119907132 PREDICTEDsimilar to Hyout protein[Bos taurus] 19.98002 1.106651 0.1329 0.113432742 3.22781 0.844503 0.0288445 3.030507 0.843197 0.031103752 gi77735539 procollagen-proline,2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase),alpha polypeptide il [Bos taurus] 27.72074 0.877814 0.101158 0.0343738 3.120213 0.498958 0.0074 2.781147 0.000376 0.012530222 gil119901144 PREDICTED:similar to malatedehydrogenase decarboxylase (NADP+) (Bos taurus] 25.42373 1.217784 0.38359 0.073445335 3.689365 0.478908 1.14E-05 3.455985 0.477728 1.36E-05 14.19878 0.900802 0.149951 0.089461939 0.956216 0.202782 0.4014582 0.952238 0.103006 0.503113248 gil1579544203 angiopoietin-like2 [Bos taurus] gi2780750 ribosomalprotein S27a [Bos taurus] 53.84616 1.229495 0.386822 0.005714112 2.449846 0.497627 1.31E-08 1.856561 0444849 0.000420791 gill 14052264 phosphoglucomutase 3 [Bos taurus] 19.18819 1.360266 0.280167 0.023770178 3.010397 0.521807 0.0025454 2.471896 0408662 0.002949993 gi|139948301 branchedchain aminotransterase1, cytosolic[Bos taurus] 20,20725 1.294087 0.252831 0.02996345 3.20688 0.406586 0.0625 2.984815 0479848 0.001737378 gi28603770 capping protein (actin filament)muscle Z-line, beta (Bnstaurus] 35.21595 1.082165 0.223792 0.314590585 2.006861 0.278274 0.0013815 2.073149 0.408887 0.004217119 gi]88853816 superoxide dismutase2, mitochondrial[Bos taurus] 34.23423 1.148276 0.284911 0.253928781 0.978402 0.619482 0.8774691 1.515731 0.010189 0.095180167 gi|119908402 PREDICTED:similar to Calsyntenin-1,partial [Bostaurus] 15.80611 0.876277 0.448436 0.540422033 0.929385 0.487573 0.6980291 0.684979 0.320106 0.104607426 gi|27806673 cathepsinL2 [Bos taurus] 30.83832 0.912492 0.447437 0.467843256 1.111225 0.269885 0.2441812 1.025236 0.440829 0.749919944 gi|119919488 PREDICTED:similar to cathepsin 0 isoform1 [Bos taurus] 28.8835 1.089541 0.614847 0.712006947 2.065618 0.225223 0.0027932 1.139716 0.280633 0.239601093 gil119894607 PREDICTED similarto peptidoglycanrecognition protein L (Bostaurus] 19.96616 3.268542 0.853115 0.000759791 0.712782 0.425171 0.0193949 0.672148 0.791011 0.099381319 gi]77404434 hypoxanthinephosphoribosyltransferase 1 [Bos taurus] 37.15596 1.17602 0.536588 0.290628484 2.803965 0.552609 0.0004158 2.658348 0.393801 7.26E-05 gil27806553 microtubule-associatedprotein 4 [Bos taurus] 23.13433 0.970701 0.110027 0.685964868 2.694891 0.528697 0.0438219 2.155718 0.753 0.121873938 gil27807445 peroxiredoxin5 precursor [Bos taurus] 29.22374 0.81151 0.749682 0.481703528 1.977168 1.606058 0.3165338 1.818467 1.701232 0.375548192 gil27806671 cathepsin B [Bos taurus] 17.61194 0.785806 0.30958 0.006301553 1.755499 0.247458 9.24E-07 0.543177 0.640833 0.002266239 gi|27807523 ribosomal protein P2 [Bos taurus] 41.73913 1.274264 0.549719 0.074405983 4.509896 0.602457 1.28E-06 4264045 0.032238 5.25E-07 gi|76610312 PREDICTED:similar to neuropilin 2 isoform 8 [Bos taurus] 19.87041 1.110245 0.176154 0.125980369 0.890141 0.292637 0.2235103 0.987084 0.162306 0.984072538 gil27806637 fibrillin1 (Bos taurus] 10.37966 0.906627 0.530205 0.478444551 0.970058 0.35082 0.6201094 0.935079 0.200966 0.425633123 gir27807209 alpha-2-plasmininhibitor [Bos taurus] 22.56097 0.599339 0.62022 0.056459368 1.117795 0.400638 0.4776424 1.292816 0.418288 0.105518239 gi}83715992 coatomer proteincomplex, subunit gamma 1 [Bostaurus] 17.04805 0.974997 0.137881 0.699598479 2.415233 0.419959 0.0026333 3.058982 0.319684 0.000334903 gi|27806139 secreted frizzled-relatedprotein 1 [Bos taurus) 31.81818 0.626649 0.33961 1.84E-07 1.024216 0.341271 0.8632156 1.230284 0.827382 0.162334404 gil119919689 PREDICTED:similar to RNA bindingmotif protein, X-linkedisoform 5 [Bos taurus] 41.68798 0.839077 0365111 0.197549662 2.282233 0.454349 0.0045123 2.470278 0.488788 0.004058435 gill 15497086 cortactin (Bos taurus] 19.88848 0.920482 0.522844 0.570787529 1.698239 0.686754 0.0286247 1.550913 008181 0.02492977 gi|134085635glyoxalase I [Bos taurus] 41.30435 1.103425 0.160067 0.116314216 4.245659 0.469967 0.0006011 3.497581 0.0319 0.001311643 gil119890932 PREDICTEDsimilar to Lamininbeta-1 chain precursor (LamininB1 chain) [Bostaurus] 16.42772 0.992998 0.340681 0.942123233 1.07211 0.349671 0.7421875 0.90306 0.378389 0.37475027 gil56710317 keratin 5 (epidermolysisbullosa simplex,Dowling-Meara/Kobner/Weber-Cockayne types) (Bos taurus] 23.96007 0.794447 0.476544 0.14970632 0.626444 0.281415 0.0017773 0.73261 0.482631 0.081605855 gi|76620378 PREDICTED:similar to cytokine-likeprotein C17 [Bostaurus] 39.41606 0.824377 0.407782 0.008142552 0.920387 0.454298 0.1532154 0.609768 0.567618 6.98E-05 gi|155372035nidogen 1 [Bostaurus] 12.65109 0.823522 0.296224 0.069126059 1.038216 0.202334 0.4375 0.723608 0.233962 0.005463519 gi|75832090 isocitrate dehydrogenase 1 (NADP+),soluble [Bos taurus] 28.26087 1.445789 0.458957 0.102862623 2.514228 0.577943 0.0201016 2.818763 0.008003 0.009451117 gi|27806447 prosaposin [Bos taurus] 15.42857 0.802455 0.309292 0.218395134 1.249937 0.244271 0.1660525 D.325786 1.488483 0.204295759 gi|78369645 SH3 domain binding glutamicacid-rich protein like[Bos taurus] 44.73684 1.253298 0.301906 0.072725986 3.625316 0.623463 0.0007949 3.338367 0.702696 0.002273803 gi|115494984 apolipoprotein0 [Bos taurus] 48.14815 0.495177 0.226909 8.27E-09 1.087048 0.277356 0.2377548 0.411653 0.378272 1.64E-07 gi|119922764 PREDICTED: similar to MSTP006 [Bos taurus] 19.82841 1.663807 0.752503 0.232522007 3.749804 0.937328 0.0733604 3.638376 0.221916 0.00459497 gil153791208immunoglobulin superfamily containing leucine-richrepeat [Bos taurus] 16.35514 0.802223 0.41405 0.296875 0.862362 0.499196 0.2610433 0.977635 0.618003 0.959597689 gil28461189 lectin, galactoside-binding,soluble, 1 (galectin 1)[Bostaurus] 35.55556 1.610276 0.374908 0.006413745 2.624683 