Proteomic Analysis of the Nuclear Membranes of Human Periodontal Ligament Fibroblast and Gingival Fibroblast Cell Types: a Comparison Study

PROTEOMIC ANALYSIS OF THE NUCLEAR MEMBRANES OF HUMAN PERIODONTAL LIGAMENT FIBROBLAST AND GINGIVAL FIBROBLAST CELL TYPES: A COMPARISON STUDY

A Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

William Patrick Kelsey, V, D.D.S.

* * * * * *

The Ohio State University 2009

Master‟s Examination Committee

Dr. Angelo J. Mariotti, Advisor Approved by

Dr. William A. Brantley

Dr. Purnima S. Kumar ______Advisor Dentistry Graduate Program

William Patrick Kelsey, V, D.D.S.

2009

ABSTRACT

Background: The role of cells derived from the periodontal ligament has always been considered an important factor in the regeneration of the periodontium. Although the genome for the periodontal ligament fibroblast (PDLF) and gingival fibroblast (GF) is the same in any individual, there have been distinct biochemical differences identified between the two cell types. The differences in behavior of these two cells, despite similar DNA content, may be explained by proteomics, specifically via the proteome of their nuclear membrane, since the nuclear membrane is arguably one of the most essential cellular organelles involved in biogenesis. The principal aim of this study was to determine if there were differences in protein content of enriched nuclear membranes from PDLF and GF.

Methods: PDLF and GF were derived from non-inflamed tissue of 3 systemically healthy subjects, and explants were grown in cell culture consisting of MEM supplemented with 10% FBS. PDLF and GF whole cell suspensions were lysed and nuclear pellets were separated via differential centrifugation using sucrose gradients until an enriched nuclear pellet was present. Samples were cleaned and quantified by DC protein quantitation assay. The same amount of protein (15 g) was taken from each sample evaluated. In addition, 2.5 g of protein was taken from each sample, respectively, and combined as an internal standard. Samples were digested with trypsin, followed by iTRAQ™ mass labeling for peptide qualitative and quantitative identification following capillary LC-MS/MS based proteomic methods for comparative analysis. Missed cleavage number was set at 4. Mass tolerance for peptides was set at 1.2Da and 0.8Da for MS/MS fragments. MASCOT provided qualitative results, and quantitative results were reported as the average ratios between the sample and internal standard. LC-MS/MS was performed multiple times, and the average ratios of protein for matched sets of GF versus PDLF were calculated with standard deviations. Differences in proteins were considered statistically significant when means were not contained within the 95% confidence interval.

Results: 44 total proteins were identified between the three samples. Proteins known to be associated with the nuclear envelope (Lamin A/C and Myoferlin) and nuclear pore complex (Myosin-lc) as well as ten other proteins known to be associated with the nucleus (Annexin A1, Fructose-bisphosphonate aldolase A, Histone H2A type 1-C, Histone H2B type 1-B, Histone H3- like, Histone H4, Neuroblast differentiation-associated protein AHNAK, Polymerase I and transcript release factor, Prohibitin, and Protein kinase C delta-binding protein) were identified but were equally expressed between the cell types. Annexin A2, Annexin A4, CD44 antigen, and Histone H3-like proteins were found to be in statistically significant greater abundance in GF than in PDLF.

Conclusion: This study constitutes the first attempt in the literature to define periodontal proteomics. The quantitative and qualitative proteomic analysis demonstrated differences in two cytoplasmic and one extracellular fibroblast protein, but no differences were found within the nuclear envelope. Concerns of proper membrane-associated protein solubilization were raised based upon the paucity of known proteins identified.

iii

DEDICATION

To Amanda, my wonderful wife, the love of my life and unquenchable font of support. To my daughter Meghan Kaylee, who won‟t remember her time in Columbus, but know that it was special. To my beautiful dog, Sophie, whose need for belly rubs put this endeavor into perspective. And to Pat, Nan, Matt and Kaitlin for their love and encouragement throughout my entire life.

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to Dr. Angelo Mariotti, my mentor and advisor, for his vision of this project, as well as his guidance and encouragement along the way.

Additional thanks goes to Deb Hooper, laboratory master, without whose help I would still be toiling.

Thank you to the residents and fellows in the Division of Periodontology, the Division of Oral and Maxillofacial Facial Surgery, and the General Practice Residency program who assisted me in obtaining samples.

Wholehearted thanks goes to Dr. Purnima Kumar and Dr. Bill Brantley who volunteered their time to serve on my thesis committee.

Finally, much appreciation goes to Dr. Kari Green-Church, Dr. Cindy James, and Dr. Liwen Zhang at The Ohio State University‟s Campus Chemical Instrument Center Mass Spectrometry and Proteomics Lab, for their multiple hours spent on our project, and for taking time out of their days to answer my basic questions.

VITA

October 29, 1979 Born, Omaha, Nebraska, United States of America

May 17, 1998 Graduation, Creighton Preparatory School, Omaha, Nebraska

May 18, 2002 Bachelor of Science, Creighton University, Omaha, Nebraska

May 13, 2006 Doctor of Dental Surgery, Creighton University, Omaha, Nebraska

July 5, 2006 – June 23, 2009 Resident in Section of Periodontology, College of Dentistry, The Ohio State University, Columbus, Ohio

PUBLICATIONS

1. Latta MA, Kelsey WP 3rd, Kelsey WP 5th. Effect of polymerization mode of adhesive and cement on shear bond strength to dentin. Am J Dent. 2006 Apr; 19(2): 96-100.

FIELDS OF STUDY

Major Field: Dentistry

TABLE OF CONTENTS

Page

ABSTRACT ii

DEDICATION iv

ACKNOWLEDGEMENTS v

VITA vi

LIST OF TABLES viii

LIST OF FIGURES ix

CHAPTERS:

1. INTRODUCTION 1

2. MATERIALS AND METHODS 16

3. RESULTS 24

4. DISCUSSION 27

APPENDIX A: TABLES 35

APPENDIX B: FIGURES 46

REFERENCES 97

vii

LIST OF TABLES

TABLE Page

TABLE 1. Summary of in vitro Differences of PDLF & GF 36

TABLE 2. Quantity & Purity of Protein Required for Different Applications 37

TABLE 3. Advantages & Disadvantages of ESI & MALDI 38

TABLE 4. Demographics of Study Patients 39

TABLE 5. Proteins Identified in This Study 40

TABLE 6. Functions & Locations of the Identified Proteins 41

TABLE 7. Known Proteins Associated with the Human Nuclear Envelope 43

viii

LIST OF FIGURES

FIGURE Page

FIGURE 1. Breakdown of Functional Genomics 47

FIGURE 2. Summary of Steps to Purify Proteins 47

FIGURE 3. Concept of ESI 48

FIGURE 4. Concept of MALDI 48

FIGURE 5. Concept of the Nuclear Membrane 49

FIGURE 6. Technique for the Enrichment of Nuclear Membranes 50

FIGURE 7. Example of 2-DE Separated PDLF Enriched Nuclear Membranes 51

FIGURE 8. Example of 2-DE Separated PDLF Crude Nuclear Membranes 52

FIGURE 9. Lamin A/C Ratios Between GF & PDLF 53

FIGURE 10. Myoferlin Ratios Between GF & PDLF 54

FIGURE 11. Myosin-lc Ratios Between GF & PDLF 55

FIGURE 12. Annexin A1 Ratios Between GF & PDLF 56

FIGURE 13. Fructose-bisphosphonate aldolase A Ratios Between GF & PDLF 57

FIGURE 14. Histone H2A type 1-C Ratios Between GF & PDLF 58

FIGURE 15. Histone H2B type 1-B Ratios Between GF & PDLF 59

FIGURE 16. Histone H3-like Ratios Between GF & PDLF 60

FIGURE 17. Histone H4 Ratios Between GF & PDLF 61

LIST OF FIGURES

FIGURE Page

FIGURE 18. Neuroblast Differentiation-Aassociated Protein AHNAK Ratios Between GF & PDLF 62

FIGURE 19. Polymerase I and Transcript Release Factor Ratios Between GF & PDLF 63

FIGURE 20. Protein Kinase C Delta-Binding Protein Ratios Between GF & PDLF 64

FIGURE 21. Prohibitin Ratios Between GF & PDLF 65

FIGURE 22. Cytoskeleton-Associated Protein 4 Ratios Between GF & PDLF 66

FIGURE 23. Actin, Alpha Cardiac Muscle Ratios Between GF & PDLF 67

FIGURE 24. Actin, Aortic Smooth Muscle Ratios Between GF & PDLF 68

FIGURE 25. Actin, Cytoplasmic 1 Ratios Between GF & PDLF 69

FIGURE 26. Actin, Gamma-Enteric Smooth Muscle Ratios Between GF & PDLF 70

FIGURE 27. Annexin A4 Ratios Between GF & PDLF 71

FIGURE 28. Annexin A5 Ratios Between GF & PDLF 72

FIGURE 29. Annexin A6 Ratios Between GF & PDLF 73

FIGURE 30. Caldesmon Ratios Between GF & PDLF 74

FIGURE 31. Filamin-A Ratios Between GF & PDLF 75

FIGURE 32. Myosin Light Polypeptide 6 Ratios Between GF & PDLF 76

FIGURE 33. Myosin Regulatory Light Chain MRLC2 Ratios Between GF & PDLF 77

FIGURE 34. Myosin Regulatory Light Polypeptide 9 Ratios Between GF & PDLF 78

FIGURE 35. Myosin-9 Ratios Between GF & PDLF 79

LIST OF FIGURES

FIGURE Page

FIGURE 36. Tropomysin Alpha-1 Chain Ratios Between GF & PDLF 80

FIGURE 37. Vimentin Ratios Between GF & PDLF 81

FIGURE 38. Cadherin-13 Ratios Between GF & PDLF 82

FIGURE 39. CD44 Antigen Ratios Between GF & PDLF 83

FIGURE 40. Cytochrome b Reductase Ratios Between GF & PDLF 84

FIGURE 41. Erythrocyte Band 7 Integral Membrane Protein Ratios Between GF & PDLF 85

FIGURE 42. Guanine Nucleotide-Binding Protien G(I)/G(S)/G(O) Subunit Gamma-12 Ratios Between GF & PDLF 86

FIGURE 43. Annexin A2 Ratios Between GF & PDLF 87

FIGURE 44. Basement Membrane-Specific Heparan Sulfate Proteoglycan Core Protein Ratios Between GF & PDLF 88

FIGURE 45. Collagen Alpha-1(I) Chain 4 Ratios Between GF & PDLF 89

FIGURE 46. Collagen Alpha-2(I) Chain Ratios Between GF & PDLF 90

FIGURE 47. Collagen Alpha-3 (VI) Ratios Between GF & PDLF 91

FIGURE 48. EMILIN-1 Ratios Between GF & PDLF 92

FIGURE 49. Extracellular Superoxide Dismutase [Cu-Zn] Ratios Between GF & PDLF 93

FIGURE 50. Fibronectin Ratios Between GF & PDLF 94

LIST OF FIGURES

FIGURE Page

FIGURE 51. Prothrombin Ratios Between GF & PDLF 95

FIGURE 52. Thrombospondin-1 Ratios Between GF & PDLF 96

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CHAPTER 1 : INTRODUCTION

BACKGROUND

The periodontal ligament is a highly specialized connective tissue, and functions to attach the tooth to the alveolus via the tooth‟s cementum. The periodontal ligament (PDL) is comprised of heterogeneous cell populations, including fibroblasts, endothelium, cell rests of

Malassez, neurons, cementoblasts and osteoblasts. Encompassing the cells is an extracellular matrix comprised of five varieties of collagen (I, III, V, VI and XII); non-collagenous proteins such as osteopontin and bone sialoprotein, along with various proteoglycans and glycoprotiens.1 The presence of multiple cell types within the PDL has led to speculation that this tissue might contain progenitor cells that maintain homeostasis and regenerate the tissues of the periodontium when lost to progressive destruction caused by inflammation.2 However, this diverse mixture of cells, matrix proteins, and neurovascular elements is enclosed by very narrow, but precisely regulated tissue. Due to this architectural complexity, directly investigating and attempting to model the biophysical attributes of the PDL is extremely difficult.

