CLINICAL RESEARCH www.jasn.org

Activation of the Endogenous -Angiotensin- System or Aldosterone Administration Increases Urinary Exosomal Sodium Channel Excretion

† † Ying Qi,* Xiaojing Wang, Kristie L. Rose,* W. Hayes MacDonald,* Bing Zhang, ‡ | Kevin L. Schey,* and James M. Luther §

Departments of *Biochemistry, †Bioinformatics, ‡Division of Clinical Pharmacology, Department of Medicine, §Division of Nephrology, Department of Medicine, and |Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee

ABSTRACT Urinary exosomes secreted by multiple cell types in the kidney may participate in intercellular signaling and provide an enriched source of kidney-specific for biomarker discovery. Factors that alter the exosomal content remain unknown. To determine whether endogenous and exogenous hormones modify urinary exosomal protein content, we analyzed samples from 14 mildly hypertensive patients in a crossover study during a high-sodium (HS, 160 mmol/d) diet and low-sodium (LS, 20 mmol/d) diet to activate the endogenous renin-angiotensin-aldosterone system. We further analyzed selected exosomal protein content in a separate cohort of healthy persons receiving intravenous aldosterone (0.7 mg/kg per hour for 10 hours) versus vehicle infusion. The LS diet increased plasma renin activity and aldosterone concentration, whereas aldosterone infusion increased only aldosterone concentration. Protein analysis of paired urine exosome samples by liquid chromatography-tandem mass spectrometry–based multidimen- sional protein identification technology detected 2775 unique proteins, of which 316 exhibited signifi- cantly altered abundance during LS diet. Sodium chloride cotransporter (NCC) and a-andg-epithelial sodium channel (ENaC) subunits from the discovery set were verified using targeted multiple reaction monitoring mass spectrometry quantified with isotope-labeled peptide standards. Dietary sodium restric- tion or acute aldosterone infusion similarly increased urine exosomal gENaC[112–122] peptide concentra- tions nearly 20-fold, which correlated with plasma aldosterone concentration and urinary Na/K ratio. Urine exosomal NCC and aENaC concentrations were relatively unchanged during these interventions. We conclude that urinary exosome content is altered by renin-angiotensin-aldosterone system activation.

Urinary measurement of exosomal gENaC[112–122] concentration may provide a useful biomarker of ENaC activation in future clinical studies.

J Am Soc Nephrol 27: 646–656, 2016. doi: 10.1681/ASN.2014111137

Urinary exosomes are small (approximately 100 nm activity are unknown. Further characterization of diameter) vesicles excreted by multiple cell types the urinary exosome protein response to these along the nephron and urogenital tract that provide a unique source of kidney-enriched pro- teins.1,2 Urinary exosomes contain RNA and pro- Received November 25, 2014. Accepted April 22, 2015. teins, including multiple sodium channels and Published online ahead of print. Publication date available at transporters, and they may contribute to physio- www.jasn.org. 1–10 logic processes in the kidney. Whether proteins Correspondence: Dr. James M. Luther, Vanderbilt University within urinary exosomes are altered by dietary fac- Medical Center2200 Pierce Avenue, 560 RRB, Nashville, TN tors or exogenous hormones and whether these 37232-6602. Email: [email protected] changes are useful in determining physiologic Copyright © 2016 by the American Society of Nephrology