0.359533 0.0002363 2.579216 0.603623 0.002451033 gi]29135329 glutathione S-transferase pi [Bs taurus] 32.38095 1.122915 0.244218 0.118041828 2.950799 0.292574 1.47E-06 3.035265 0.277031 7.23E-07 gi|115497078 splicing factor, arginine/serine-rich1 (splicing factor 2, alternate splicing factor) [Bostaurus] 50 1.234643 0.305858 0.058368077 2.890116 0.370763 0.0001726 2.536704 0.49682 0.001104549 gi|119924055 PREDICTED:similar to p167 isoform4 {Bostaurus] 23.32849 1.314471 0.187168 0.067196595 2.246554 0.525588 0.0633805 1.833652 0.80870 0.212801969 gi|119879765 PREDICTEDsimilar to eukaryotictranslation initiation factor 4 gamma, 1 [Bos taurus] 22.98739 0.852552 0.354759 0.287069522 1.537511 0.545863 0.1152917 1.263803 1.216474 D.597450382 gi|116004039 peptidylprolylisomerase C (cyclophilinC) [Bostaurus] 38.20755 0.684331 0.373857 2.47E-09 1.062364 0.299366 0.2160807 0.916782 0.310311 0.064609318 gil27806783 thioredoxin[Bos taurus] 51.42857 1.40006 0.110957 5.D9E-06 2.949452 0.495424 4.97E-05 2.918305 0390989 9.37E-06 gi]78369458 proteasome (prosome, macropain) subunit, alpha type, 1 {Bostaurus] 38.78327 1.12676 0.505423 0.399398202 2.98806 0.524133 0.0002231 2.769028 0031731 0.000309952 gil119908208 PREDICTED:similar to Calpain 2, (m/l1)large subunit isoform 3 [Bos taurus] 16.09589 1.294738 0.547471 0.511199959 2.865098 0.986682 0.1192677 2.405706 0.79040 0.106135047 gi|77736071 heterogeneous nuclearribonucleoprotein K (Bostaurus] 28.23276 1.162442 0.27411 0.209559792 2.827074 0.658521 0.007377 2.580231 0407060 0.002457434 gi|84000347 interleukinenhancer bindingfactor 2 [Bos taurus] 28.71795 118807 0.413414 0.248192181 2.591724 0.911163 0.0293373 2.416142 0.893766 0.031827392 gi|41386760 CD14 antigen [Bos taurus] 29.49062 D.718284 0.426758 0.067077633 0.447034 0.82842 0.0332025 0.487415 0.716581 0.033797768 gi|115495555 endoplasmicreticulum protein 29 [Bos taurus] 32.17054 1.063647 0.500749 0.650900277 3.895887 0.816752 0.0002715 3.665525 0.67863 0.000492021 gi|78369232 phosphorylase,glycogen, brain [Bos taurus] 1957295 1.124417 0.46718 0.592429287 2.372325 0.962257 0.1580827 1.868395 0.848406 0.200478112 gi|27806081 peroxiredoxin1 [Bostaurus] 3969849 1.013692 0.364535 0.894837687 2.996943 0.608624 0.0014924 3.170503 0.670037 0.001640537 gi|77736371 actin related protein203 complex subunit 2 [Bos taurus] 24 0.846992 0.409937 0.419687408 1.586726 0.414133 0.114149 1.658406 0.7137240.210314559 gi|41386798 poly(A)binding protein, cytoplasmic1 [Bos taurus] 22.64151 1.04511 0.544222 0.80341858 2.595454 0.010263 0.0076688 2.809743 0.724413 0.012307 gi}61888866 tropomyosin1 alpha chain [Bos taurus] 49.29577 1.269786 0.233070 0.123991012 2.19207 0.041845 0.0004718 2.20171 0.611100 0.081511689 gi|78042498 phosphoglyceratedehydrogenase [Bos taurus] 17.82364 0.927041 0.397486 0.623947485 2.364541 0.493862 0.0052285 2.892111 0013386 0.004805503 gil27806751 alpha-2-HS-glycoprotein[Bos taurus] 21.72702 0.322124 0.472372 0.006198951 0.51348 0.379411 0.0139899 0311114 0940334 0.03899761 gir77736343 ErbB3-bindingprotein 1 [Bostaurus] 15.98985 1.0047090.064191 0.890964497 2.208606 0.707947 0.267018 1.713963 0.8866600.424095043 gi|27807377 glutamic-oxaloacetctransaminase 2, mitochondrial(aspartate aminotransferase 2) [Bos taurus] 26.97674 1.118437 0.227507 0.341451802 2.947352 0.228243 0.0072522 2.502367 0.367001 0.02398561 gi]62177164 tumor protein, translationally-controlled1 [Bos taurus] 3662791 1.140074 0.510119 0361989591 2.504788 0.731534 0.0032679 2.915557 07084 0.00158855 gij41386729 high-mobilitygroup box 1 [Bostaurus] 29.30233 0.975179 0.471119 0.875466602 3.528455 0.59746 0.0625 4.561559 0.60021 0.0625 gi(164420709retinol binding protein 4, plasma [Bostaurus] 33.33333 0.420745 0.338166 0.023614332 0.636046 0.096835 0.0068255 0.244006 0.090417 0.027453193 gi]27806487 serine (or cysteine) proteinase inhibitor,clade F (alpha-2 antiplasmin, pigment epitheliumderived factor), member 1(Bos taurus] 30.04808 0.687616 0.308951 0.039742456 1.631605 0.34124 0.0276504 1.053292 0.297749 0.567994518 gi|119924945PREDICTED: similar to agrin [Bos taurus] 12.58035 0.882582 0.100124 0.037338157 D.694748 0.256876 0.0238678 0.660421 0.106879 0.001688625 gi]82524405 US01 homolog,vesicle docking protein[Bos taurus] 14.77627 0818211 0.58442 0.612123221 1.900213 0.742402 0.3344105 1.486134 0.797999 0.484810234 52.20588 0.753026 0.441014 0.072713355 1.281729 0.001102 0.2272909 2.63243 0.898001 0.004206016 gi|76651183 PREDICTEDsimilar to histone H3 [Bos taurus) 0.0013022 0.83957 0.22989 gi|27806881 epididymalsecretory proteinE1 [Bos taurus] 30.87248 0.356994 0.193381 6.75E-05 2.180407 0.310342 0.086816037 0.922926019 0.828111 0.340358 0.184089 0.643004 0.8993780.263761812 gi|119912531 PREDICTED:similar to mannose receptor,C type 2 {Bostaurus] 14.20839 1.017853 0.309243 0.318717364 1.176168 0.550588 0.3855219 0.864672 0403676 0.4375 gi|114051730 cell growthregulator with EF-hand domain 1 [Bos taurus] 37.40458 1.220882 0.639517 2.413955 0.230209 0.001667 2.38417 0.368222 0.006189962 gi|77735425 proteasome alpha 7 subunit [Bos taurus] 39.51613 1.034125 0.284267 0.750738059 2.958899 0.768915 0.0078125 1.918399 1011161 0.031271826 gi|119905546 PREDICTED:similar to Cu/Zn superoxide dismutase [Bos taurus] 39.14894 1.184763 0.734416 0.1484375 gil27807147 C-type lectindomain family3 member A [Bostaurus] 26.39594 0.483096 0.281244 0.004993909 0.597662 0.235224 0.0074339 0.660605 0.314855 0.034987006 gil119892706 PREDICTEDsimilar to chondroitn 4-0-sulfotransferase [Bos taurus] 30.04622 0.885377 0.267538 0.218332299 0.79729 0.238971 0.0625 0.776221 0.380156 0.112181895 gil119906216 PREDICTED:similar to Eukaryotic translation elongationfactor 1 delta (guanine nucleotide exchange protein) isoform 1 [Bos taurus] 