Periodontal disease is an infectious process that results in inflammation of the supporting tissues of the teeth. It is characterized by destructive changes, specifically loss of bone and PDL,3 the combination of which may lead to tooth loss. The major goals of periodontal therapy are to eliminate inflammation and restore the fibrous attachment and lost osseous components. This requires reconstitution of connective tissue, formation of new bone and root cementum, and attachment of new connective tissue fibers.4,5 Techniques that

achieve these results have been described, but their success is unpredictable.6-12 More frequently, formation of tissues that do not fully restore the original architecture or the function of the periodontium are the result.13-16

In 1976 Melcher was the first to propose that the periodontal ligament played a central role in periodontal regeneration.2 His previous work in the late 1960‟s and early 1970‟s on the contribution of the periodontal ligament regarding wound healing17-21 introduced several theories regarding regenerative therapy to treat the effects of periodontal disease. Up until that time, the main goal of periodontal defect regeneration was to obturate osseous craters created by inflammatory periodontal disease with bone grafting materials. Melcher believed that instead of focusing the aim of therapy on only one of the periodontium‟s constituent tissues, the objective should be to recreate the entire organ that supports the tooth, namely bone, periodontal ligament, cementum and connective tissue. Consequently, he reasoned that if one were to achieve true periodontal regeneration, the first tissue to be involved would have to be the cells of the periodontal ligament. He, and other researchers of the Medical Research Council (MRC)

Group in Periodontal Physiology at the University of Toronto, tested the hypothesis that if one were to isolate a periodontal defect from the overlying connective tissue, and regeneration were to occur, one would be able to identify the location of the cells that instigated regeneration of lost tissue.22 This study utilized a small piece of mylar film as the barrier membrane, and was one of the first to employ the concept of integrity of a cellular domain, a model created by

Pritchard,23 in the oral cavity. This idea formed the basis of the techniques developed by the groups based out of Göteborg, Sweden and Aarhus, Denmark. Their work with millipore

filters24,25 included the first reports utilizing guided tissue regeneration on root surfaces exposed due to periodontal disease. The formation of new attachment of connective tissue inserting into new cementum was a breakthrough moment in periodontal regeneration, as previous studies in animals had only demonstrated this phenomenon on denuded, healthy root surfaces.26 The combined efforts of these groups were the seminal pieces of information that defined the origin, function and regulation of distinct cell populations in the periodontium.

The most prevalent cell type in gingival connective tissue and the periodontal ligament is the fibroblast.27 Initial attempts to describe their primary characteristics, in order to discover a link to regeneration of the periodontium, were based upon their variable physical morphologies and ability to synthesize interstitial collagens and fibronectin. Beginning in the late 1970‟s, while describing potential mechanisms for phenytoin influenced gingival enlargement, Hassell described “subpopulations” of fibroblasts that potentially presented with various capacities for metabolism and response to environmental stimuli.28 Since that time, with the work performed regarding regeneration of the periodontium, it has become evident that fibroblasts from distinct physical areas of the periodontal tissues differ in their biologic functions. The term that has come to describe this phenomenon is heterogeneity, and it refers not only to the differences between gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF), but also within the groups as well. First coined by Limeback, et. al., heterogeneity was described after primary cultures of periodontal ligament cells were shown to be capable of not only synthesizing different levels of collagen, but also different types of collagen.29 In their study, 14C-labeled collagen was compared to total protein synthesis in two independent periodontal ligament

isolations. One clone synthesized 32% to 47% collagen to total protein while the second group ranged from 55% to 70%. And although each clone synthesized types I, III, and V collagens, the amounts of each type of collagen, as revealed by fluorography, varied widely.

Numerous laboratories have evaluated PDLF and GF individually, but more compelling are the reports that compare the two heterogeneous subsets directly. In the earlier comparison studies, often times the different cell types were stimulated in the same way, then characterized by their response via production of certain metabolic products. The first of these studies demonstrated that populations of periodontal ligament fibroblast-like cells were more homogenous than GF with respect to collagen and fibronectin production in vitro.30 Specifically,

PLDF cultures were 99% collagen positive/fibronectin positive while the GF cultures were 57% collagen/fibronectin positive and 42% collagen positive/fibronectin negative using double label immunofluorescence techniques on porcine cells.

Several laboratories have utilized in vitro models offering more evidence that fibroblasts found in the gingiva or periodontal ligament are different. A review of the literature resulted in nineteen studies that directly contrasted the PDLF and the GF with regard to biologic function in vitro (TABLE 1)31-59.

It is apparent from these numerous in vitro reports characterizing dissimilarities in behavior between the PDLF and GF that these cells are heterogeneous. However, the aforementioned reports only demonstrate the differences between the two cell types and do not explain the biologic basis by which these variations exist. Nor is there understanding, at the molecular level, of how the expression patterns of these heterogeneous cell types in the periodontium interact and / or are integrated with each other and the other cell types present. 4

One could surmise that a good place to begin to understand these molecular differences would be to obtain a total expressed set of proteins for the structures being studied. The traditional approach to garnering this information would be to isolate single genes, then establish the function of genes one after another. This method is effective in providing a potential list of the proteins available from an organism‟s DNA, but is extremely time consuming, and may not be feasible in higher order organisms where several genes are responsible for protein synthesis.

Nor does it give an exact definition of the proteins expressed. Since this information is at the genomic level, it only provides a putative list of proteins that could be transcribed. A modern approach to this quandary would utilize proteomic analysis.

PROTEOMICS

With the human genome having been fully sequenced and published in the public domain,60,61 the next great challenge for scientists is to relate each gene to its function(s). The desire to understand how this intricate relationship unfolds is driving entirely new and diverse sub-specialties of biology. These related, but separate, divisions of research are genomics

(DNA), transcriptomics (mRNA), proteomics (proteins), and metabolomics (metabolites)

(FIGURE 1).62 Functional genomics represents the combined efforts of these four areas, and seeks to elucidate the function of genes as revealed by complete gene sequences.

Proteomics is the dynamic arm of post-genomic science that “aims to unravel the biological complexity encoded by the genome at the protein level.”63 Initially proteomics referred to the study of the expressed proteins of a genome using two-dimensional gel

electrophoresis (2-DE) and mass spectrometry (MS) to separate and identify proteins, followed by sophisticated informatics approaches for working through the data gathered. This technique now refers to “expression” or “global profiling” proteomics, and it represents one of the two approaches taken by researchers when elucidating protein composition.62 The other approach is targeted proteomics. This variety attempts to detect the dynamic expressions of proteins, so as to define a gene‟s physiologic function, specifically with protein expression within a particular cell type and / or subcellular organelle.63 Regardless of the approach, the key elements in proteomic analysis remain the same: availability of high-quality biologic samples, mass spectrometry for peptide fragment identification, and powerful informatics software to combine the discovered peptides into relevant proteins. Our group believes that by isolating the proteins of subcellular structures of PDLF and GF via expression proteomics, differences will exist between the subpopulations of those cells which may begin to explain why each has distinct aspects of phenotype expression.

SAMPLE PREPARATION

Prior to utilizing analytical techniques, an essential component is possession of a high- quality biologic sample. Attaining this end presents several technical challenges for two reasons. One, is that cellular proteomes are, by nature, complex interacting networks. The second is that there is considerable dynamic range of cellular proteins in any sample analyzed, with some estimates ranging between 105 and 106 protein abundance in each.62 When one considers examples of biologic matrices, such as a cell or tissue extract, along with the overarching goal of achieving a high-quality biologic sample, plus the fact that many proteins of

biologic interest are found in extremely low abundance, often making up less than 0.1% of the total cellular protein content,62 it comes as no surprise that there are massive variations in the techniques involved in separating protein content. The first question an investigator must ask is, ”What are the end goals and the purpose for purifying a protein in the first place?”

According to Simpson,” the scale of protein purification depends on the amount of material required to perform a particular task” (TABLE 2).62 Bonnerjea, et. al.64 reported an average of four steps were necessary to achieve a high-quality, homogenous sample with an overall yield of 28% protein. These four or more steps can be generalized into stages (FIGURE 2).65

Typically in the first step, one is attempting to fraction a crude sample of intracellular proteins, lipids, RNA, DNA, carbohydrates, etc. Conventionally, differential centrifugation is the primary means selected. Other means include coarse filtration, fractional precipitation using reagents specific for what one is attempting to isolate, and ultrafiltration with a membrane, or combinations of the above.66 Sample fractionation, by any means, is achieved by separating proteins from each other and their environment based upon the intrinsic properties of the proteins involved. Whether it be a protein‟s molecular weight, exposed side chains, or stability in different environments (i.e. pH, temperature, etc.), these are the parameters identified and then utilized to purify a sample.

In order to access the protein content of a cell, the external cellular membrane must be disrupted. This can be achieved in a number of ways, either individually or in combination.

Whichever method selected will also depend on the type of cell or tissue being broken down, and the targeted protein(s) sought after. Common methods applied include an osmotic

gradient, freeze-thaw cycling, detergent lysis, enzymatic lysis, pressure, and homogenization with glass beads or a rotating blade.65,67,68 Once the cell is lysed, there exist contaminating cell products that must be removed or inactivated. Specifically, the presence of proteolytic enzymes, salts, lipids, nucleic acids, carbohydrates, etc, must be identified and understood to plan for their elimination. Another enormous challenge during the clarification stage is to keep the remaining proteins in solution in order to progress to the protein / peptide separation phase.

Again, depending upon the nature of the target protein, specific solubilization buffers will be created and used.

The next two steps are essentially the same, with a goal of enriching the target protein(s) in the greatest concentration without losing less abundant peptides along the way.

Final polishing removes any minor contaminants, and prepares the mixture for storage.

Because several reagents are used to arrive at this point, it is important to understand the nature of the protein being targeted. If one is fortunate, protocols will be published to assist the choices that must be made, however, most likely one will be forced to discern an appropriate set of procedures to achieve those ends.

ISO ELECTRIC FOCUSING

With gel-based proteomic analyses, once a purified sample of protein has been obtained, isoelectric focusing (IEF) of the sample must occur to satisfy the first dimension of protein/peptide separation via charge. This is accomplished by applying the sample to a strip of

material that has a pH gradient spanning its length, and subjecting the strip to a charge of electricity over an extended period of time.

During the early days of protein separation utilizing 2-DE, reproducibility of results was a large issue due to unreliable means of establishing a pH gradient during IEF.69 Since the late-1980‟s these problems have been ameliorated by the commercial fabrication of strips with immobilized pH gradients (IPG) using stable weak acids and weak bases.70,71 Each strip can have varying breadths of pH gradients ranging from 3 to 7 pH units, up to pH 10 for a wide IPG, and 1 to 1.5 pH units for narrow IPG strips.65 As with each previous step, the choice of pH gradient depends upon the targeted protein(s).