646 ISSN : 1046-6673/2702-646 J Am Soc Nephrol 27: 646–656, 2016 www.jasn.org CLINICAL RESEARCH modifying factors would inform their use in future biomarker We previously identified .3000 unique proteins from hu- discovery efforts. man urinary exosomes using multidimensional protein iden- Urinary exosomes are formed in cells lining the nephron by tification technology (MudPIT).5 In the present study, we formation of endocytic vesicles that may eventually be released tested the hypothesis that RAAS activation during a low- into the urinary space by the process of exocytosis.11 The exo- sodium (LS) diet and during exogenous aldosterone infusion some content includes membrane and soluble proteins as well alters the urinary exosome proteomic profile. We extended these as RNA, all of which may be relatively protected from degra- findings using a more targeted, sensitive, and quantitative dation in the urine by the vesicular lipid bilayer. The role of approach to investigate the profile of ENaC and NCC, which exosomes in human physiology is an area of intense investi- are known to play an essential role in renal sodium and po- gation, but within the kidney they may transport their con- tassium homeostasis. tents intercellularly, signal fibrotic responses, and perform innate immune functions.7–9 Much interest has focused on urinary exosomes as a source for biomarker discovery in hu- RESULTS mans due to the relative enrichment of membrane proteins.11 Validation of a urinary biomarker would be supported by Participant Characteristics and Effects of Dietary predictable alterations during physiologic stimulation or in- Sodium Restriction hibition. Potential approaches in humans could use dietary, We assessed the urinary exosome protein cargo using matched pharmacologic, or hormonal modification. urine samples from 14 patients during a high-sodium (HS) The renin-angiotensin-aldosterone system (RAAS) is acti- and a low-sodium (LS) diet to activate the endogenous RAAS vated in response to dietary sodium restriction, which helps and increase renal sodium reabsorption. In a separate crossover maintain long-term BP by modifying renal sodium and water study, aldosterone (0.7 mg/kg per hour) and vehicle were in- handling.12 The RAAS stimulates sodium reabsorption in part fused intravenously overnight (10 pm–8 am) as described pre- via aldosterone, leading to epithelial sodium channel (ENaC) viously,14 and urine was collected from 1 am to 7 am for and sodium chloride cotransporter (NCC) activation, which exosome isolation and analysis. Participant characteristics can be inhibited by potassium sparing (e.g., amiloride) and are presented in Table 1. During the LS diet, plasma renin thiazide diuretics, respectively.13 Because no direct measures activity and plasma aldosterone increased and urinary so- of renal ENaC activity exist in humans, it has been estimated dium excretion (198.5621.0 for HS diet versus 18.662.1 by urinary sodium-to-potassium ratio in prior studies. So- mmol/d for LS diet; P,0.001) and urinary sodium-to-potas- dium channel peptides and novel sodium channel phosphor- sium ratio (2.2060.18 versus 0.2960.05, respectively; ylation sites have been identified by proteomic analysis of P,0.001) decreased as anticipated. Urinary creatinine con- urinary exosomal proteins,1 but no studies have investigated centration (0.8460.12 mg/ml for HS diet versus 0.9560.17 their physiologic role or the dynamic changes during RAAS mg/ml for LS diet; P=0.24), creatinine excretion rate (1.596 activation in humans. 0.10 g/d versus 1.6360.13 g/d; P=0.77), urinary exosomal

Table 1. Participant characteristics and physiologic effects of dietary sodium restriction Dietary Study Aldosterone Characteristic P Value P Value HS LS HS+Vehicle HS+Aldosterone Screening measurements Age (yr) 42.963.0 44.967.8 Men/women (n/n)7/72/2 Race (white/black) (n/n) 10/4 4/0 Height (m) 1.7460.033 1.7165.5 Weight (kg) 90.967.8 98.366.4 Body mass index (kg/m2) 29.561.6 33.862.1 Creatinine (mg/dl) 0.8960.04 0.7960.09 Serum sodium (mEq/L) 139.260.49 139.560.65 Serum potassium (mEq/L) 4.060.08 3.860.10 Pre- and post-dietary measures Systolic BP (mmHg) 136.064.1 131.463.4 0.32 111.865.5 108.668.2 0.56 Diastolic BP (mmHg) 81.563.3 79.162.0 0.36 60.563.9 58.163.4 0.47 rate (beats/min) 64.562.4 67.862.8 0.004 60.365.5 57.964.7 0.27 Plasma aldosterone (ng/dl) 7.6960.81 16.161.8 ,0.001 7.561.2 83.5627.9 0.06 Plasma renin activity (ng AngI/ml per hour 0.8760.20 2.9260.49 ,0.001 1.0860.22 1.8760.31 0.04 Unless otherwise noted, values are the mean6SD.