18.8383 0.803381 0.427767 0.330534308 1.414743 0.412788 0.1815515 1.70436 0.454989 0.095161529 0.357271 gi|148232266 fibulin1 [Bos taurus] 14.58924 0.972503 0.354609 0.817151886 1.165717 0.360374 0.280848 1.207589 0.141190722 gi|166706929 spectrin, alpha, non-erythrocytic1 (alpha-fodrin)[Bos taurus] 23.94822 0.939537 0.537312 0.801387909 1.476593 0.107719 0.0129203 1.626208 0.983398 0.33146398 gil1 15497888 nuclear distributiongene C homolog [Bos taurus] 24.6988 1.61261 0.33356 0.069751071 2.734684 0.214804 0.007428 3.269898 0.363047 0.014371437 gi]62460514 angiopoietin-like7 [Bos taurus] 16.56977 1.117718 0.442446 0.69642184 0.417986 0.0939 0.0330213 0.153246 1.0067D6 0.164939633 3.798151 0468633 0.00074897 gi]84370163 SET translocation (myeloidleukemia-associated) [Bos taurus] 13.71841 1.242814 0.444574 0.251830874 3.8849 0.7417 0.0042834 gi|62751847 ribosomal protein LlOa [Bos taurus] 42.39631 1.0630420.213483 0.352668292 3.533629 0.263291 2.46E-07 3.520818 0.237968 1.07E-07 gi|119919151PREDICTED similarto AHNAKnucleoprotein isoform 1 [Bostaurus] 38.43826 1.183134 0.141004 0.247244968 3.471307 0.304387 0.0768193 2.609466 0275736 0.087842949 gil14052350 WD repeat domain 1 [Bos taurus] 14.35644 0.775361 0.032555 0.00262712 2.19931 0.270689 0.003729 2657109 0427772 0.006822752 gi|125630701collagen triplehelix repeat containing 1(Bos taurus] 22.63374 0.668077 0.351419 0.004703672 0.813613 0.270199 0.0207383 0.721987 0409756 0.041342839 gi|76613946 PREDICTED:similar to Histone H1.5[Bostaurus] 54.42478 0.830506 0.365389 0.33330418 1.270872 0.533668 0.4016913 4.620782 0450048 0.013324411 gill 19904280 PREDICTED:similar to collagen type 5 alpha 1 [Bos taurus] 24.63235 0.976096 0.373942 0.7421875 0.939595 0.336983 0.3852008 0.954205 0.272693 0.616667519 gill 16003917 far upstream element-bindingprotein [Bos taurus] 20.52877 D.903883 0.95509 0.817537461 1.770837 0.953737 0.2817003 1.737182 0.991753 0.289734467 gil148228060 stem cell growthfactor [Bos taurus] 28.7037 0.612661 0.22486 0.015625 0.920192 0.111661 0.046875 0.762631 0.273554 0.011575853 gil28461187 myosin, lightchain 6, alkali, smooth muscle and non-muscle [Bos taurus] 33.77483 1.249014 0.265085 0.093465616 3.086608 0.230374 00007995 2.771789 0.180657 0.00048544 gil134085825 carboxypeptidase Z[Bos taurus] 13.35404 0.58929 0.278876 0.041876022 1.020271 0.15032 0.9308931 0.98617 0.253463 0.999005344 10.46622 -0.99926 1 -1 2.150729 0.20013 0.0826136 2.183436 0.366483 0.141469321 gil1198935367 3 PREDICTED:similar to KIAA0755protein [Bos taurus] giJ773582 ribosomal protein S20 [Bos taurus] 31.93277 0.795183 0.275014 0.096156675 1.667681 0.361371 0.0285578 1.855457 0.333426 0.012005374 gil1 19920777 PREDICTED:similar to collagen,type IX, alpha 2 [Bos taurus] 39.27227 0.779581 0.414449 0.0078125 0.917289 0.262812 0.1626 0.754799 0.336487 0.014238535 gi|157785567calnexin [Bostaurus] 12.98482 0.865918 0.226559 0.165656654 2.926706 0.8834 0.0406038 2.265914 0.661015 0.035943935 gi|125991882phospholipase C, delta 1 [Bostaurus] 17.32804 0.849466 0.162996 0.291038812 1.955129 0.245251 0.0216417 1.547054 0.383386 0.099083505 gill 14326274 crystallin, zeta (quinone reductase) [Bos taurus] 26.66667 1.236331 0.482611 D.292766628 2.943605 0.583895 0.0133519 2.743991 0.591988 0.015489679 gi77735583 S-adenosylhomocysteine hydrolase [Bostaurus] 29.86111 0.875234 0.171927 0.112621037 2.778236 0.556254 0.0136238 2.111045 0.59152 0.033963277 gil119923009PREDICTED similar to put. HMG-17protein [Bostaurus] 66.66667 0.833021 0.303443 0.182038529 3.169982 0.864344 0.0319157 3.391513 1.0386290.041581134 0.278015 gi|119907521PREDICTED: similar to NuMAprotein [Bostaurus] 20.08487 0.739806 0.47084 0.416795077 1.398908 0.748488 0.3954241 1.169949 0.264062386 0.225493 gil158937293fibroblast growth factor receptor 1 [Bos taurus] 12.31707 1.26736 0.31132 0.026835321 0.6248 0.249036 0.0003068 0.983398 0.962779865 0.675848 gil114052384heterogeneous nuclear ribonucleoproteinAl [Bostaurus] 45.9375 1.06482 0.652055 0.76892942 1.86465 0.820084 0.0747428 1.983742 0.028937035 gi]77736141 extracellular matrixprotein 2, female organ and adipocyte specific [Bos taurus] 20.58824 0.874987 0.217415 0.266463338 1.600423 0.362142 0.0878642 1.15332 0.34888 0.378310561 gi|27807389 bucentaur [Bos taurus] 18.24324 1.310154 0.236449 0.257355307 1.678449 0.069102 0.0030006 2.018276 0.348796 0.036133204 2.973906 0.56251 0.003186068 gil27807481 ubiquitin-likeprotein SMT3B[Bos taurus] 47.36842 1.26D646 0.3885340.125911276 3047138 0.350288 0.0005344 37.62376 1.433824 0.428222 0093076211 2.753903 0.491145 0.0098709 3.182135 0.573603 0.009751096 gir27807075226 92 55 9 S100 calcium-bindingprotein A4 [Bostaurus] gill oncostatin Mreceptor [Bos taurus] 18.82716 1.216908 0.41015 0.116358817 0.710875 0.427083 0.0191566 0.713548 0.247116 0.002462396 gi]91680535 proteasome activatorsubunit 1 [Bostaurus] 25.70281 1.269151 0.308154 0.192091906 2.101795 0.621285 0.0993864 2.040854 0.920073 0.186858663 gil76633778 PREDICTED:similar to syndecan 4 [Bostaurus] 19.59799 0.534016 0.478089 0.032408073 0.415467 0.598041 0.0230515 0.435597 0.465745 0.014886286 0.039043115 gi]45430017 apolipoproteinA- bindingprotein [Bos taurus] 18.75 1.454892 0.430942 0.161253804 2.919583 0.152417 0.0033118 2.524393 0.47828 gi|164448628carboxypeptidase X [Bos taurus] 15.47945 0.606999 0.088434 0.00050569 1.335219 0.315531 0.0866305 0.558246 0.492702 0.04466754 gi|115496724 heat shock protein, alpha-crystallin-related,B6 [Bos taurus] 21.34146 1.035913 0.613646 0.896815108 2.426789 1.124426 0.1921033 2.486681 1.12189 0.175536569 gil27807275 matrixGta protein[Bos taurus] 24.27184 0.913055 0.251079 0.074444217 0.785242 0.412548 0.005232 0.651858 0.573383 0.00012207 gi|155372143 arginine-rich.mutated in early