The amount of protein necessary to complete the IEF is an important value, which underscores the importance of proper sample preparation as discussed in previous sections.

ELECTROPHORESIS

Due to its superior protein resolution power, reproducibility, and simplicity 2-DE with immobilized pH gradients (IPGs) is by far the most common protein separation technique in proteomics.69,71 2-DE is a combination of isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which aims to separate proteins first by isoelectric point (pI), then by molecular weight. In this manner, 2-DE may be able to resolve greater than 5000 proteins simultaneously, and may detect less than 1 nanogram of protein per spot.65 This is obviously advantageous considering the aforementioned complexity and dynamic range of a biological matrix. It is important to note that, conventionally, when 2-DE is

utilized entire proteins are being separated and not digested peptides. Once the proteins are separated on the gel, sophisticated instrumentation identifies and excises the protein spots which are then enzymatically digested into peptide fragments for proteomic analysis.

Prior to gel excision, proteins in the sample that have been separated, first by isoelectric point during IEF, then by molecular weight, must have detection methods applied in order to reveal their position. There are five major types of gel detection methods which include anionic dyes, negative staining with metal cations, silver staining, radioactive isotopes, and fluorescence.65

The most popular anionic dye is Coomassie blue. It works by noncovalently binding to the lysyl residues of proteins. The typical dye solution is 0.1% Coomassie blue, 40% ethanol and 10% acetic acid and it can account for as little as one microgram of protein per spot.65

Because protein bound metal cations are more reactive in a gel than in free solution, this difference can be utilized to discern protein spots in a “negative” staining method. In this way, the proteins remain transparent and the background of the gel becomes stained.65 Due to their ability to stain quickly, reliably, and simply with a detection limit of greater than 50 nanograms per spot, zinc ion or imidazole-zinc ion are the most commonly used stains with this method.72

Silver staining methods are extremely sensitive with detection limits nearing 0.1 nanogram per spot, however they have a very limited dynamic range.65 Also, because

preparing the gel and actually staining it is a subjective, non-automated procedure, the results can be less reproducible.

Prior to electronic detection of degrading radioisotopes, autoradiography was utilized to visualize radiolabeled proteins. This method took a long time to gather data and had a limited dynamic range of film response.65 Electronic detection via phosphorimaging and scanning with a helium-neon laser has sped up the process and has increased the sensitivity of detection to that of, or even better than, silver staining.73

Fluorescent dyes applied to 2-DE analysis can be added before IEF or after separation by molecular weight by coating the proteins in the gel.74 Cyanine based labels (Cy) are typically used prior to electrophoresis, whereas the SYPRO Ruby stain is utilized post-electrophoresis.

Both of these techniques can be automated, increasing the reproducibility, and both have detection limits of nearly 1 to 2 nanograms of protein per spot.65 An advantage of fluorescence techniques is the ability to then perform computer aided image analysis. Created in 1997,75 a method called fluorescent difference gel electrophoresis (DIGE) simplified this process even further. Prior to IEF, samples are labeled individually with different fluorescent cyanine dyes that possess separate excitation and photon emission wavelengths. Once labeled, the samples are mixed and separated via 2-DE. The gel is subjected to the specific energy levels associated with the fluorescent labels and an image is taken. After all wavelength-associated images are accounted for, the combination of images are overlaid and subtracted so that only the differences can be seen.

MASS SPECTROMETRY

When coupled with cutting edge mass analysis, ion detection, and data processing technologies, MS is the fundamental platform technology of protein identification.76 Prior to MS, the gold standard for this process was the tedious Edman degradation procedure (ED), where a peptide‟s amino-terminal residue is labeled and removed from the peptide without breaking the covalent bonds between the remaining amino acid residues.77 By working more efficiently, MS analyzes and identifyies gel-separated proteins by their molecular weight and charge (m/z ratio), via each one‟s constituent peptides, not the entire molecule. There exist several types of mass spectrometers, however, each variety consists of three key elements: an ion source, a mass analyzer and a detector.

When dealing with high molecular weight molecules like proteins, the ionization sources one may utilize are limited, as in order to be effective, the molecule must be kept intact and transferred to the gas-phase.78 This process is colloquially known as “soft ionization”. Several years of investigation have yielded two principal methods of creating “soft ions” of large biomolecules via vaporization without decomposing the original protein‟s structure.79,80 In fact, the 2002 Nobel Prize for Chemistry was awarded to scientists for their work in developing these methods.

The two means of accomplishing “soft ionization” are electrospray ionization (ESI)81 and matrix assisted laser desorption ionization (MALDI).82 ESI forms ions by creating a fine spray of charged droplets through a strong magnetic field (FIGURE 3). The charged droplets are

desolvated by the application of a countercurrent flow of gas and/or heat causing the droplet to evaporate. Once this occurs ions are created as the droplet changes state from a liquid to a gas. Unique to ESI, is that molecules with multiple charges can be created, known as a “charge envelope”. For large molecules with several moieties that can be charged, like a protein, this can lower the m/z ratio such that the range that can be measured is now at a level that several types of mass analyzers can handle.

MALDI is executed by embedding substances to be analyzed in a matrix with a specific wavelength absorption pattern. This matrix is then dried to form a co-crystallized mixture.65

Ions are produced by subjecting the mixture with short duration pulses of UV light from a nitrogen laser. This interaction results in ionization of both the matrix and the sample molecule

(FIGURE 4). The ions that are created are accelerated through an electric field through a portal into the mass analyzer portion of the MS.94-96

The following table lists common advantages and disadvantages of ESI and MALDI types of MS (TABLE 3).

PROTEIN IDENTIFICATION

Once again, it is important to note that, in most cases, after 2-DE occurs and protein spots on gels are identified, MS analysis does not commence right away. Protein spots are enzymatically digested, typically by trypsin, to break the proteins into peptide fragments. The resulting solution in made up of peptide fragments that are able to undergo soft ionization to be identified by the mass analyzer portion of the MS instrument. Once peptides in the sample

protein are defined by their mass to charge ratio, a “fingerprint” of each peptide is created. This is also known as the “peak list”. The masses of these peptides are then added and compared to theoretical peptide masses calculated from known sequence databases using parameters from one‟s experiment. The results are statistically analyzed and are given as top one or two protein possibilities. Researchers then discern if there is any correlation to the result and previously reported evidence on the structure investigated.

NUCLEAR MEMBRANE

The specific subcellular structure of PDLF and GF investigated in this report is the nuclear membrane. The nuclear membrane is the interface between the nucleus and cytoplasm and is fundamental in upholding the unique biochemical identity of the individual spaces

(FIGURE 5). It is comprised of several elements, the most prominent of which are the inner nuclear membrane (INM) and the outer nuclear membrane (ONM). In vertebrates, these structures are separated by a perinuclear space (PNS) of about 30-50 nm.87 The INM and PNM are connected at several junctions that form aqueous channels between the nucleoplasm and cytoplasm. These connections are called nuclear pore complexes (NPC), and are responsible for regulating the trafficking of macromolecules across the nuclear membrane. In addition to its connections with the INM, the ONM is also seamlessly attached to the endoplasmic reticulum

(ER). The last major feature of the nuclear envelope is the nuclear lamina (NL). This is a thin

20-50 nm lattice of proteins associated with the INM and the underlying chromatin. The NL provides sites of anchorage for chromatin at the nuclear periphery, in addition to being a scaffold for the nuclear membrane.

Until recently, the nuclear membrane has been regarded as a passive structure, essentially serving as a selectively permeable structure for the cell‟s genetic material. However it is now known that the NE operates as a key player in the cell cycle, and that deficiencies in the proteins of the NL have been linked to activities as diverse as transcription,88-91 DNA replication,92-93 nuclear anchoring and migration within the cell,94 and signaling cascades.95

Additionally, at least 15 inherited diseases and syndromes have been linked to deficiencies in

NL components and certain NE transmembrane proteins.96-99 With this in mind, the implication that the NE provides a dynamic link between the nucleus and the cytoplasm may be of paramount importance when describing the heterogeneity of PDLF and GF.

The present investigation hopes to expand upon the knowledge in the literature and to contribute, for the first time to our knowledge, a report on the proteome of cells derived from oral tissues. Specifically our aims are to isolate human PDLF and human GF from matched pairs of individuals, separate each type of fibroblasts‟ nuclear membrane, and characterize the proteins expressed via proteomic analysis utilizing 2-DE. We believe that the proteins derived from the

NE of human PDLF differ in composition and amount from proteins derived from the NE of human GF.

The hypothesis of this study is that there exist variations in the protein content of the nuclear membranes of PDLF and GF that could begin to explain differences in behavior of these periodontal cells.

CHAPTER 2 : MATERIALS & METHODS

Isolation and Culture of Human Gingival and Periodontal Ligament Fibroblasts

Fibroblasts were collected from individuals seeking extraction of impacted third molars in the Division of Oral and Maxillofacial Surgery at The Ohio State University‟s College of

Dentistry. Teeth sought after were impacted in soft tissue and/or bone, and did not have any communication with the oral environment. Interested subjects who met the inclusion criteria were required to provide signed informed consent approved by the Institutional Review Board of

The Ohio State University. Inclusion criteria were healthy patients without any systemic disease, 16 years of age or older, a patient of record at The Ohio State University College of

Dentistry, and signed treatment plan for extraction procedure. All subjects who consented to participate were adults between 17 and 31 years of age (TABLE 4).

Human Gingival Fibroblasts

Human GF were isolated from the connective tissue of the overlying flap of the impacted third molars. All connective tissue samples taken were from gingival tissues that appeared clinically healthy (firm, non-edematous, pink tissue without any signs of inflammation).

Upon extraction of the tooth and harvesting of connective tissue, each tissue explant was immediately placed in 10 ml of biopsy media whose stock solution was comprised of alpha-

Minimum Essential Medium (Mediatech, Manassas, VA), 10% Fetal Bovine Serum (Invitrogen,

Carlsbad, CA), Penicillin G (100 IU/ml) / streptomycin (100 g/ml) (Mediatech, Manassas, VA),

L-glutamine (350 g/ml) (Mediatech, Manassas, VA), and fungizone (0.5 g/ml) (Mediatech,

Manassas, VA).

Using aseptic laboratory techniques under a laminar flow hood, the connective tissue sample was cut into small pieces using a sterile #10 blade (Miltex, York, PA), and pipetted along with a small amount of biopsy media into 25 ml flasks. Excess media was removed and the tissue explants were allowed to attach to the bottom of the flask for a minimum of five minutes. Once seated, 1.5 ml of 10% fetal bovine serum (growth medium) was added, and the fibroblasts were allowed to grow from the explants until confluency was reached in a 5% carbon dioxide, water-jacketed incubator at 37°C.

Human Periodontal Ligament Fibroblasts

Human PDLF were isolated only from the mid-root surface of the extracted, impacted third molars. Upon extraction, each tooth was immediately placed in a separate tube of the aforementioned biopsy media. Utilizing aseptic laboratory techniques, the periodontal ligament tissues were teased from the mid-root surface with a #10 blade, then cut into small pieces, and pipetted along with a small amount of biopsy media into 25 ml flasks. Excess media was removed, and the tissue explants were allowed to attach to the bottom of the flask for a minimum of five minutes. 1.5 ml of 10% fetal bovine serum was added to the flasks and samples were allowed to grow from the explants until confluency was reached in a 5% carbon dioxide, water-jacketed incubator at 37°C.