J Am Soc Nephrol 27: 646–656, 2016 RAAS Alters Urinary Exosomes 647 CLINICAL RESEARCH www.jasn.org protein excretion (50.467.2 mg protein versus 54.266.2 mg and -related peptidase 10, among others. These pro- protein; P=0.58), diastolic BP, and systolic BP were not sig- teins are responsible for the enriched GO terms, extracellular nificantly changed by LS diet. matrix organization, extracellular structure organization, and regulation of ectodomain proteolysis in Dietary Sodium Restriction Alters Urinary Exosomal the biologic process category; serine-type peptidase activity, Protein Expression extracellular matrix structural constituent, serine Paired urine exosome samples from 14 patients during the LS activity, and serine-type activity in the molec- and HS diets were analyzed by MudPIT. We identified a total of ular function category; and extracellular-related terms in the 1,514,909 tryptic peptides, representing 34,208 unique pep- cellular component category. For the downregulated proteins, tides with scores above the minimum peptide identity thresh- the abundance of ribosomal proteins observed at lower levels olds (see Concise Methods section). After removal of proteins in the HS samples are responsible for the endoplasmic retic- with low total spectral counts (,10 spectral counts across 28 ulum (ER)- and -related GO terms and the macro- samples), 2775 proteins remained (Supplemental Table 1). molecular complex and non-membrane bound organelle Dietary sodium restriction significantly altered the expression terms in Figure 2. of 316 of 2775 (11.4%) urinary exosome proteins after adjust- ment for multiple comparisons. Of these, 113 (4.1%) in- creased and 203 (7.3%) decreased during LS diet. Hierarchical clustering demonstrated a visible distribution pattern of pro- teins clustered with dietary sodium intake (Figure 1). A high- resolution figure with protein ID annotation is available in the Supplemental Material.

Dietary Sodium Restriction Does Not Markedly Alter Urinary Exosomal Marker Abundance Intracellular vesicle trafficking proteins are enriched in exo- somes and have been proposed as specific vesicular orexosomal markers.1,6,15 As anticipated, all of the major exosome mark- ers, including multivesicular body marker TSG101 and several tetraspanin proteins (e.g., CD9, CD63, CD81, and CD82), along with proteins consistent with exosome biosynthesis, were detected in our preparations (Table 2). After correcting for multiple comparisons we found no statistically significant difference in these urinary exosome proteins during the LS diet, although several common exosomal proteins (CD9, charged multivesicular body protein 1a, charged multivestic- ular body protein 6) were altered in uncorrected analysis. Overall, our data suggest that dietary sodium intake does not markedly alter urinary exosomal protein markers, al- though the abundance of a few endosomal sorting complex required for transport III complex proteins tended to change with diet (Table 2). Therefore, we normalized protein abun- dances in each sample according to the amount of total protein injected and total spectral count from each MudPIT analysis.

Pathway Analysis Identifies Biologic Processed Altered During Dietary Sodium Restriction To examine systems associated with exosomal proteome changes during the LS diet, we performed ontology Figure 1. Low salt diet alters urinary exosomal protein expres- (GO) enrichment analysis for the 316 proteins with significant sion. Heat map of urine exosome protein expression during LS versus HS diet. The change in expression within each participant alterations. Figure 2 depicts the significantly enriched biologic (using LS as reference) for 316 proteins (vertical axis) from 14 processes, molecular functions, and cellular components as- paired samples demonstrates clustering during the HS versus the sociated with these proteins. Up- or downregulated proteins in LS diet. The change in expression within each participant is shown these GO categories are listed in the Supplemental Table 2. Of as a color-coded data point for each protein (green, decrease particular note is the upregulation of peptidase and during the HS versus LS; red, increase). A high-resolution anno- activities, as represented by upregulation by , , tated version is available in the Supplemental Material.

648 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 646–656, 2016 www.jasn.org CLINICAL RESEARCH