stage tumors [Bos taurus] 23.46369 0.946534 0.167793 0.356197163 3.697732 0.617168 0.0024956 3.852789 0701618 0.00330936 gi|164420791transforming growthfactor, beta 2 [Bos taurus] 26.08696 0.802539 0.131196 0.016999756 1.643629 0.367663 0.0322574 2.280779 0.323806 0.004963112 gil28603812 epsilon subunit of coatomer protein complex [Bostaurus] 35.06494 1.116745 0.06763 0.184486441 2.278645 0.134782 0.0518435 1.701141 0378442 0.208708142 gi|119917542 PREDICTED:similar to tolloid-like2 proteinisoform 2 [Bos taurus] 1803922 0.465451 0.239334 0.015355155 0.770373 0.053673 0.006043 0.813512 0.133918 0.069635347 gi|27806419 seryl-tRNAsynthetase [Bos taurus] 18.67704 1.579072 0.659546 0.1390788 4.16785 0.93634 0.0223702 3.146452 0.975216 0.041443138 gi|78042520 eukaryotictranslation initiation factor 48 [Bos taurus] 25.40984 1.490223 0.17479 0.029291852 5.005955 0.732354 0.0320889 4.290955 0.754442 0.039660058 gi|61097917 cystatin E/M [Bos taurus] 51.00671 1.57135 1.391916 0.417271146 3.610196 0.825578 0.0099289 1.308161 0.734717 0.34790038 gil27806743 alpha-1-microglobuhn/bikunin[Bos taurus] 13.06818 1.087494 0.33666 0.462935933 1.002925 0.259727 0.8955853 0.827057 0.227758 0.087467767 gill 14051347 cytidinemonophosphate (UMP-CMP)kinase 1, cytosolic[Bos taurus] 26.31579 1.139562 0.222354 0.18674671 2.471173 0.39826 0.0075525 2.346831 0.40005 0.008248 gil27806279 caldesmon 1 [Bos taurus] 24.85981 1.420384 0.346329 0.126510497 2.308451 0.428326 0.0408018 2.519663 0.386029 0.026058352 gi|29135295 aspartate aminotransferase 1 [Bos taurus] 26.87651 1.147065 0.296698 0.516777071 2.780204 0.085909 0.0265684 2.384269 0.018312 0006471147 gi|155371881 alanyt-tRNAsynthetase [Bos taurus] 22.52066 0.958442 0.235416 0.701296239 3.613826 0.280184 0.0076978 3.364019 0202327 0.004324917 gill 19908206 PREDICTED:hypothetical protein [Bos taurus] 16.47727 1.231063 0.120557 0.049539842 2.732936 0.643642 0.0613781 2.462334 0822684 0.108715161 gi|115496906 acidic ([eucine-rich)nuclear phosphoprotein 32 family,member E [Bos taurus] 11.78707 0.672156 0.366952 0.271090308 2.641361 0.649759 0.066527 2.137621 0.529138 0.067409869 gill 19901449 PREDICTED:similar to PDNP1 [Bos taurus] 13.84462 1.300379 0.384962 0.060269605 2.584342 0.728107 0.0061718 2.987183 0951432 0.009201833 21.80665 1.303009 0.469639 0.201449286 2.485554 1.304786 0.1417659 2.090313 1.226407 0.175255314 gil114051291 6 4 tissue specifictransplantation antigen P35B [Bos taurus] gi|780425 CNDP dipeptidase 2 [Bos taurus] 16.84211 0.74381 0.091638 0.096115311 1.5355320.276254 0.0642906 1.152352 0.232898 0.236409376 gil116004251 granulin [Bostaurus] 14.65077 0.808832 0.513145 0.320042318 1.213373 0.46849 0.3502469 0.588216 0459439 0.047694216 gil76614948 PREDICTED similarto pepbdyl-Pro cis trans isomerase isoform 1 [Bos taurus] 37.19512 0.497807 0.459888 0.022140592 1.146593 0.566521 0.5234901 3.541769 0.290595 4.95E-07 gi]77736257 protein phosphatase 1, catalytic subunit, beta isoform [Bostaurus] 30.88685 1.007481 0.422626 0.965747982 3.146577 0.696114 0.0555497 2.366375 0.76722 0.104143124 gi|l 19909677 PREDICTED:similar to MGC84382protein [Bos taurus] 30.8 0.795847 0.531392 0.396532407 1.931222 0.562766 0.1036383 1.717631 0.502826 0.128879012 gi|164448698 chaperonin containingTCP1, subunit 3 [Bos taurus] 21.65138 0.826817 0.391769 0.350435816 1.537338 0.629118 0,2403691 1.614271 0.320710 0.061593675 gi|165905571 leucine aminopeptidase 3 [Bos taurus] 36.41618 1.197426 0.605365 0.533044454 2.139903 0.580549 0.0847148 1.724988 0.6974980.183003339 gi|156120555 glyoxalase domain containing 4 [Bos taurus] 22.81879 1.175848 0.129091 0.087720645 3.282099 0.12729 0.0018764 3.123256 0.180034 0.00388226 gi|123858766 prefoldinsubunit 2 [Bos taurus] 27.92208 1.080303 0.153165 0.331178713 2.247358 0.716627 0.109088 2.620447 0622145 0.059184844 18.4669 0.939144 0.602384 0.820956401 1.255821 0.354889 0.2714627 1.839502 0.426399 0.06740268 gij990289637 9 5' nucleotidase,ecto [Bos taurus] gil77735 6 complement component 1, r subcomponent [Bos taurus] 13.19797 0.371729 0.255753 0.010543925 0.445584 0.224777 0.0117492 0.307881 0407087 0023818009 gil148233312 dermatan 4 sulfotransferase 1 [Bostaurus] 30.05319 0.643613 0.144348 0.101515473 0.693874 0.487356 0.3575317 0.805619 0287647 0.387807689 0.974748 giJ77736509 S-phase kinase-associated protein 1 [Bos taurus] 22.699390.669089 0.127617 0.098484838 1.6058430.831632 0.3014508 1.694182 0.299648314 gil115495701 pentraxin 3 [Bos taurus] 21.98953 1.513781 0.30821 0.078004648 1.861369 0.236121 0.0233769 1.546302 0.1621990.020184636 gi|116812902 hemoglobin alpha chain [Bos taurus] 39.43662 1.943351 0.46772 0.010124121 1.317848 0.288852 0.0430657 1.21806 0.282345 0.073192769 gil156120327thrombospondin 3 [Bos taurus 12.76151 0.748922 0.148359 0.01120273 0.935365 0.315655 0.5127564 0.869454 0.310559 0.326842981 gi|27806825 peptidylglycinealpha-amidating monooxygenase [Bos taurus] 13.06584 0.685843 0.618738 0.267882545 1.056921 0.400266 0.8211274 0.710438 0.250022 0.08188384 gi|154152039scrapie responsive protein 1 [Bos taurus] 50 0.985846 0.423782 0.943757758 0.439114 0.347235 0.0264629 0.146196 0.83028 0.017017848 gil119904972 PREDICTED:similar to Collagenalpha-2(iv) chain [Bostaurus] 17.6681 0.923236 0.251127 0.636275225 1.109448 0.339989 0.6856706 0.838427 0.54257 0.676592696 gi|77735597 FK506-bindingprotein 4 [Bos taurus] 19.17211 -0.99926 1 -1 1.461249 0.842797 0.5391877 1.978104 1.585442 0.53689562 18.70324 0.983074 0.527972721 2.405346 0.348523 0.0056359 2.328281 0.27950 0.003021512 gil 19914266 PREDICTED:similar to cartilage-associated protein(CASP) [Bos taurus] 0.053878 01103968 2543983 0.145043 g0122692537 damage-specific DNAbinding protein 1, 127kDa [Bos taurus] 19.38597 0988155 0.074826 0.811770675 