Once each flask of GF and PDLF were confluent, the growth media was removed and the cells were washed first with phosphate buffered saline (PBS) 7.4 pH, then with trypsin/EDTA to remove them from the bottom of the flask. The fibroblasts were transferred to sequentially larger flasks and plates, with the final plate being 150 mm in diameter. Cells for the following experiments were used between the second and fifth passage. Cells were frozen in liquid nitrogen until their imminent use was required.

Fibroblast Nuclei Purification

Fibroblast nuclei were purified from cell culture using a nuclei isolation kit (NUC-201,

Sigma, St. Louis, MO). Growth medium was aspirated from the 150 mm plate and fibroblast cells were rinsed with 10 ml PBS. The rinse was aspirated and 5 ml of ice-cold lysis solution

(dithiothreitol (DTT) and Triton X-100) was added to each plate. Lysed fibroblasts were harvested with a small bladed scraper. The entire cell lysate was transferred to a 15 ml centrifuge tube and incubated on ice. Purification of nuclei occurred through centrifugation through a 1.8 M sucrose cushion in Beckman ultracentrifuge tubes. Samples were centrifuged for 45 minutes at 30,000 X g at 4°C.

Once complete, whole nuclei were visible as a “V” shaped deposit on the side of the centrifuge tube. The supernatant was carefully removed via aspiration. Nuclei were suspended in solution using 5 ml cold Nuclei PURE Storage Buffer (Sigma) then subjected to another round of centrifugation, this time at 500 X g for 5 minutes at 4°C. The supernatant was completely removed via aspiration, and 200 μl cold Nuclei PURE Storage Buffer (Sigma, St. Louis, MO)

was added to suspend the nuclei. The final suspension was transferred to a microcentrifuge tube for nuclear envelope enrichment.

Fibroblast Nuclear Membrane Enrichment

Nuclear envelope enrichment protocols were utilized as described by Matunis100

(FIGURE 6). The supernatant of the microcentrifuge tube containing whole fibroblast nuclei was carefully removed and 1 ml of room temperature lysis buffer (0.1mM MgCl2, 1 mM DTT, 1x protease inhibitors [leupeptin/pepstatin A, PMSF and aprotinin]), 5 g/ml DNase I, 5 g/ml

RNaseA) was added in a drop-wise fashion while gently vortexing. The resuspended nuclei were immediately transferred to a 15 ml round-bottomed tube and 4 ml of room temperature extraction buffer (10% sucrose (weight/volume), 20mM triethanolamine (pH 8.5), 0.1 mM MgCl2,

1 mM DTT, 1x protease inhibitors (leupeptin/pepstatin A, PMSF and aprotinin) was added drop- wise while vortexing. This solution was incubated at room temperature for 15 minutes. The extracted nuclei were then underlaid with 4 ml of ice-cold sucrose cushion solution (30% sucrose (weight/volume), 20 mM triethanolamine (pH 7.5), 0.1 mM MgCl2, 1 mM DTT, 1x protease inhibitors (leupeptin/pepstatin A, PMSF, and aprotinin). The crude nuclear envelopes were pelletized by centrifugation at 4000 X g for 15 minutes at 4°C using a swinging bucket rotor.

The supernatant and sucrose cushion were aspirated, and the nuclear envelope pellet was resuspended in 1.0 ml of ice-cold extraction buffer, added drop-wise while vortexing.

Immediately 0.5 ml of ice-cold extraction buffer containing 0.3 mg/ml heparin was added to the

resuspended nuclear envelope solution. This was then underlaid with 4 ml of ice-cold sucrose cushion solution, and centrifuged for 15 minutes at 4000 X g at 4°C using a swinging bucket rotor. Once the supernatant and sucrose cushion were removed, the resulting pellet corresponded to enriched nuclear envelopes that were stripped of ribosomes and contaminating chromatin. Each sample pellet was either placed in liquid nitrogen for storage, or sent to The

Ohio State University Campus Chemical Instrument Center Mass Spectrometry and Proteomics

Facility.

Fibroblast Nuclear Membrane Protein Solubilization & Labeling

Sample pellets were resuspended in 100 l of 2xRIPA buffer and incubated on ice for twenty minutes. This solution was sonicated gently, and then centrifuged at 13,000 rpm for thirty minutes to remove any insoluble matter. The supernatant was removed, and proteins were precipitated from the RIPA using 25 l of 100% trichloroacetic acid (TCA). Samples were incubated on ice for 30 minutes and centrifuged at 4°C for another 30 minutes at 13,000 rpm.

Pellets were washed with 1.0 ml of iced acetone to remove the proteins from the TCA and incubated at -20°C for one hour. Pellets were then centrifuged at 4°C for another half hour at

13,000 rpm, and allowed to air dry on the bench top for 10 minutes. Pellets were resuspended in RESOL (5M urea, 2M thiourea, 2% CHAPS, and 0.05% ASB-14) solution and vortexed to aid in solubilization.

Samples were cleaned and quantified by DC protein quantitation assay. In preparation for labeling, 15 g of protein from each sample was used. In addition, 2.5 g of protein was

taken from each sample and mixed as an internal standard. Samples were digested and labeled according to the isobaric tag for relative and absolute quantitation (iTRAQ™) reagent –

8plex manufacturer protocol (Applied Biosystems Inc. Foster City, CA) with some minor changes. Samples were first reduced by adding reducing buffer (2.0 µl, 50 mM tris-(2- carboxyethyl) phosphine solution) and incubated at 60°C for 1 hour before the addition of cysteine blocking reagent (2.0 µl, 200 mM methyl methanethiosuifonate solution in isopropanol).

The mixture was further incubated for 10 minutes in the dark at room temperature, and then digested with promega sequencing grade trypsin (1:20 enzyme:substrate) for 1 hour at 37°C. A

200 µl aliquot of isopropanol was added to each of the iTRAQ™ labeling reagents – 8plex vials, and the enitre labeling solution was added to a fibroblast sample. The labeling reaction was carried on at room temperature for 2 hours. Labeled samples of the same mixing group were carefully pooled together and cleaned with a cation exchange cartridge following the instruction provided by manufacturer. The final elute was concentrated to ~50 µl for capillary LC MS/MS experiments.

Peptide Separation via Liquid Chromatography & Tandem Mass Spectrometry

Capillary-liquid chromatography-nanospray tandem mass spectrometry (Capillary-

LC/MS/MS) of global protein identification was performed on a Thermo Finnigan LTQ Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA) equipped with a nanospray source operated in positive ion mode. Samples were separated on a capillary column [(0.2X150mm

Magic C18AQ 3µ 200A, (Michrom Bioresources Inc, Auburn, CA)] using an UltiMate™ 3000

HPLC system (LC-Packings, Sunnyvale, CA). Each sample was injected into the trapping 21

column (LC-Packings, Sunnyvale, CA), and desalted with 50 mM acetic acid for 10 minutes.

The injector port was then switched to inject, and the peptides were eluted off the trap onto the column. Mobile phase A was 0.1% formic acid in water, and 0.1% formic acid in acetonitrile was used as mobile phase B. Flow rate was set at 2.0 µl/min. Typically, mobile phase B was increased from 2% to 50% in 90-250 minutes, depending on the complexity of the sample, to separate the peptides. Mobile phase B was increased from 50%-90% in 5 minutes and kept at

90% for another 5 minutes before being brought back quickly to 2% in 1 minute. The column was equilibrated at 2% of mobile phase B (or 98% A) for 30 minutes before the next sample injection. The MS/MS was acquired with a nanospray source operated with a spray voltage of 2

KV and a capillary temperature of 175°C. The scan sequence of the mass spectrometer was based on the data dependant TopTen™ method (Thermo Scientific, Waltham, MA). Moreover the analysis was programmed for a full scan recorded between 300 – 2,000 Da and a MS/MS scan to generate product ion spectra to determine amino acid sequence in consecutive scans of the ten most abundant peaks in the spectrum. The resolution of full scan was set at 30,000 to achieve high mass accuracy MS determination. The PQD fragmentation energy was set to

32%. Dynamic exclusion was enabled with a repeat count of 30 seconds, exclusion duration of

350 seconds, and a low mass width of 0.50 and high mass width of 1.50 Da. Multiple MS/MS detection of the same peptide was excluded after detecting it three times. The results were searched by MASCOT

(http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=MIS) for protein identification and quantitation. Missed cleavage number was set at four. Mass tolerance for peptides was set at 1.2 Da and 0.8 Da for MS/MS fragments. MASCOT gave the quantitation 22

results as the ratios between the sample and internal standard (114/113, 115/113, 116/113,

117/113, 118/113 and 119/113). A comparison between samples GF8and PDLF8 was calculated as (114/113)/(115/113). Similarly, the comparison between samples GF9 and PDLF9 was calculated as (116/113)/(117/113), while the comparison between sample GF11 and

PDLF11 was calculated as (119/113)/(121/113).

STATISTICS

The mean, standard deviation, and 95% confidence interval (CI) for all proteins were determined using the data from the three matched GF and PDLF nuclear membrane pellets.

Differences between GF and PDLF were determined when a protein ratio (a 1.0 corresponds to an equal amount of protein for GF and PDLF) was outside of the 95% CI. This methodology was used since confidence intervals are closely related to statistical significance testing. More specifically, by calculating the point estimate of a protein with confidence interval [a,b] at a confidence level of 95%, any value outside of the interval [a,b] will be significantly different from the mean of the protein ratio at significance level  = 1 – 0.95, assuming the same distributional assumptions that were made to generate the confidence interval. In other words, for an average ratio, an observed value less than a or greater than b would require rejection of the null hypothesis that the true value of this protein equlaled 1.0 and the 0,05 () level of significance.

CHAPTER 3 : RESULTS

The initial study design called for peptide separation via 2-DE. In order to test that protocol, enriched nuclear membranes of one PDLF sample were isolated and a Bradford assay demonstrated that 30 g of protein was present. These nuclear envelope proteins were solubilized and submitted for 2-DE analysis. The stained gel produced discernible protein spots, but certainly not the quantity expected. Nor could the spots be identified as distinct, instead they had a smudged appearance (FIGURE 7). A second attempt using another sample of PDLF nuclear envelope protein occurred, however the protocol for nuclear membrane enrichment was terminated after the removal of the DNA and RNA material using DNase and

RNase. Elimination of the second step of nuclear membrane enrichment quadrupled the protein yield, resulting in approximately 80 g of protein. When this sample of crudely enriched nuclear envelope material was subjected to 2-DE, more peptide spots on the gel appeared, but the smudging issues were more apparent and there seemed to be an agglutination of peptides located near the IEF strip near the top of the gel (FIGURE 8). This was interpreted to mean peptide separation issues remained with the reagents necessary for solubilization for 2-DE. It was decided to attempt another method for peptide separation, and a liquid chromatography

(LC) approach was selected.