Table 2. Effect of diet sodium on common exosome trafficking proteins UniProtB UniProtB Corrected Protein Description Accession NSC HS NSC LS Ratio LS/HS P Value Entry Name P Value Number Tetraspanins CD9 antigen P21926 CD9 4638.32 8156.91 1.76 0.02 0.09 CD63 antigen P08962 CD63 705.26 564.59 0.80 0.89 0.96 CD81 antigen P60033 CD81 875.83 1380.07 1.58 0.15 0.36 CD82 antigen P27701 CD82 135.01 142.70 1.06 0.49 0.71 Programmed cell death 6-interacting protein Q8WUM4 PDC6I 9920.87 14,056.16 1.42 0.03 0.15 Programmed cell death protein 10 Q9BUL8 PDC10 206.55 209.94 1.02 0.78 0.90 Programmed cell death protein 6 O75340 PDCD6 1068.33 795.78 0.74 0.51 0.72 CD151 antigen P48509 CD151 90.33 40.86 0.45 0.05 0.19 ESCRT I complex Tumor susceptibility gene 101 protein Q99816 TS101 1641.37 1335.51 0.81 0.67 0.84 Vacuolar protein sorting-associated protein 28 Q9UK41 VPS28 1054.33 1314.67 1.25 0.34 0.58 homolog Vacuolar protein sorting-associated protein 37B Q9H9H4 VP37B 427.54 564.71 1.32 0.34 0.59 Vacuolar protein sorting-associated protein 37C A5D8V6 VP37C 116.08 144.65 1.25 0.34 0.59 Vacuolar protein sorting-associated protein 37D Q86XT2 VP37D 271.12 344.92 1.27 0.20 0.44 ESCRT II complex Vacuolar protein-sorting-associated protein 25 Q9BRG1 VPS25 248.01 242.40 0.98 0.90 0.96 Vacuolar protein-sorting-associated protein 36 Q86VN1 VPS36 444.21 508.17 1.14 0.33 0.57 Vacuolar-sorting protein SNF8 Q96H20 SNF8 154.65 201.85 1.31 0.16 0.38 ESCRT III complex Charged multivesicular body protein 1a Q9HD42 CHM1A 121.00 45.91 0.38 0.008 0.06 Charged multivesicular body protein 1b Q7LBR1 CHM1B 502.96 284.11 0.56 0.11 0.30 Charged multivesicular body protein 2a O43633 CHM2A 1085.69 1077.26 0.99 0.91 0.96 Charged multivesicular body protein 2b Q9UQN3 CHM2B 448.23 272.71 0.61 0.26 0.51 Charged multivesicular body protein 3 Q9Y3E7 CHMP3 104.94 195.58 1.86 0.25 0.50 Charged multivesicular body protein 4a Q9BY43 CHM4A 57.63 30.33 0.53 0.04 0.17 Charged multivesicular body protein 4b Q9H444 CHM4B 335.18 231.16 0.69 0.45 0.67 Charged multivesicular body protein 4c Q96CF2 CHM4C 59.69 79.18 1.33 0.15 0.37 Charged multivesicular body protein 5 Q9NZZ3 CHMP5 749.17 892.09 1.19 0.53 0.75 Charged multivesicular body protein 6 Q96FZ7 CHMP6 85.29 163.88 1.92 0.01 0.08 NSC, normalized spectral count; ESCT, endosomal sorting complex required for transport.

Dietary Sodium Restriction Alters Abundance of Solute (e.g., SGK-1, GILZ1, and Nedd4-2)16 were not detected in Transporter Proteins Relevant to Sodium and our MudPIT analysis. Multiple serine known to Electrolyte Transport proteolytically activate ENaC (furin, , Proteins specific to each nephron segment of the nephron and kallikrein-10)16 were detectable, and all significantly were readily detectable.5 Selected proteins with functional increased during the LS diet, whereas prostasin was un- relevance to renal electrolyte transport are presented in Ta- changed (Table 4). /plasminogen also tended to in- ble 3. Expression of proteins specifictotheproximaltubule crease, although the peptides identified do not differentiate was relatively unchanged during the LS diet, except for in- between the proenzyme and active form. Among all signifi- creased carbonic anhydrase IV abundance. Several notable cantly altered proteins, mannan-binding lectin serine proteins related to distal nephron segments were signifi- protease 2 (MASP2) was most markedly increased, al- cantly affected. During the HS diet a, b,andgENaC subu- though this protein has no previously known role in sodium nits were detected in low abundance in MudPIT analysis, homeostasis. with total spectral counts ,28 for all 28 samples. The abun- dance of a, b, and gENaC subunits increased during the LS Validation of Exosomal Protein Expression Using diet compared with the HS diet, whereas aquaporin 2 was Multiple-Reaction Monitoring Mass Spectrometry unchanged (corrected P=0.23). The thiazide-sensitive so- Because ENaC and NCC are known to be activated by RAAS dium chloride cotransporter (NCC) also increased to a lesser activation and aldosterone administration in rodents and in extent during the LS diet. Other aldosterone-responsive pro- vitro, we developed targeted multiple reaction monitoring teins known to regulate ENaC trafficking and activation (MRM) mass spectrometry assays for these solute transporters

J Am Soc Nephrol 27: 646–656, 2016 RAAS Alters Urinary Exosomes 649 CLINICAL RESEARCH www.jasn.org

Figure 2. Low salt diet alters multiple pathways as determined by WebGestalt GO enrichment analysis. GO enrichment is presented for upregulated (A) and downregulated proteins (B) by three terms: biologic processes, molecular functions,andcellular components.