2.488301 0.320618 0.047660548 0.03822 0.199785382 gi|115497736 olfactomedin-like3 [Bos taurus] 16.25616 0.557809 0.109217 0.05829103 1.515813 0.0296611 0.729204 0.199797 0.005659271 gi|119888815 PREDICTED:similar to GALEprotein isoform2 [Bos taurus] 20.64394 0.986212 0.602951 0.967447781 3.903813 0.381253 0.0125738 2.981569 0.208893 9184370199 6-phosphogluconolactonase [Bostaurus] 32.55814 1.08729 0.010834 0.039985804 1.630192 0.384234 0.2405187 1.707107 0.290343 0.161500826 gi82697343 cystatin B (stefin B) [Bos taurus] 51.02041 1.190769 0.389515 0.172527176 2.16493 0.309373 8.46E-05 1.8108 0.315601 0.000324255 0.0625 gi|29135293 farnesyl diphosphatesynthase [Bos taurus] 16.71388 0.97314 0.518484 0.877111348 2.074039 0.496513 0.0625 1.993744 0.479779 0.01096698 gi|77736061 NSFLI (p97)cofactor (p47)[Bos taurus] 29.18919 1.296683 0.801161 0.417270753 2.111991 1.432306 0.236042 3.13167 0.59002 gi[115495441hexosaminidase A[Bostaurus 15.12287 0.999343 0.237049 0.999520984 1.681304 0.472008 0.2732886 1.00883 0.481833 0.938567978 gi[115497820parvalbumin [Bos taurus] 40.90909 0.99826 0.482785 0.995001014 2.075344 0.571627 0.0625 2.21725 0.735683 0.023727802 gil119915943 PREDICTED:similar to serine (or cysteine) proteinaseinhibitor, clade B (ovalbumin),member 1 isoform 1 [Bostaurus] 21.17647 1.113376 0.319955 0.401371937 2.752236 0.26127 0.0015985 2.825008 0.253982 0.00125798 gil149642605 drebrin-like[Bos taurus] 23.64066 1.187723 0.140487 0.241424229 3.737837 0.158998 00379909 3.369437 0.205367 0.052031586 gil119925682 PREDICTEDsimilar to adlican [Bos taurus] 13.23736 1.086712 1.000353 0.89210617 0.928769 0.237273 0.5873691 0.876498 0.048336 0.126394813 gill 54152127 olfactomedin-tike21 [Bos taurus] 11.46245 0.814567 0.347086 0.441956589 0.920045 0.215988 0.5261371 0.596049 0.438993 0.257159665 gi83035095 high-mobilitygroup box 2 [Bos taurus] 29.1866 0.905687 0.5384 0692930167 1.715068 0.347504 0.0634901 2.901278 0.334401 0.015030236 gi|27806555 legumain [Bostaurus] 24.94226 1.28063 0.311188 0.350910137 1.305949 0.188029 0.2225851 0.989315 0.947051 0.993323057 gi|122692375 hypotheticalprotein LOC783871[Bos taurus] 35.46512 0.421234 0.665214 0.229761274 1.151609 0.049037 0.119084 1.221621 0.251098 0.331938612 gi|27805927 heat shock lOkDaprotein 1 [Bos taurus] 42.15686 1.352487 0.680836 0.289690003 3.832442 0.82003 0.0184602 3.778411 0.686969 0.01101608 gi|119901068 PREDICTED:similar to Collagen, type IX,alpha 1. partial [Bos taurus] 37.22628 0.589893 0.184696 0.003749994 0.787361 0.425489 0.1844166 0.624327 0.487785 0.073576372 gi|27805981 ribosomal protein SA [Bostaurus] 28.81356 0.874403 0.276509 0.351077261 3.869427 0.407906 0.01454053.470368 0.498863 0.024258935 gi77735725 cathepsin H [Bos taurus] 14.32836 0.753775 0.331318 0.091258679 2.119895 0.421949 0.0150507 1.40763 0.189489 0.012317972 gi|84000203 esophageal cancer related gene 4 protein [Bos taurus] 17.68707 0.905053 0.397999 0.312978304 2.037898 0.923839 0.0113757 1.524122 1.032493 0.10535932 gil1 19895711 PREDICTEDsimilar to cytokine-likeprotein ZCYTD7 isoform 1 [Bos taurus] 21.88841 0.636762 0.502956 0.154553615 0.932442 0.397235 0.649541111.128668 0.230758 0.28099731 gil119914936 PREDICTEDsimilar to histone Hlx [Bostaurus] 43.45794 0.922147 0.373228 0.644629952 1.853161 0.283439 0.0336409 2.989851 0.319115 0.012995765 gil27806677 connective tissue growthfactor [Bos taurus] 12.03438 0.504088 0.520528 0.0321576 0.747283 0.5165610. 1864789 0.13033 0.991603 0.0097754 gil119901084 PREDICTED:similar to CD109 [Bos taurus] 26.02041 0.80244 0.306074 0.03393584 0.742776 0.361113 0.0165521 0.805539 0.294339 0.039489852 gi|119914223 PREDICTED:similar to KIAA2019protein [Bos taurus] 28.78556 1.307125 0.774543 0.608019143 3.38677 1.199377 0.2887992 2.90209 0.92197 0.252382552 gi|115497124 asparagine synthetase [Bos taurus] 16.22103 0.730472 0.983032 0.632936793 1.49994 0.490593 0.3508227 1.346408 0.543093 0.450155858 gi|77735669 lysyl-tRNAsynthetase [Bos taurus] 19.10112 1.413725 0.478522 0.378409805 3.158358 0.731462 0.1946331 2.786114 0.882606 0.251223404 gil119907290 PREDICTED:hypothetical protein [Bos taurus] 16.90722 1.546651 0.400086 0.268612245 0.261942 0.578954 0.1315333 0.111001 1.49149 0.205612544 gil119896208 PREDICTED:similar to PRDX3 protein [Bos taurus] 38.7234 1.626509 0.450798 0.27106167 4.012125 0.423475 0.0148501 4.140285 0756945 0.041796827 gil119919315 PREDICTED:similar to latent transforming growthfactor beta binding protein3 [Bos taurus] 15.59829 0.624843 0.001418 0.000942204 1.436095 0.219236 0.1907879 0.819444 0.760568 0.70648233 gi[78045531 aspartyl-tRNAsynthetase [Bos taurus] 18.16367 1.29131 0.109172 0.130976267 1.720934 0.282647 0.1631461 1.881404 0.139305 0.067028078 gi|114051185programmed cell death 5 [Bos taurus] 48 1.10207 0.263788 0.587298948 1.787634 0.644411 0.3236882 1.535001 0.633431 0.389420646 gij149773586lectin, mannose-binding, 1 [Bos taurus] 12.35521 1.530055 0.083158 0.60717466 1.704893 0.262832 0.0386577 1.71455 0.266353 0.03517643 gi]77736560 stathmin l/oncoprotein 18 [Bos taurus] 57.64698 1.413739 0.619969 0.296846802 1.2346790.367858 0.313355 1.667756 0.552267 0.140513744 gi[77736489 high-mobilitygroup nucleosome binding domain 1 [Bos taurus] 33.66337 0.749563 0.504437 0.198595925 2.461898 0.432901 0.0096732 3.105833 0.402257 0.003755483 gi|84000165 complementfactor I[8os taurus] 12.13592 1.154973 0.2097890.393053895 0.893997 0.199503 0.4195872 0.9416720.169139 0.677325398 gil119919951 PREDICTED:similar to glucose-6-phosphate dehydrogenase G6PD [Bostaurus] 15.13513 1.008522 0.522136 0.977048396 2.595589 0.545915 0.1766078 2.907788 0.443163 0.126183586 gil27805805 fatty acid bindingprotein 5 [Bostaurus] 17.03764 1.228877 0.078394 0.11692543 4.054718 0.075811 0.0170667 2.344424 0.482357 0.169642131 gi[77735927 3-hydroxybutyratedehydrogenase, type 2 [Bos taurus] 17.55102 1.032032 0.198887 0.67154207 2.979959 0.207751 0.0006472 1.967751 0.358372 0.011459212 gi1149642873hyaluronan and proteoglycanlink protein 3 [Bos taurus] 33.33333 0.975716 0.37877 0.649016468 0.921851 0.139075 0.084606 0.888852 0.190702 0.154086587 gil115432055 tyrosine 3-monooxygenaseltryptophan5-monooxygenase activation protein, beta polypeptide [Bos taurus] 45.93496 1.067162 0.281426 0.617276161 2.581333 0.370805 0.0243184 2.088619 0.081218 0.00186993 gil119906919 PREDICTED:similar to ribosomal protein L6-tike[Bos taurus] 27.52613 0.758 0.14045 0. 