A total of 44 proteins were identified from the three samples analyzed via LC-MS/MS, and the average ratios for GF8/PDLF8, GF9/PDLF9 and GF11/PDLF11 were calculated

(TABLES 5, 6; FIGURES 9-52). Of these 44 proteins, two were known to be associated with the nuclear envelope (Lamin A/C and Myoferlin) and one with the nuclear pore complex 24

(Myosin-lc). Ten other proteins were known to be associated with the nucleus (Annexin A1,

Fructose-bisphosphonate aldolase A, Histone H2A type 1-C, Histone H2B type 1-B, Histone H3- like, Histone H4, Neuroblast differentiation-associated protein AHNAK, Polymerase I and transcript release factor, Prohibitin, and Protein kinase C delta-binding protein). One protein was associated with the endoplasmic reticulum, which is confluent with the ONM (Cytoskeleton- associated protein 4). The remaining proteins were derived from the cytoplasm, the cytoskeleton, the external cell membrane or could be found in the extracellular matrix. From the isolated proteins, four were found to be significantly different between GF and PDLF nuclear envelope pellets. These proteins were Annexin A2, Annexin A4, CD44 antigen and Histone H3- like protein. These proteins were expressed in greater amounts in GF when compared to PDLF nuclear membranes.

Annexin A2 is a member of the annexin family which are calcium-dependent phospholipid-binding proteins that assist cellular growth and transduction pathway regulation. It is primarily found in the extracellular matrix. Specifically, annexin A2 functions as an autocrine factor that increases osteoclast formation and bone resorption.101

Annexin A4 is also a member of the annexin family. It is mainly found in the cytoplasm or cytoskeleton. Specifically ANXA4 functions as a promoter for membrane fusion during exocytosis.102

CD44 Antigen is an extracellular receptor for the glycosaminoglycan hyaluronic acid that mediates cell-cell and cell-matrix interactions through its affinity for hyaluronic acid, and possibly also through its affinity for other ligands such as osteopontin, collagens, and matrix metalloproteinases (MMPs). Adhesion with hyaluronic acid has a putative role in cell migration, 25

tumor growth and progression. CD44 antigen also has a relationship with the immune system in that it may be involved in lymphocyte activation, recirculation and homing.103

Histone H3-like protein is an example of a histone. Histones are core component of the nucleosome which wraps and compact DNA into chromatin, limiting DNA accessibility.

Histones thereby play a central role in transcription regulation, DNA repair and replication, as well as chromosomal stability. H3L protien binds to the inner surface of the nuclear membrane.104

No statistically significant differences were found with proteins associated with the nuclear envelope, nuclear pore complex, endoplasmic reticulum or nucleus.

CHAPTER 4 : DISCUSSION

This study constitutes the first attempt in the literature to define the proteome of any structure associated with the periodontium. Nuclear envelopes were isolated from whole cell lysates of human GF and PDLF, then analyzed via liquid chromatography / tandem mass spectrometry (LC-MS/MS). Forty-four proteins were discovered, and comparisons of each protein between GF and PDLF were reported with four proteins demonstrating significantly greater expression in GF versus PDLF.

A review of the literature and protein databases has revealed 110 known proteins associated with the human nuclear envelope, which includes the inner nuclear membrane, the outer nuclear membrane, and nuclear pore complexes (TABLE 7). Categorization of these proteins did not typically occur from direct proteomic analysis, like the current study. Rather, definition of proteins from humans occurred via chromosomal mapping from cDNA clones of human tissues, then comparison to known genes associated with nuclear protein(s) from another species, followed by Northern blot analysis to identify the presence or absence of the desired gene.105-109 The previous reports on non-human, mammalian nuclear membrane proteins occurred with rat liver nuclei100, 110-113, xenopus laevis and xenopus tropicalis eggs,114-

117 and mouse neuroblastoma neuro 2a cells118,119 as the source of nuclear material. Although instances of direct human nuclear membrane protein identification are found in the literature.120-

123

It is significant that fourteen proteins from the human GF and PDLF nuclear complexes were cataloged, but it is unfortunate that none of them were found to be statistically different.

Additionally, it is of concern that our efforts yielded only three proteins found specifically in the nuclear envelope, and that high standard deviations were seen within the comparisons resulting in few proteins reaching a statistically significant difference. This was the case because although eleven subjects donated PDLF and GF samples, four experienced contamination during culturing and had to be discarded, while another four could not be brought up after freezing in liquid nitrogen. This meant that only three samples could be submitted for nuclear envelope enrichment, and because of significant human heterogeneity, there was high variance among the samples. Greater numbers of samples analyzed would have resulted in smaller standard deviations and potentially more proteins that fell into the statistically significant range.

When it became apparent that solubilization of proteins was an issue when the 2-DE method was utilized, switching to a LC-based approach was considered. Instead of attempting to separate entire proteins, LC first digests the sample proteins into peptides which increases the polarity and exposes more charged groups on constituent biomolecules, thus theoretically increasing the ability to enter solution. The change in technology certainly translated into a significant increase in the number of proteins identified in general, but not those necessarily associated with the nuclear envelope. This finding suggests that the fractionation protocol was not adequate in selectively eliminating cytosolic or plasma membrane associated proteins.

The fractionation protocol was a version of the one first proposed by Blobel and

Potter102 and utilized a 1.8 M sucrose gradient and centrifugation, which separates whole nuclei based on density. In theory, the nucleus, being the heaviest organelle, migrates toward the bottom of the tube while the other parts of the cell remain suspended in the sucrose solution.

This method has been demonstrated to be successful with rat liver nuclei, and is also 28

advantageous because it has been shown to remove most of the endoplasmic reticulum, which is contiguous with the outer nuclear membrane, without damaging the nuclear envelope.102

This protocol has never been attempted on human fibroblast cells, and it appears that the sucrose gradient is not sufficient enough to prevent cytoskeletal, cytoplasmic and cellular membrane associated proteins from passing through. Why this protocol works for rat liver nuclei and not fibroblast nuclei is unknown. Other protocols for separating whole nuclei exist,124,125 but it is unknown if these methods would work for nuclear membrane isolation in fibroblasts from the periodontal apparatus.

Because the NPCs and many of the other known nuclear membrane proteins have hydrophobic domains, our assumption as to why we did not see them in our analysis is because of poor solubilization. The agents used for solubilization are determined based upon the peptide separation method used for the proteomics portion of the analysis, and because we used a reverse-phase liquid chromatography approach, stronger solubilization agents could not be used.

LIMITATIONS OF METHODS EMPLOYED

Reverse phase high performance liquid chromatography (RP-HPLC) was chosen for several reasons. First, it possesses the capacity to analyze digested peptides instead of entire proteins which is advantageous because peptides are more soluble and easier to separate than the parent proteins. This becomes especially important because we anticipated a large number of hydrophobic membrane proteins involved, and peptides of hydrophobic proteins enter solution much easier. Second, RP-HPLC possesses high sensitivity regarding the resolution of

peptides with small concentrations.124 Finally, the entire proteomic analysis could be performed online (in a single injection step) with a tandem mass spectrometer. However, just as there are advantages to this method, there, too, exist limitations. RP-HPLC columns are sensitive to extremes in pH and exposure to organic solvents. High salt concentrations work well, but cannot be used because will then unfold or denature.124 Although it is adept at reading low concentrations of protein, too low of a concentration combined with the wrong packing material in the column can result in the protein being adsorbed onto the matrix.65 Finally,once a sample has entered the column, there is never another opportunity to analyze the sample again for verification purposes.

Tandem mass spectrometry (MS/MS) with an electrospray ionization source (ESI) was utilized mainly because the sample was arriving off the chromatography column as a liquid and not in a gel, so ionization of the sample material was most practical by simply creating a spray and subjecting it to a voltage. But even when compared to the other “soft” ionization source,

MALDI (matrix assisted laser desorption ionization), no superiority between the two can be decided.125-133 In fact, there is some consensus that ESI and MALDI are complementary ionization techniques that when combined, lead to the highest protein identification rates.125-133

Regardless, ESI does have its limitations in that as a flowing technique, it completely consumes the entire amount of sample, that the sheer enormity of the amount of ion data it creates complicates analysis, and that it does have an effective upper limit of 100,000 atomic mass units.133 MS/MS renders the most detailed structural features of the peptides analyzed, since after the initial identification step via mass determination, because specific ions (i.e. via the Top

Ten™ Method) are subjected to fragmentation by collision whereby the fragments are again 30

analyzed by mass. This experiment utilized the most state-of-the-art mass analyzing techniques, via an orbitrap. Orbitrap mass analyzers separate ions in an oscillating electric field which allows their sensitivity and detection limits to meet or exceed those of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometers (a proteomics gold standard) in the analysis of complex mixtures.134 Additionally, the cost of these instruments is less than that of a FTICR as an orbitrap does not require expensive electromagnets. The largest drawback with LC-MS/MS analysis is the inability to generate highly reproducible patterns and to match related patterns reliably with software tools available.135-136 This can be seen in the data from this study in one instance where the protein, Prohibitin, was not located in the GF8/PDLF8 sample. Quantitative analysis also presents a challenge for routine LC-MS/MS analyses, but methods using stable isotope labeling,137-139 or isobaric labeling140-148. have been described.

Once mass spectrometry analysis is complete the mass peak lists are submitted to a protein database. The theory behind protein data banks is that when the experimental data is compared with either known or calculated peptide fragment mass values that are derived from known cleavage rules, those peptides that have the closest matching mass values will be selected. If an unknown protein is present, the entries in the database with the closest values, often equivalent proteins from related species, will be listed with a disclaimer.149 Several protein databases exist, but this study utilized MASCOT.149 The greatest limitation of these databases is that the assigned peptide sequences in the database are given without any respect to the actual quality of the mass spectrum given to it. LC-MS/MS has the ability to generate a large number of these bad quality spectra that contain too little, irrelevant, or even ambiguous information, and is even more common when samples are used with a paucity of material to be 31

analyzed.150 When these “poor data” are entered, errors in the database occur, resulting in much confusion mainly via false positives.151-1154 Thus, search-engine dependent techniques should be employed to verify results, a measure that was not undertaken in this study. But, it is important to consider the prevalence of these error in the databases. Rejtar, et. al.155 calculated the false positive identifications by the use of a protein database to be from 2.7% to 3.9%.

Protein identifications can also be erroneous in the uncommon event that different peptides share the exact same mass. MS/MS attempts to correct these errors, but it is plausible that proteins of less abundance, or mis-cleaved or nonspecifically cleaved proteins could fall below the mass abundance range the instrument scans, and errors are incorporated. Without question, MS analysis will continue to be the workhorse of peptide identification in proteomic analysis, however when considering results, the technology‟s limitations must be taken into consideration.

The organelle selected for subproteome categorization, namely the nuclear envelope, also comes with its own set of limitations. Any proteomic analysis of hydrophobic proteins, specifically those that have high insolubility coefficients, is extremely challenging. This is primarily because missed cleavages and nonspecific cleavages at various sites occur, which may lead to generation of fewer peptides, short peptides, and most likely, critical errors in protein identification. Because the nuclear membrane consists of two membrane regions (inner and outer), one of which is seamlessly associated with another major organelle (the endoplasmic reticulum), in addition to a third region of complex pore complexes, it may have been too grandiose of a scheme to characterize all of the proteins present. Examples of published isolation techniques focus on either the nuclear pore complexes,100-103 or attempt to 32

isolate the proteins associated with the hydrophobic membrane.156,157 We believe that the presence of this dichotomy in solubility of nuclear membrane constituent proteins and the global approach taken with regards to isolation complicated the ability to solubilize the proteins present. This was plainly evident in the 2-DE separations performed, and apparently followed into the LC method even when proteins were digested into peptides where hydrophobicity theoretically should have been less of a problem. Future endeavors of this lab will certainly narrow the focus of attempting to discover differences between fibroblast nuclear membrane constituent proteins and utilize solubilization techniques specific for the proteins in the region.