(Supplemental Table 1). Protein expression levels in urine including ACE1, ACE2, and angiotensinogen, did not change exosomes were quantified using stable isotope-labeled peptide in abundance (data not shown). standards for these proteins. Four sample pairs during the HS/LS diets and four sample pairs during vehicle/aldosterone infusion were used in the DISCUSSION MRM validation study (participant characteristics shown in Table 1). The selected bENaC target peptide was not reliably Urine exosomal proteins provide a rich source of potential detected in the validation sample set. Although subject-to- biomarkers, but the effect of activation of the endogenous subject variability was significant, most results agreed with RAAS on exosomal protein cargo has not been previously MudPIT spectral counting results. The target peptide for investigated in humans. We now demonstrate that RAAS NCC[200–209] was unchanged during the LS diet or aldosterone activation modifies urinary exosome protein cargo using mass infusion (Figure 3A). The abundance of aENaC tended to in- spectrometry–based methods. Furthermore, urinary biomark- crease during the LS diet and aldosterone infusion, although ers for renal sodium channel and transporter activity in humans this effect was not statistically significant (Figure 3B). Dietary are lacking. We developed a sensitive and specific MRM mass sodium restriction significantly increased the gENaC[112–122] spectrometry method to measure a gENaC peptide which is peptide (Figure 3C), which also correlated positively with markedly altered under conditions of avid renal sodium reab- plasma aldosterone levels (Figure 3D). Other target peptides, sorption, including LS diet and aldosterone infusion.

650 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 646–656, 2016 www.jasn.org CLINICAL RESEARCH

Table 3. Selected proteins of potential relevance to renal electrolyte transport UniProtB Region/Cell Type UniProtB Corrected Accession NSC HS NSC LS Ratio LS/HS P Value Protein Description Entry Name P Value Number mTAL Solute carrier family 12 member 1 (NKCC2) Q13621 S12A1 3909.2 4059.4 1.04 0.56 0.76 DCT Solute carrier family 12 member 3 (NCC) P55017 S12A3 4836.0 7431.4 1.54 0.09 0.26 Serine/-protein kinase OSR1 O95747 OXSR1 127.5 109.0 0.86 0.76 0.89 Transient receptor potential cation channel subfamily Q9NQA5 TRPV5 97.8 80.9 0.83 0.73 0.87 V member 5 Transient receptor potential cation channel subfamily Q9H1D0 TRPV6 80.3 91.5 1.14 0.16 0.39 V member 6 CD-PC Amiloride-sensitive sodium channel, a subunit (aENaC) P37088 SCNNA 41.7 49.0 1.18 0.003 0.03 Amiloride-sensitive sodium channel, b subunit (bENaC) P51168 SCNNB 41.7 47.1 1.13 0.04 0.17 Amiloride-sensitive sodium channel, g subunit (gENaC) P51170 SCNNG 43.6 60.7 1.39 0.008 0.06 Aquaporin-2 P41181 AQP2 184.5 85.5 0.46 0.07 0.23 Solute carrier family 12 member 2 (NKCC1) P55011 S12A2 451.4 231.0 0.51 0.04 0.15 Sodium/potassium-transporting ATPase subunit a-1 P05023 AT1A1 1615.0 1469.3 0.91 0.89 0.96 Sodium/potassium-transporting ATPase subunit a-3 P13637 AT1A3 825.2 801.5 0.97 0.62 0.81 Sodium/potassium-transporting ATPase subunit b-1 P05026 AT1B1 297.2 335.8 1.13 0.20 0.44 14–3-3 protein b/a P31946 1433B 727.4 623.4 0.86 0.39 0.62 14–3-3 protein « P62258 1433E 1270.9 1399.8 1.10 0.30 0.55 CD-IC Sodium-independent sulfate anion transporter Q86WA9 S2611 62.5 66.9 1.07 0.35 0.59 Solute carrier family 12 member 2 (NKCC1) P55011 S12A2 451.4 231.0 0.51 0.04 0.15 Band 3 anion transport protein P02730 B3AT 100.6 66.0 0.66 0.36 0.60 Pendrin O43511 S26A4 1023.6 2925.8 2.86 0.004 0.04 Sodium-independent sulfate anion transporter Q86WA9 S2611 62.5 66.9 1.07 0.35 0.59 NSC, normalized spectral count; mTAL, medullary thick ascending limb; DCT, distal convoluted tubule; CD-PC, collecting duct- principal cell; CD-IC, collecting duct-intercalated cell.