155436407 2.262101 0.56771 0.2125714 1.437656 0.813034 0.517568894 gi|155371891ADAM metallopeptidase with thrombospondin type I motif,1 [Bos taurus] 15.6701 1.07832 0.340438 0.510480475 1.607625 0.208313 0.002043 1.636837 0.455804 0.023020513 gi|76642911 PREDICTED:similar to IL-104nduciblechemokine [Bos taurus] 40 0.774014 0.398277 0.161353081 0.983356 0.298251 0.7840996 0.94777 0.377036 0.781783441 gill 15496496 matrix-remodellingassociated 8 [Bos taurus] 21.81426 0.609769 0.30D607 0.184549596 0.671404 0.651627 0.4193901 0.472828 0.554075 0.225327496 gil27806001 matrixmetalloproteinase 14 [Bos taurus] 11.85567 0.908796 0.955072 0.87267598 1.541102 0.826353 0.4897151 1.205964 1.212126 0.791974892 gil114052322 alpha-N-acetytgalactosaminidase[Bos taurus] 16.54501 0.831156 0.063374 0.018435872 1.521106 0.031548 0.0009691 0.887839 0.160644 0.244335628 gil157279907 parathymosin [Bostaurus] 33.33333 1.188807 0.59957 0.311175001 2.415955 0.851656 0.00807761.976041 0.82029 0.017803395 gi]41386699 heat shock 70kDa protein2 [Bos taurus] 25.1981 0.904301 0.552239 0.515898683 2.14605 0.332899 1.03E-05 2.719939 0.384057 3.09E-06 gil115496734 proteasome (prosome, macropain) 260 subunit, non-ATPaose,5 [Bostaurus] 21.47117 1.075324 0.911732 0.896403774 2.130784 1.273523 0.4451057 1.9598641.61162 0.544310075 gi]62461611 ribosomal protein012 [Bos taurus) 54.54546 0.819442 0.214485 0.075765251 2.19407 0.797832 0.6683928 2.034711 0.447271 0.018504045 gil119904908 PREDICTED:similar to CDEP[Bos taurus] 19.40727 -0.99926 1 -1 2.688501 0.80084 0.0932213 1.690321 0.385162 0.214474083 gil27806703 CD44 antigen [Bos taurus] 13.6612 1.645602 0.128848 0.384738395 0.489597 0.367648 0.010623 0.591614 0.323209 0.019768668 gill 14051578 histidyl-tRNAsynthetase [Bos taurus] 22.59332 1.122038 0.220312 0.477515858 2.002624 0.115951 0.0530599 2.129274 0.645424 0.248204185 23.10469 1.079881 0.611607 0.838749351 2.202377 0.247301 0.0159043 2.870485 0.965361 0.023359158 gil2780594507 inositol(myo)-l(or4)-monophosphatase 1 [Bos taurus] gi|147902 9 glpcyl-tRNAsynthetase [Bos taurus] 23.81597 1.606374 0.0542D6 0.000135907 4.327654 0.211279 0.0002809 3.225096 0.367893 0.002636469 gi|62460568 eukaryotictranslation elongationfactor 1 beta 2 [Bostaurus] 30.22222 1.222485 0.401154 0.079414648 2.816158 0.49834 1.79E-07 2.871123 0.488319 8.67E-08 gi|169658384 non-metastaticcells 1, protein (NM23A)expressed in [Bostaurus] 55.92105 1.213016 0.389997 0.340100794 3.35112 0.806727 0.5658469 3.685255 0.630437 0.03472345 gi|155372073 cullin-associatedand neddylation-dissociated 1 [Bos taurus] 12.27642 0.910426 0.466138 0.753663294 2.446792 0.225866 0.0796139 2.721135 0.165432 0.050742599 gi|28461173 elastin [Bostaurus] 13.25301 0.46035 0.302444 0.005116389 0.50723 0.122546 0.00050090.512771 0.180011 0.000303379 gi|157074012 EF-hand domain family,member D2 [Bos taurus) 28.92562 1.479102 0.407377 0.0691202 2.330451 0.438356 0.0119723 2.591688 0.797542 0.039588963 gi|62751650 poly(rC)binding protein1 [Bos taurus] 15.44944 0.951504 0.060815 0.181876504 2.491774 0.406024 0.031161 2.44942 0.659596 0.074831083 gij47564074 hypotheticalprotein LOC407769[Bos taurus] 31.03448 1.760139 0.026391 0.014542702 6.579779 0.508267 0.0843201 4.937296 0.418019 0.080537143 gill25991938 cadherin 11,type 2 [Bos taurus] 9.798995 1.029332 0.216288 0.826355089 1.269545 0.29725 0.3664875 1.191328 0.456152 0.55299561 gil119909757 PREDICTED:similar to E-selectin ligand-1 [Bos taurus] 11.81435 0.968227 0.130439 0.708197466 1.711253 0.62285 0.3363318 1.469913 0.734501 0.467101739 gil119909718 PREDICTED:similar to KIAA0015[Bos taurus] 18.33031 1.054522 0.1086 0.496408999 1.355215 0.146074 0.1541969 1.3775060.111768 0.103388318 gil134085807 capping protein (actinfilament) muscle Z-line,alpha 1 [Bos taurus] 27.27273 1.106632 1.132295 0.884212225 -1.01428 1 -1 2.25468 0.090837 0.034236561 gil114051892 UDP-N-acteylglucosaminepyrophosphorylase 1 [Bos taurus] 9.386973 1.639564 0.121794 0.076378038 2.416769 0.233459 0.0834175 2.558425 0.299703 0.097314623 gi|70778754 adenine phosphoribosyltransferase[Bos taurus] 22.22222 1.071031 0.77651 0.844289155 3.287405 0.451077 0.0227638 3.177302 0.330875 0.012565068 gill 19890624 PREDICTED:similar to insulin responsive sequence DNA bindingprotein-1 [Bostaurus] 15.90361 0.751705 0.061726 0.007446011 0.877509 0.164043 0.1581004 0.748 0.248511 0.108900501 gil62460548 sushi-repeat-containingprotein. X-linked2 [Bos taurus] 10.75269 0.778538 0.412148 0.433162454 1.022105 0.381825 0.9738584 0.7615490.222237 0.254028191 gil27806265 leukocytecell derived chemotaxin 1 precursor [Bostaurus] 18.50746 0.873375 0.345613 0.271256636 1.728857 0.13948 0.0625 0.7777750.21868 0.024898749 gil27806093 endothelialprotein C receptor [Bos taurus] 22.82158 0.902405 0.0565790.020129491 1.03466 0.29098 0.9117492 0.93193 0.241818 0.717119088 gil 14051423 ubiquitincarboxyl-terminal esterase L1 [Bos taurus] 22.22222 1.641466 0.274248 0.69276435 1.565913 0.352563 0.03797481.468517 0.759323 0.227343469 gi|87196507 unactive progesterone receptor, 23 kD[Bos taurus] 32.5 -0.99926 1 -1 2.612931 0.782133 0.0942004 