FUTURE OF PERIDOTONAL APPARATUS FIBROBLAST PROTEOMICS

This study has opened the door to proteomic research in the field of dentistry and the specialty of periodontology. The data obtained were proof in principle that proteins from nuclear envelope of gingival fibroblasts and periodontal ligament fibroblasts can be isolated. However, the limitations of this study, most evident after interpreting the results, calls for protocols to be revamped and reagents to be revisited and tailored to the specific qualities of fibroblast cells.

To begin, it is possible that human fibroblast nuclei may be more dense than those of other nuclei separated in previous isolation attempts. Assuming this to be true, adjustment of the sucrose gradient may allow for more nuclei to be harvested and enriched leading to more protein being submitted for analysis. Our samples maxed out near 80 g of protein whereas the minimum protein content after rat liver nuclei enrichment was greater than 300 g.109,110 An increased amount of nuclear envelope protein will certainly lend itself to a greater chance of discovering proteins and seeing differences between the cell types if they exist. 33

Because our group relied on proven techniques for different cell types, we assumed that our enrichment process ended with a concentrated amount of nuclear envelope material.

Those previous studies utilized scanning electron microscopy, immunofluorescence and radioactive labeling to positively identify that, in fact, nuclear envelope material was present. It may be in the best interest of our group to follow that lead and seek confirmation of this type as it can only lend credence to the final results of future studies.

Finally, it is of the utmost importance to discover an effective means of solubilizing the proteins and / or peptides we do isolate so they can be put through the proteomic analysis protocols. A recent paper suggests158 that when samples are digested with trypsin, as was the case in our experiment, the amount of hydrophobic peptides extracted can be increased by the use of other agents. This may be one of several steps that we can alter in the hope of attaining better results.

APPENDIX A : TABLES

TABLE 1. Summary of the Literature Available that Discusses the in vitro Differences Between Periodontal Ligament Fibroblasts and Gingival Fibroblasts.

Author Summary of Differences between PDLF and GF Sommerman, et. al. 1988 PDLF produced more protein, collagen, & alkaline phosphate. Mariotti & Cochran GF had more hyaluronic acid and heparin, but less chondroitan sulfate 1990 A & C.

Ogata, et. al. 1995 PDLF had greater alkaline phosphatase activity, & less PGE2 release. Giannopoulou, et. PDLF attached to well plates with gelatin, laminin, & vitronectin. GF al. 1996 with collagens. Bocato-Bellemin, et. al. 2000 PDLF & GF express mRNA for MMP & TIMP differently, except TIMP-2. Joe, et. al. 2001 PDLF expressed a greater amount of ICAM-1. van der Pauw, et. No differences between the instrinsic phagocytic capacities of these al. 2001 cells. Ohshima, et. al. 2001 PDLF & GF secrete different chemotactic factors for gingival epithelium. Palaiologou, et. al. PDLF adhered well to all root surface proteins, while GF did not adhere 2001 with laminin. Ivanovski, et. al. RT-PCR comparison of mRNA expression of hard tissue proteins stained 2001 weakly for both GF & PDLF. GF possesses a greater ability to synthesize fibrillins-1 & -2 than PDLF, Tsurga, et. al. 2002 & tropoelastin not made by PDLF. An analysis of the expressed genes of PDLF & GF revealed several Han & Amar, 2002a differences between GF & PDLF. Apoptosis was constitutively reduced in PDLF compared to GF when Han & Amar, 2002b stimulated by IGF-1. Hatakeyama, et. al. PDLF & GF heterogeneity exists with resepect to CD-14 Toll-Like 2003 Receptor expression. Discovered several molecular marker genes that distinguished bone Ishii, et. al. 2005 marrow stem cells from PDLF & GF. de Vries, et. al. 2006 Discussed the relationship between RANKL and OPG with PDLF & GF. Discovered several molecular marker genes that distinguished bone Fujita, et. al. 2007 marrow stem cells from PDLF & GF. GF were twice as mobile as PDLF when grown on ECM protein Lallier, et. al. 2007 scaffolds.

TABLE 2. Quantity and Purity of Protein Required for Different Applications.65

TABLE 3. Advantages and Disadvantages of Electrospray Ionization and Matrix-Assisted Laser Desorption Ionization Techniques.62

TABLE 4. Demographic Description of Patients Enrolled in the Present Study. Samples 001, 002, 003, 004, 005, 006, 009, and 010 were discarded either due to contamination or inability to reach confluency after liquid nitrogen preservation.

No. SUBJECT TOBACCO PDL AGE SEX MEDICATIONS TEETH GF NUMBER USE F USED

001 17 M NO NO 2 YES YES

002 23 F NO Tylenol 1 YES YES

003 17 F NO NO 2 YES YES

004 28 M NO NO 1 YES YES

005 21 M NO NO 2 YES YES

006 21 F NO NO 2 YES YES

007 20 F NO NO 2 YES YES

008 17 M NO NO 3 YES YES

009 20 F NO NO 2 YES YES

010 17 M NO NO 2 YES YES

011 25 M NO NO 2 YES YES

M : F TOTALS 20.5 0 / 11 1 /11 1.9 11/11 11/11 6 : 5

TABLE 5. Identified Protein Results. Gingival / Periodontal Ligament Fibroblast Ratios, Reported by Location

Protein Name Protein Abbreviation GF8 / PDLF8 GF9 / PDLF9 GF11 / PDLF11 Ave. Ratio STD 95% CI Lamin-A/C LMNA_HUMAN 1.82 1.41 0.75 1.33 0.54 0.61 Myoferlin MYOF_HUMAN 1.14 1.23 0.67 1.01 0.30 0.34 Myosin-Ic MYO1C_HUMAN 0.82 0.65 1.61 1.03 0.51 0.58 Annexin A1 ANXA1_HUMAN 1.01 1.65 3.62 2.09 1.36 1.54 Fructose-bisphosphate aldolase A ALDOA_HUMAN 0.59 2.32 1.70 1.54 0.87 0.99 Histone H2A type 1-C H2A1C_HUMAN 0.39 2.24 2.15 1.59 1.04 1.18 Histone H2B type 1-B H2B1B_HUMAN 0.77 1.17 1.56 1.17 0.39 0.44 Histone H3-like H3L_HUMAN 2.00 1.60 1.89 1.83 0.21 0.23 Histone H4 H4_HUMAN 0.84 2.22 1.70 1.59 0.70 0.79 Neuroblast differentiation-associated protein AHNAK AHNK_HUMAN 1.30 0.70 1.69 1.23 0.50 0.56 Polymerase I and transcript release factor PTRF_HUMAN 1.31 0.89 1.77 1.32 0.44 0.50 Protein kinase C delta-binding protein PRDBP_HUMAN 0.99 0.93 1.58 1.16 0.36 0.41 Prohibitin PHB_HUMAN 0.00 0.93 1.13 0.69 0.60 0.68 Cytoskeleton-associated protein 4 CKAP4_HUMAN 0.65 1.71 0.97 1.11 0.55 0.62

Actin, alpha cardiac muscle ACTC_HUMAN 1.88 1.10 0.86 1.28 0.53 0.60 Actin, aortic smooth muscle ACTA_HUMAN 2.21 1.20 0.78 1.40 0.73 0.83 Actin, cytoplasmic 1 ACTB_HUMAN 1.89 1.21 0.80 1.30 0.55 0.62 Actin, gamma-enteric smooth muscle ACTH_HUMAN 1.50 0.98 0.68 1.05 0.42 0.47 *** Annexin A4 ANXA4_HUMAN 1.26 1.37 2.27 1.63 0.56 0.63 Annexin A5 ANXA5_HUMAN 1.62 2.54 11.53 5.23 5.48 6.20 *** Annexin A6 ANXA6_HUMAN 1.19 2.68 1.53 1.80 0.78 0.88 Caldesmon CALD1_HUMAN 2.12 1.61 0.73 1.49 0.71 0.80 Filamin-A FLNA_HUMAN 0.70 1.63 0.90 1.08 0.49 0.55 Myosin light polypeptide 6 MYL6_HUMAN 1.28 3.36 0.68 1.77 1.41 1.59 Myosin regulatory light chain MRLC2 MRLC2_HUMAN 1.74 1.83 0.39 1.32 0.80 0.91 Myosin regulatory light polypeptide 9 MYL9_HUMAN 1.98 3.02 0.53 1.84 1.25 1.41 Myosin-9 MYH9_HUMAN 1.90 1.17 0.88 1.32 0.53 0.60 Tropomyosin alpha-1 chain TPM1_HUMAN 1.49 0.84 0.60 0.97 0.46 0.52 Vimentin VIME_HUMAN 1.23 1.22 0.62 1.03 0.35 0.39 Cadherin-13 CAD13_HUMAN 1.08 0.86 0.95 0.97 0.11 0.12 CD44 antigen CD44_HUMAN 1.09 1.43 1.64 1.39 0.28 0.32 Cytochrome b reductase CYBR1_HUMAN 0.66 0.83 2.38 1.29 0.94 1.07 Erythrocyte band 7 integral membrane protein STOM_HUMAN 0.67 1.11 1.75 1.18 0.54 0.61 Guanine nucleotide-binding protein G(I)/G(S)/G(O) GBG12_HUMAN subunit gamma-12 1.05 0.95 0.23 0.74 0.45 0.50

*** Annexin A2 ANXA2_HUMAN 1.02 3.09 2.83 2.31 1.13 1.28 Basement membrane-specific heparan sulfate proteoglycan PGBM_HUMAN core protein 2.34 0.88 1.17 1.46 0.77 0.87 Collagen alpha-1(I) chain 4 CO1A1_HUMAN 1.33 1.04 0.39 0.92 0.48 0.55 Collagen alpha-2(I) chain CO1A2_HUMAN 1.19 0.73 0.54 0.82 0.33 0.38 Collagen alpha-3(VI) chain CO6A3_HUMAN 2.28 1.05 4.29 2.54 1.63 1.85 EMILIN-1 EMIL1_HUMAN 2.03 0.84 1.51 1.46 0.60 0.68 Extracellular superoxide dismutase [Cu-Zn] SODE_HUMAN 2.18 0.50 6.44 3.04 3.06 3.46 Fibronectin FINC_HUMAN 1.66 1.23 1.00 1.29 0.33 0.38 Prothrombin THRB_HUMAN 0.29 0.45 20.57 7.10 11.66 13.20 Thrombospondin-1 TSP1_HUMAN 0.44 1.31 1.64 1.13 0.62 0.70

TABLE 6. Identified Proteins and Their Functions, Reported by Location.

Protein Name Protein Location and Function Nuclear membrane. Found on the inside of INM, provides a Lamin-A/C framework for the NE and interacts with chromatin. Myoferlin Nuclear membrane. Function at nuclear membrane unknown. Nuclear pore complex & cytoplasm. At the NPC colocalizes with Myosin-Ic ribosomal protein 6.