1.88813 0.26381 0.125071838 gi[76641864 PREDICTED:hypothetical proteinisoform 1 [Bostaurus 20.33898 0.804241 0.024704 0.035467011 2.201792 0.360675 0.1427529 1.627975 0.02169 0.013495405 gi[27807173 S100 calcium bindingprotein A10 [Bos taurus] 37.1134 0.836268 0.428373 0.408045367 2.869626 0.661961 0.002605 4.900832 0.220089 1.65E-06 gill 14053237 GDP-mannose pyrophosphorylaseB [Bos taurus] 9.722222 1.080336 0.475313 0.721457043 2.67214 0.42058 0.028887 2.71291 0.503566 0.037364144 gi)64370167 ADP-ribosylationfactor-like 3 [Bos taurus] 19.78022 -0.99926 1 -1 3.079884 0.177022 0.0496288 2.920123 0.200703 0.05752439 gi176691395 PREDICTED:similar to mammaryserum amyloidA3.2 [Bos taurus] 76.33588 0.288428 0.456861 5.79E-07 0.321994 0.462015 1.25E-06 0.321651 0.470007 1.77E-06 gi[77735515 leukotrieneA4 hydrolase [Bos taurus] 17.18494 1.394948 0.087069 0.010734163 3.895395 0.414711 0.0148731 3.50586 0.562383 0.030065774 gi[76615127 PREDICTEDsimilar to Dihydrolipoamidedehydrogenase isoform2 [Bos taurus] 12.37721 1.717126 1.088033 0.495190248 2.184036 0.52265 0.2052057 2.318915 0.371718 0.133631271 12.83293 0.931581 0.253872 0.561370934 1.071063 0.199089 0.5647226 1.443218 1.0218470.449906825 von Willebrandfactor A domain containing1 [Bos taurus] gil157073982 27.41936 1.130883 0.10817 0.10369116 2.25123 0.848307 0.1433237 1.947455 0.7378770.147658919 gil77735861 vacuolar proteinsorting 29 [Bostaurus] 0.17445 2.910279 1.505746 0.214312616 gi77735805 leucinezipper transcriptionfactor-like 1 [Bos taurus] 14.71572 1.322494 0.505015 0.4605583593.320728 1.432508 0.309989 0.0598521 0.776968 0.220637 0.052742037 gil27806849 melanomainhibitory activity [Bos taurus] 23.07692 0.896063 0.274897 0.335599871 0.738496 0.625445 0.0570018 1.785884 0.940802 0.075396343 gil27806297 biliverdinreductase B (flavinreductase (NADPH))[Bos taurus] 25.24272 1.19426 0.338181 0.225598036 1.94962 1.043652 0.411732 0.8339538 10.8504 1.518189 0.007001955 gil145279649tumor necrosisfactor alpha[Bos taurus] 8.974359 10.26339 1.608471 0.009506114 4.705827 0.879107 0.0150849 3.128766 1.0002520.044667797 gi|119888578 PREDICTED:hypothetical protein [Bos taurus] 33.33333 1.255328 0.529528 0.302118766 2.901684 0.109101 0.0017209 2.61229 0.1785690.005336706 gi|115497946 LanClantibiotic synthetase componentC-like 1 [Bos taurus] 8.270676 1.213854 0.158229 0.091615507 1.070718 0.1477175 2.464121 0997691 0.148761828 giJ84000233 hypotheticalprotein LOC517857 [Bos taurus] 23.751.143714 0.520116 0.583065838 2.722416 0.613421 0.5701 0.0842976 0.703241 0456227 0.122131253 gi|77735537 ephrin-A1[Bos taurus] 14.146340.810966 0.52612 0.335263055 2.49032 0.336657 1.22E-05 3.027809 0401923 9.02E-06 gi|119884774 PREDICTEDsimilar to profilin1 [Bostaurus] 26.42857 0.88314 0.23018 0.0390625 0.050452 0.0615547 0.379411 0511780 0.163511214 gi|27806161 tissue inhibitorof metalloproteinase1 [Bos taurus] 28.98551 0.970092 0.035717 0.3398223321.308818 3 84 0.707693 0.0923627 1.983863 0.0763760.184405477 gi|119921 PREDICTEDsimilar to Oxysterol-bindingprotein 1 [Bostaurus] 14.10891 1.432519 1.117848 0.629770703 2.411433 4 2.651395 0.412343 0.0282469 2.304034 0434077 0.039430197 giJ780 2510 haloaciddehalogenase-like hydrolase domain containing 2 [Bostaurus] 16.98842 0.785426 0.23742 0.126766471 0.229025 0.010853 2.109202 0.1350490.005036076 gi]77735687 proteasome(prosome, macropain) subunit,beta type 6 [Bostaurus] 22.17573 0.902672 0.145471 0.222907663 2.427291 0.36153 0.4925552 1.63145 0484437 0.057707358 gi|41386683 beta-2-microglobulin[Bos taurus] 19.49152 1.609752 0.326146 0.024347998 1.118331 0.278919 0.0003113 7.332573 0.235170.000146953 gi|164414726 thymosin beta 4 [Bos taurus] 72.72727 0.937717 0.310371 0.596139681 6.453733 0.403245 0.1434794 2.50919 0.089830.029986705 gi|27806365 UDP-glucose dehydrogenase [Bos taurus] 16.39676 0.944 0.546123 0.86668958 2.401798 0.558286 0.0877555 2.606138 0.4373 0.030923861 gi|77735683 S100 calcium bindingprotein A2 [Bostaurus] 27.83505 0.876233 0.48295 0.566780294 2.049911 0.1567652 3.584035 0.4171960.100036468 gi|75812956 GTP bindingprotein Rabla [Bostaurus] 34.10405 0.943646 0.094966 0.434522862 3.356788 0.413392 0.124386 1.9765 003727 0.016719328 gi|77735697 threonyl-tRNAsynthetase [Bostaurus] 14.79945 0.859343 0.104989 0.209293288 1.913765 0.256355 0.028603 3.904594 068587 0.015547378 gi|114053301 receptoraccessory protein 5 [Bostaurus] 19.57672 1.081227 0.165518 0.509091202 4.331859 0.133176 0.660564 0.1069098 2.142663 0.897016 0.108541195 gil126722829 BCL2-associatedathanogene 3 [Bos taurus] 14.52992 0.840595 0.256045 0.233579823 2.130448 0.471729 0.0224042 3.865134 0.400497 0.019243706 gi|148235020 cold shock domain proteinA [Bos taurus] 22.07447 1.009469 0.221745 0.919238372 3.504716 0.5671233 2.483742 0627781 0.204676729 gil114053087 glutathioneS-transferase M3 [Bos taurus] 49.77778 1.017097 0.070453 0.69849654 3.077448 0.239657 0.1259579 3.455764 0031901 0.007936662 gi|149642641 D-dopachrome tautomerase [Bostaurus] 32.20339 1.07376 0.895094 0.896568411 1.611407 0.189353 2.492973 0.258773 0.086514746 gi]77404290 ribosomal proteinS3 [Bos taurus] 27.98354 0.588744 0.4677 0.260310714 2.273715 0.805692 0.2243478 0.3605628 1.418432 0.292037 0.089630587 gil123959772 leucine rich repeat neuronal 1 [Bos taurus] 10.19553 0.864345 0.2882 0.335496087 1.181397 0.324037 0.0043993 0.568377 0.537349 0.124535298 gil77735465 D component of complement (adipsin) [Bostaurus] 9.65251 0.563551 0.595564 0.138162472 0.585334 0.091417 5.23E-05 0.5641 0.214774 0.03125 giJ76608584 PREDICTED:similar to KIAA0653protein isoform1 [Bos taurus] 5.688623 1.004959 0.25053 0939136611 0.334446 0.30689 0.45052 0.189858 0.0726815 0.310432 0352916 0.09457953 gi[27806273 chemokine (C-C motif)ligand 20 [Bos taurus] 18.75 0.175335 