Nucleus & cytoplasm. Promotes membrane fusion and is involved Annexin A1 with exocytosis. Fructose-bisphosphate aldolase Nucleus & cytoplasm. Enzyme involved in glycolysis. A Nucleus. It is the core component of the nulceosome which wraps Histone H2A type 1-C and compacts DNA into chromatin. Nucleus. It is the core component of the nulceosome which wraps Histone H2B type 1-B and compacts DNA into chromatin. Nucleus. It is the core component of the nulceosome which wraps Histone H3-like and compacts DNA into chromatin. Nucleus. It is the core component of the nulceosome which wraps Histone H4 and compacts DNA into chromatin. Neuroblast differentiation- Nucleus. It is required for neuronal cell differentiation. associated protein AHNAK Polymerase I and transcript Nucleus, ER, Cell membrane, Cytoplasm. Involved with the release factor termination of transcription by RNA polymerase I. Protein kinase C delta-binding Nucleus. Enzyme that negatively regulates the cell cycle. protein Prohibitin Nucleoplasm. Inhibits DNA synthesis.

Cytoskeleton-associated protein Endoplasmic reticulum - Golgi intermediate compartment membrane. 4 Function is unknown.

Cytoplasm, cytoskeleton. Involved in types of cell motility. Actin, alpha cardiac muscle Ubiquitously expressed in all eukaryotic cells. Cytoplasm, cytoskeleton. Involved in types of cell motility. Actin, aortic smooth muscle Ubiquitously expressed in all eukaryotic cells. Cytoplasm, cytoskeleton. Involved in types of cell motility. Actin, cytoplasmic 1 Ubiquitously expressed in all eukaryotic cells. Actin, gamma-enteric smooth Cytoplasm, cytoskeleton. Involved in types of cell motility. muscle Ubiquitously expressed in all eukaryotic cells. Cytoplasm, cytoskeleton. Promotes membrane fusion and is Annexin A4 involved in exocytosis. Annexin A5 Cytoplasm. It is an anticoagulant protein. Cytoplasm. Regulates the release of calcium ions from intracellular Annexin A6 stores. Cytoplasm, cytoskeleton. In non-muscle cells plays essential role Caldesmon during cellular mitosis and receptor capping. continued

TABLE 6 continued.

Protein Name Protein Location and Function Cytoplasm, cytoskeleton. Promotes branching of actin Filamin-A filaments and links actin to membrane glycoproteins. Myosin light polypeptide 6 Cytoplasm, cytoskeleton. Regulatory light chain of myosin. Cytoplasm, cytoskeleton. Involved in cytokinesis, cell shape Myosin regulatory light chain MRLC2 receptor capping and cell locomotion. Cytoplasm, cytoskeleton. Involved in cytokinesis, cell shape Myosin regulatory light polypeptide 9 receptor capping and cell locomotion. Cytoplasm, cytoskeleton. Involved in cytokinesis, cell shape, Myosin-9 secretion and receptor capping. Cytoplasm, cytoskeleton. Binds to actin filaments and plays a Tropomyosin alpha-1 chain central role in stabilization of cytoskeleton. Vimentin Cytoplasm. It is a type of intermediate filament.

Cell membrane. It is a calcium dependent cell adhesion Cadherin-13 protein involved in connecting cells. Cell membrane. Involved with cell adhesion, lymphocyte CD44 antigen activation. Cytochrome b reductase Cell membrane. Specifically reduces ions of iron. Erythrocyte band 7 integral membrane Cell membrane, cytoplasmic side. Associates with Golig- protein derived vesicles. Guanine nucleotide-binding protein Cell membrane, cytoplasmic side. Involved in GTPase activity G(I)/G(S)/G(O) subunit gamma-12

Extracellular matrix. It is a Ca2+ membrane binding protein Annexin A2 that may be involved in heat-stress response. Basement membrane-specific heparan Extracellular matrix. It is responsible for the basement sulfate proteoglycan core protein membrane's fixed negative electrostatic charge. Extracellular matrix. It is a member of group I collagen which Collagen alpha-1(I) chain 4 forms fibrils of tendon, ligaments and bone. Extracellular matrix. It is a member of group I collagen which Collagen alpha-2(I) chain forms fibrils of tendon, ligaments and bone. Extracellular matrix. Collagen VI acts as a cell-binding Collagen alpha-3(VI) chain protein. Extracellular matrix. Involved with elastic fiber formation and EMILIN-1 cell adhesion. Extracellular superoxide dismutase [Cu- Extracellular matrix. Enzyme that is anchored to heparan Zn] sulfate proteoglycans, neutralizes superoxide radicals. Extracellular matrix. Involved in cell adhesion, cell motility, Fibronectin opsonization, wound healing and maintain cell shape. Prothrombin Extracellular matrix. It is an anticoagulant protein. Extracellular matrix. It is an adhesive glycoprotein that Thrombospondin-1 mediates cell-to-cell and cell-to-matrix interactions.

TABLE 7: Known Proteins Associated with the Human Nuclear Envelope.

Entry name Protein names AKP8L_HUMAN A-kinase anchor protein 8-like (AKAP8-like protein) (Neighbor of A-kinase-anchoring protein 95) (Neighbor of AKAP95) (Homologous to AKAP95 protein) (HA95) (Helicase A-binding protein 95) (HAP95) ANKL2_HUMAN Ankyrin repeat and LEM domain-containing protein 2 ATF6A_HUMAN Cyclic AMP-dependent transcription factor ATF-6 alpha (cAMP-dependent transcription factor ATF-6 alpha) (Activating transcription factor 6 alpha) (ATF6-alpha) [Cleaved into: Processed cyclic AMP-dependent transcription factor ATF-6 alpha] B2CL1_HUMAN Bcl-2-like protein 1 (Bcl2-L-1) (Apoptosis regulator Bcl-X) B3KQU6_HUMAN cDNA PSEC0207 fis, clone HEMBA1002981, highly similar to Homo sapiens nurim (nuclear envelope membrane protein) (NRM), mRNA BAF_HUMAN Barrier-to-autointegration factor (Breakpoint cluster region protein 1) BIK_HUMAN Bcl-2-interacting killer (Apoptosis inducer NBK) (BP4) (BIP1) BNI3L_HUMAN BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (NIP3-like protein X) (NIP3L) (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3A) (Adenovirus E1B19K-binding protein B5) BNIP2_HUMAN BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 BNIP3_HUMAN BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 BOREA_HUMAN Borealin (Dasra-B) (hDasra-B) (Cell division cycle-associated protein 8) (Pluripotent embryonic stem cell-related gene 3 protein) CBX3_HUMAN Chromobox protein homolog 3 (Heterochromatin protein 1 homolog gamma) (HP1 gamma) (Modifier 2 protein) (HECH) CBX5_HUMAN Chromobox protein homolog 5 (Heterochromatin protein 1 homolog alpha) (HP1 alpha) (Antigen p25) CENPF_HUMAN Centromere protein F (Kinetochore protein CENP-F) (Mitosin) (AH antigen) DD19A_HUMAN ATP-dependent RNA helicase DDX19A (EC 3.6.1.-) (DEAD box protein 19A) (DDX19-like protein) DD19B_HUMAN ATP-dependent RNA helicase DDX19B (EC 3.6.1.-) (DEAD box protein 19B) (DEAD box RNA helicase DEAD5) DESP_HUMAN Desmoplakin (DP) (250/210 kDa paraneoplastic pemphigus antigen) EMD_HUMAN Emerin GLE1_HUMAN Nucleoporin GLE1 (hGLE1) (GLE1-like protein) GNAZ_HUMAN Guanine nucleotide-binding protein G(z) subunit alpha (G(x) alpha chain) (Gz-alpha) IMB1_HUMAN Importin subunit beta-1 (Karyopherin subunit beta-1) (Nuclear factor P97) (Importin-90) KAZRN_HUMAN Kazrin LAP2A_HUMAN Lamina-associated polypeptide 2, isoform alpha (Thymopoietin isoform alpha) (TP alpha) (Thymopoietin-related peptide isoform alpha) (TPRP isoform alpha) [Cleaved into: Thymopoietin (TP) (Splenin); Thymopentin (TP5)] LAP2B_HUMAN Lamina-associated polypeptide 2, isoforms beta/gamma (Thymopoietin, isoforms beta/gamma) (TP beta/gamma) (Thymopoietin-related peptide isoforms beta/gamma) (TPRP isoforms beta/gamma) [Cleaved into: Thymopoietin (TP) (Splenin); Thymopentin (TP5)] LEMD2_HUMAN LEM domain-containing protein 2 (hLEM2) LMNA_HUMAN Lamin-A/C (70 kDa lamin) (Renal carcinoma antigen NY-REN-32) LMNB1_HUMAN Lamin-B1 LMNB2_HUMAN Lamin-B2 LORI_HUMAN Loricrin LPPRC_HUMAN Leucine-rich PPR motif-containing protein, mitochondrial (130 kDa leucine-rich protein) (LRP 130) (GP130) MAN1_HUMAN Inner nuclear membrane protein Man1 (LEM domain-containing protein 3) MARH6_HUMAN E3 ubiquitin-protein ligase MARCH6 (EC 6.3.2.-) (Membrane-associated RING finger protein 6) (Membrane-associated RING-CH protein VI) (MARCH-VI) (RING finger protein 176) (Protein TEB-4) (Doa10 homolog) MVP_HUMAN Major vault protein (MVP) (Lung resistance-related protein) MYO1C_HUMAN Myosin-Ic (Myosin I beta) (MMI-beta) (MMIb) NAV3_HUMAN Neuron navigator 3 (Steerin-3) (Pore membrane and/or filament-interacting-like protein 1) (Unc-53 homolog 3) (unc53H3) NDC1_HUMAN Nucleoporin NDC1 (hNDC1) (Transmembrane protein 48) NPIP_HUMAN Nuclear pore complex-interacting protein (NPIP) NPL4_HUMAN Nuclear protein localization protein 4 homolog (Protein NPL4) NRM_HUMAN Nurim (Nuclear rim protein) (Nuclear envelope membrane protein) continued

TABLE 7 continued.

Entry name Protein names NU107_HUMAN Nuclear pore complex protein Nup107 (Nucleoporin Nup107) (107 kDa nucleoporin) NU133_HUMAN Nuclear pore complex protein Nup133 (Nucleoporin Nup133) (133 kDa nucleoporin) NU153_HUMAN Nuclear pore complex protein Nup153 (Nucleoporin Nup153) (153 kDa nucleoporin) NU155_HUMAN Nuclear pore complex protein Nup155 (Nucleoporin Nup155) (155 kDa nucleoporin) NU160_HUMAN Nuclear pore complex protein Nup160 (Nucleoporin Nup160) (160 kDa nucleoporin) NU188_HUMAN Nucleoporin NUP188 homolog (hNup188) NU205_HUMAN Nuclear pore complex protein Nup205 (Nucleoporin Nup205) (205 kDa nucleoporin) NU214_HUMAN Nuclear pore complex protein Nup214 (Nucleoporin Nup214) (214 kDa nucleoporin) (Protein CAN) NUP37_HUMAN Nucleoporin Nup37 (p37) NUP43_HUMAN Nucleoporin Nup43 (p42) NUP50_HUMAN Nucleoporin 50 kDa (Nuclear pore-associated protein 60 kDa-like) NUP53_HUMAN Nucleoporin NUP53 (Nuclear pore complex protein Nup53) (Nucleoporin Nup35) (35 kDa nucleoporin) (Mitotic phosphoprotein 44) (MP-44) NUP54_HUMAN Nucleoporin p54 (54 kDa nucleoporin) NUP62_HUMAN Nuclear pore glycoprotein p62 (62 kDa nucleoporin) NUP85_HUMAN Nucleoporin NUP85 (Nuclear pore complex protein Nup85) (85kDa nucleoporin) (Nucleoporin Nup75) (Pericentrin-1) (FROUNT) NUP88_HUMAN Nuclear pore complex protein Nup88 (Nucleoporin Nup88) (88 kDa nuclear pore complex protein) NUP93_HUMAN Nuclear pore complex protein Nup93 (Nucleoporin Nup93) (93 kDa nucleoporin) NUP98_HUMAN Nuclear pore complex protein Nup98-Nup96 [Cleaved into: Nuclear pore complex protein Nup98 (Nucleoporin Nup98) (98 kDa nucleoporin); Nuclear pore complex protein Nup96 (Nucleoporin Nup96) (96 kDa nucleoporin)] NUPL1_HUMAN Nucleoporin p58/p45 (Nucleoporin-like 1) NUPL2_HUMAN Nucleoporin-like 2 (NLP-1) (hCG1) (NUP42 homolog) NXF2_HUMAN Nuclear RNA export factor 2 (TAP-like protein 2) (TAPL-2) (Cancer/testis antigen 39) (CT39) NXNL1_HUMAN Nucleoredoxin-like protein 1 (Thioredoxin-like protein 6) OIT3_HUMAN Oncoprotein-induced transcript 3 protein (Liver-specific zona pellucida domain-containing protein) OPRS1_HUMAN Sigma 1-type opioid receptor (Sigma1-receptor) (Sigma1R) (SIG-1R) (hSigmaR1) (SR31747-binding protein) (SR-BP) (Aging-associated gene 8 protein) P121A_HUMAN Nuclear envelope pore membrane protein POM 121 (Pore membrane protein of 121 kDa) (Nuclear envelope pore membrane protein POM 121A) P121B_HUMAN Putative nuclear envelope pore membrane protein POM 121B P121C_HUMAN Nuclear envelope pore membrane protein POM 121C (Pore membrane protein of 121 kDa) (Nuclear pore membrane protein 121-2) (POM121-2) PARP1_HUMAN Poly [ADP-ribose] polymerase 1 (PARP-1) (EC 2.4.2.30) (ADPRT) (NAD(+) ADP-ribosyltransferase 1) (Poly[ADP-ribose] synthetase 1) PDIA1_HUMAN Protein disulfide-isomerase (PDI) (EC 5.3.4.1) (Prolyl 4-hydroxylase subunit beta) (Cellular thyroid hormone-binding protein) (p55) PE2R3_HUMAN Prostaglandin E2 receptor EP3 subtype (PGE receptor, EP3 subtype) (PGE2-R) (Prostanoid EP3 receptor) PEPL_HUMAN Periplakin (195 kDa cornified envelope precursor protein) (190 kDa paraneoplastic pemphigus antigen) PO210_HUMAN Nuclear pore membrane glycoprotein 210 (POM210) (Nuclear pore protein gp210) PTGDS_HUMAN Prostaglandin-H2 D-isomerase (EC 5.3.99.2) (Lipocalin-type prostaglandin-D synthase) (Glutathione-independent PGD synthetase) (Prostaglandin-D2 synthase) (PGD2 synthase) (PGDS2) (PGDS) (Beta-trace protein) (Cerebrin-28) RAB14_HUMAN Ras-related protein Rab-14 RAGP1_HUMAN Ran GTPase-activating protein 1 (RanGAP1) RBP17_HUMAN Ran-binding protein 17 RBP2_HUMAN E3 SUMO-protein ligase RanBP2 (Ran-binding protein 2) (Nuclear pore complex protein Nup358) (Nucleoporin Nup358) (358 kDa nucleoporin) (p270) REC1_HUMAN HERV-K_12q14.1 provirus Rec protein REC15_HUMAN HERV-K_3q21.2 provirus Rec protein (HERV-K(I) Np9 protein) continued

TABLE 7 continued

Entry name Protein names REC16_HUMAN HERV-K_10p14 provirus Rec protein REC17_HUMAN HERV-K_11q22.1 provirus Rec protein REC2_HUMAN HERV-K_7q22.1 provirus Rec protein (HERV-K(HML-2.HOM) Rec protein) (HERV-K108 Rec protein) (HERV-K(C7) Rec protein) (Central open reading frame) (cORF) (c-orf) (Rev-like protein) (Rev/Rex homolog) (K-Rev) REC3_HUMAN HERV-K_19q12 provirus Rec protein (HERV-K(C19) Rec protein) REC4_HUMAN HERV-K_6q14.1 provirus Rec protein (HERV-K109 Rec protein) (HERV-K(C6) Rec protein) REC5_HUMAN HERV-K_19p13.11 provirus Rec protein (HERV-K113 Rec protein) REC6_HUMAN HERV-K_8p23.1 provirus Rec protein (HERV-K115 Rec protein) REC9_HUMAN HERV-K_5q13.3 provirus Rec protein (HERV-K104 Rec protein) RGPD8_HUMAN RANBP2-like and GRIP domain-containing protein 8 (Ran-binding protein 2-like 3) (RanBP2L3) (Fragment) RNF8_HUMAN E3 ubiquitin-protein ligase RNF8 (EC 6.3.2.-) (RING finger protein 8) RTN4_HUMAN Reticulon-4 (Neurite outgrowth inhibitor) (Nogo protein) (Foocen) (Neuroendocrine-specific protein) (NSP) (Neuroendocrine-specific protein C homolog) (RTN-x) (Reticulon-5) S10A6_HUMAN Protein S100-A6 (S100 calcium-binding protein A6) (Calcyclin) (Prolactin receptor-associated protein) (PRA) (Growth factor-inducible protein 2A9) (MLN 4) SEC20_HUMAN Vesicle transport protein SEC20 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 1) (Transformation-related gene 8 protein) (TRG-8) SEH1L_HUMAN Nucleoporin SEH1-like (SEC13-like protein) SENP2_HUMAN Sentrin-specific protease 2 (EC 3.4.22.-) (Sentrin/SUMO-specific protease SENP2) (SMT3-specific isopeptidase 2) (Smt3ip2) (Axam2) SIRT1_HUMAN NAD-dependent deacetylase sirtuin-1 (hSIRT1) (EC 3.5.1.-) (SIR2-like protein 1) (hSIR2) SRBP1_HUMAN Sterol regulatory element-binding protein 1 (SREBP-1) (Sterol regulatory element-binding transcription factor 1) [Cleaved into: Processed sterol regulatory element-binding protein 1] SYNE1_HUMAN Nesprin-1 (Nuclear envelope spectrin repeat protein 1) (Synaptic nuclear envelope protein 1) (Syne-1) (Myocyte nuclear envelope protein 1) (Myne-1) (Enaptin) SYNE2_HUMAN Nesprin-2 (Nuclear envelope spectrin repeat protein 2) (Synaptic nuclear envelope protein 2) (Syne-2) (Nucleus and actin connecting element protein) (Protein NUANCE) SYNE3_HUMAN Nesprin-3 TERA_HUMAN Transitional endoplasmic reticulum ATPase (TER ATPase) (15S Mg(2+)-ATPase p97 subunit) (Valosin-containing protein) (VCP) TM109_HUMAN Transmembrane protein 109 (Mitsugumin-23) (Mg23) TMM43_HUMAN Transmembrane protein 43 (Protein LUMA) TNKS1_HUMAN Tankyrase-1 (TANK1) (EC 2.4.2.30) (Tankyrase I) (TNKS-1) (TRF1-interacting ankyrin-related ADP-ribose polymerase) TOIP1_HUMAN Torsin-1A-interacting protein 1 TOR1A_HUMAN Torsin-1A (Torsin family 1 member A) (Dystonia 1 protein) TPR_HUMAN Nucleoprotein TPR UFD1_HUMAN Ubiquitin fusion degradation protein 1 homolog (UB fusion protein 1) UN84A_HUMAN Protein unc-84 homolog A (Sad1/unc-84 protein-like 1) UN84B_HUMAN Protein unc-84 homolog B (Sad1/unc-84 protein-like 2) (Rab5-interacting protein) (Rab5IP) UNC50_HUMAN Protein unc-50 homolog (Uncoordinated-like protein) (Periodontal ligament-specific protein 22) (PDLs22) (Protein GMH1 homolog) (hGMH1) XPO7_HUMAN Exportin-7 (Exp7) (Ran-binding protein 16)

APPENDIX B : FIGURES

FIGURE 1. Breakdown of Functional Genomics.65

FIGURE 2. Summary of Steps Required to Purify Proteins.65

FIGURE 3. Diagram of the Concept of Electrospray Ionization (ESI).65

FIGURE 4. Diagram of the Concept of Matrix-Assisted Laser-Desorption Ionization (MALDI).65

FIGURE 5. Concept of the Nuclear Membrane.100

FIGURE 6. Laboratory Technique by Matunis for the Isolation of Enriched Nuclear Membranes.100

FIGURE 7. Two-Dimensional Gel Electrophoresis of Enriched Nuclear Membrane proteins from a PDLF Sample. Nuclear Envelope Protein (@30 g) on IEF 3-7 pH 7cm stained with Lavapurple and imaged by Typhoon.

FIGURE 8. Two-Dimensional Gel Electrophoresis of Crude Nuclear Membrane proteins from a PDLF Sample. Samples were treated with Cy3 dye and run on 3-10 pH 7cm strips with 12% acryl SDS-PAGE gel at 150 V. Imaging is with Typhoon phosphor imager.

Figure 9. Lamin A/C ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 10. Myoferlin ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 11. Myosin-lc ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 12. Annexin A1 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 13. Fructose-bisphosphonate aldolase A ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 14. Histone H2A type 1-C ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 15 . Histone H2B type 1-B ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 16. Histone H3-like ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 17. Histone H4 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 18. Neuroblast differentiation-associated protein AHNAK ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 19. Polymerase I and transcript release factor ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 20. Protein kinase C delta-binding protein ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 21. Prohibitin ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 22. Cytoskeleton-associated protein 4 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 23. Actin, alpha cardiac muscle ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 24. Actin, aortic smooth muscle ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 25. Actin, cytoplasmic 1 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 26. Actin, gamma-enteric smooth muscle ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 27. Annexin A4 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 28. Annexin A5 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 29. Annexin A6 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 30. Caldesmon ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 31. Filamin-A ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 32. Myosin light polypeptide 6 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 33. Myosin regulatory light chain MRLC2 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 34. Myosin regulatory light polypeptide 9 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 35. Myosin-9 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 36. Tropomyosin alpha-1 chain ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 37. Vimentin ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 38. Cadherin-13 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 39. CD44 antigen ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 40. Cytochrome b reductase ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 41. Erythrocyte band 7 integral membrane protein ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 42. Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-12 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 43. Annexin A2 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 44. Basement membrane-specific heparin sulfate proteoglycans core protein ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 45. Collagen alpha-1(I) chain 4 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 46. Collagen alpha-2(I) chain ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 47. Collagen alpha-3(VI) chain ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 48. EMILIN-1 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 49. Extracellular superoxide dismutase [Cu-Zn] ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 50. Fibronectin ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 51. Prothrombin ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

Figure 52. Thrombospondin-1 ratios between gingival fibroblast and periodontal ligament fibroblasts. Nuclear membrane pellets were analyzed using Capillary LC-ESI/MS/MS and proteins were identified using the MASCOT database. Data were expressed as ratios for strains 8 (GF8/PDLF8), 9 (GF/PDLF9) and 11 (GF11/PDLF11) as well as the average of all three strains with the corresponding 95% confidence interval (CI). Differences between GF and PDLF protein production were considered significant when the 95% CI exceeded 1.0.

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