0.04058 0.007275412 1.023182 0.00042 0.0149939 1.082923 0.216119 0.538829433 gil119927320 PREDICTED:similar to lamininB2, partial [Bostaurus] 1390729 1.077214 0.122751 0.429894005 0.1904942 1.983858 0086821 0.038702029 gij78042550 glutaredoxin3 [Bos taurus] 20.65868 0.99249 0.344049 0.9743642581.734352 0.33765 0.369555 0.1170882 3.068018 0.063427 0.017431026 gil119879173PREDICTED: similar to putativeMAPK activating proteinisoform 2 [Bos taurus] 13.49353 0.846562 0.390174 0.5452257042.685494 0.351494 0.080679787 gil76652407 PREDICTED:similar to ChainA, CrystalStructure Of HumanRangap1-Ubc9 isoform2 [Bostaurus] 34.17721 1.264753 0.169236 0.215488391 3.871949 0.129965 0.0302497 3.81154 0.513389 0.1457719 3.458487 0.495D49 0.121591531 gil125991756heterogeneous nuclear ribonucleoproteinU [Bos taurus] 18.30303 1.130716 0.155434 0.35165662 2.985644 6 3 3 0.4713422 1.674737 0.88536 0.436910788 gil2780 5 tryptophanyl-tRNAsynthetase [Bostaurus] 15.57895 1.018586 0.414788 0.940178567 1.71781 0.9823 0.658952 0.6305649 1.01386 0.358876 0.944212072 gi[76635398 PREDICTED:similar to DDX6protein isoform 1 [Bos taurus] 17.59834 0.821274 0.587027 0.619061102 1.253473 1.041339 0.128786 0.7484005 1.24845 0.16535 0.210607741 gil29135323 sialyltransferase 1 [Bos taurus] 18.76543 0.784499 0.47048 0.48468537 0.015662464 1.009076 0.024297 0.6498587 1.105178 0.097416 0.099640677 gi[76631783 PREDICTED:similar to chondroitin6-sulfotransferase [Bostaurus] 17.53653 1.314842 0.086855 1 -1 1.082899 0.063705 0.204752902 gill 14052973 glypican 1 [Bos taurus] 20.75134 1.186429 0.907828 0.765556137 -1.01428 0.191887 0.0613723 2.119185 0.583501 0.227727609 gil122692293chloride intracellularchannel 4 [Bos taurus] 20.1581 1.138384 0.095079 0.218574049 2.682452 1.473169 0.450713 0.0625 1.205623 0.31191 0.106078454 gi77735939 proliferatingcell nuclear antigen [Bos taurus] 25.28736 0.847545 0.3874 0.242201857 0.255512 0.1743667 1.446848 0.20476 0.162961893 gi|119910239 PREDICTED:similar to gamma-adaptin[Bos taurus) 11.0303 1.533588 1.213802 0.60272586 1.583943 0.1275546 2.591611 0.731529 0.226181408 gi|47564098 cell divisioncycle 10 [Bostaurus] 12.91866 0.901104 0.578761 0.777492668 2.627683 0.394435 0.139293 0.0316113 1.895719 0.379887 0.050063854 gi|84000371 H2Ahistone family,member V [Bos taurus] 45.3125 0.767522 0.154416 0.050718956 1.377428 0.20276 0.0556207 3.931079 0.189636 0.002966252 gi|134085671 histone cluster 1, Hi c [Bos taurus] 65.25822 0.817959 0.208153 0.138142118 1.410168 0.202559 0.1029734 1.569368 0.584936 0.351888895 gi78042544 galactosidase, beta 1 [Bos taurus] 14.24196 0.725925 0.431444 0.372150115 1.863763 0.0045842 2.703866 0.315668 0.015323242 gi[84000197 FK506 bindingprotein 3,25kDa [Bostaurus] 25.44643 0.867531 0.398158 0.46858875 2.704596 0.166508 7 0.0597197 3.040205 0.392492 0.107723999 gil2780584 crystallin,lambda 1 [Bostaurus] 12.14953 1.169827 0.457059 0.609700183 2.80394 0.195191 0.4399357 0.834813 0.550225 0.647913246 gil115497422 glucosamire (N-acetyl)-6-sufatase [Bos taurus] 18.21429 0.760507 0.335275 0.34492051 1.45D658 0.603864 0.466993694 2.348802 0.322914 0.1185965 2.643298 0.322171 0.101084937 gi|77736031 chaperonincontaining TCP1, subunit 6A [Bostaurus] 19.77401 0.772838 0.47251 1.229275 0.289036 0.4042864 1.127218 0.1983 0.399893253 gil122692521 DEAH(Asp-Glu-Ala-His) box polypeptide 36 [Bos taurus} 16.83168 0.732151 0.151561 0.149252811 2.368498 1.027467 0.3411324 2.815247 0.667527 0.192449106 gi|76635454 PREDICTED:similar to OAFhomolog (Drosophila) [Bos taurus] 25.17731 1.175187 0.27629 0.442918937 3.667679 0.075 0.0182012 3.574233 0.325252 0.0784065 gi|77736207 growthfactor receptor-bound protein 2 [Bos taurus] 24.42396 1.372247 0.069782 0.068380505 3.522881 0.52881 0.1306551 3.805666 0.110959 0.025621412 gi|77735815 rrbosomalprotein L30[Bos taurus] 38.26087 1.181253 0.764009 0.732484837 -1.01428 1 -1 2.15683 0.14969 0.059500004 giJ62751458 endoplasmicreticulum thioredoxin superfamily member. 18 kDa [Bos taurus] 22.67442 1.089121 0.718946 0.847410241 0.0467477 2.827037 0908164 0.25454044 gi45430019 ribosomal protein L12[Bos taurus] 38.78788 0.945167 0.038057 0.205892189 3.691938 0.193913 0.0750611 1.171559 0.3561150.349964129 gil27806083 peroxiredoxin3 [Bos taurus) 22.56809 0.768652 0.942605 0.558666155 1.50584 0.286954 1.281265 0.5215266 1.576041 1.554514 0.64873546 gi62751982 proteasome (prosome, macropain) subunit, alphatype. 5 [Bostaurus] 31.95021 -0.99926 1 -1 1.824061 2.110537 0.796037 0.311478 2.028654 0.5566810.230181937 gi|155371989 methionineadenosyltransferase 11,alpha [Bos taurus] 15.6962 1.392466 0.503745 0.407245916 1.884966 0.307009 0.1516395 1.661418 051494 0.089996198 gi|1 14052601 hypotheticalprotein LOC530784[Bos taurus] 10.79365 1.074493 0.419049 0.783715444 2.052029 0.715259 0.1328952 2.407481 0.1011430.002049768 gi|149642987 S100 calcium binding proteinAl1 (calgizzarin)[Bos taurus] 9.708738 0.927106 0.459521 0.723099805 0.682088 0.77761 0.3303916 0.577906 0.4494980.097124639 gil76669880 PREDICTED:similar to LRTS841 [Bos taurus] 20.49608 0.790905 0.611498 0.578211681 0.482928 0.1149155 3.478751 0.8899 0.152654038 gi|77736139 proteasome (prosome, macropain) subunit, beta type,3 [Bos taurus] 20.4878 1.336283 0.347604 0.337542728 3.710544 1.149164 0.097311 0.2322929 1.044494 0.056572 0.035564114 gi|119901198PREDICTED: similar to Man9-mannosidase[Bos taurus] 19.42337 0.704295 0.233639 0.201401163 0.257946 0.0913148 0.629888 0480153 0.307839705 gil122692459ABI gene family,member 3 (NESH)binding protein[Bos taurus] 11.89802 0.943457 0.538466 0.863524662 0.422662 0.4636148 1411991 1.011494 1.349311 0.519302 0600944594 gil114051602KH domain containing,RNA binding,signal transduction associated 1 [Bostaurus] 21.21896 0.582443 0.70544 0.36285151