Urinary exosomes are an attractive source of renal bio- The observed urinary exosome protein content in the marker proteins because they are noninvasively obtained present study generally reflects anticipated protein expression and the isolation process concentrates the proteins and changes in the nephron, with some exceptions. In particular, reduces potential interfering substances commonly encoun- the gENaC[112–122] peptide abundance increased markedly tered in urinary proteomics. The relationship between during the LS diet and after aldosterone infusion, whereas exosomal protein expression and renal protein expression the increase in aENaC was not significant and NCC expres- remains undefined and cannot be directly addressed in the sion did not change appreciably. Aldosterone can increase re- present study. The analysis of ENaC and NCC proteins may nal ENaC activity by increasing a-subunit expression and provide some insight, however, because RAAS activation is increasing cell surface localization. The aENaC and gENaC known to increase protein expression of aENaC and subunits can also be activated via protease-dependent re- NCC.16,17 moval of inhibitory peptides, and the cleaved forms increase

Table 4. Proteins and proteases potentially involved in sodium channel activation UniProt UniProt Corrected Protein Description HS NSC LS NSC Ratio LS/HS P Value Accession Number Entry Name P Value 14–3-3 protein sigma P31947 1433S 1417.8 630.3 0.44 6.4E-4 0.01 Prostasin Q16651 PRSS8 361.9 315.5 0.87 0.97 0.99 Furin P09958 FURIN 45.8 66.3 1.45 1.5E-3 0.02 Neutrophil elastase P08246 ELNE 610.3 1144.4 1.88 2.8E-3 0.03 Mannan-binding lectin 2 O00187 MASP2 432.5 2352.2 5.44 5.4E-8 4.8E-5 Kallikrein-10 O43240 KLK10 64.0 108.5 1.70 2.4E-3 0.03 NSC, normalized spectral count.

J Am Soc Nephrol 27: 646–656, 2016 RAAS Alters Urinary Exosomes 651 CLINICAL RESEARCH www.jasn.org

proteases, MASP2 increased most significantly. MASP2 has been identified as 1 of 13 active serine in human urine but has not been linked to activation of sodium chan- nels.22 MASP2 activates the innate immune system by associating with mannan-binding lectin and activating the terminal complement cascade, and MASP2 deficiency in hu- mans is associated with severe pneumococcal infection and autoimmune disease.23,24 Because the RAAS also increases inflammatory and profibrotic mediators such as inhibitor-1,25 MASP2 may provide an additional link between the RAAS and inflammatory pathways. In patients with hyperaldosteronism, NCC and phosphor- ylated NCC are increased in urinaryexosomes, and in rats acute aldosterone administration increases phosphorylated NCC, NCC, and prostasin excretion.21 In our study, NCC abundance increased during the LS diet in our MudPITanalysis, although this finding was not validated using MRM methods or during aldosterone infusion. We also did not observe any change in urinary prostasin during LS diet. Although we searched for Figure 3. Endogenous renin-angiotensin-aldosterone system NCC phosphopeptides in the MudPIT analysis, none were activation and exogenous aldosterone increase urinary exosomal detected. Identification of phosphorylated peptides gENaC excretion. Effect of LS and HS diet on urinary exosome requires a much larger starting urine volume, urine collection peptide abundance for NCC (A), aENaC (B), and gENaC (C). with a phosphatase inhibitor, and phosphoenrichment to in- gENaC abundance in urinary exosomes correlated strongly with crease the sensitivity of this method.1 Our model of RAAS , plasma aldosterone. P 0.001 for linear correlation (D). Aldo, al- activation differs significantly from primary aldosteronism dosterone. with regard to species and pathophysiology and duration of activation, which could explain differences in NCC or prosta- in abundance during aldosterone administration.17–19 The sin. For example, short-term RAAS activation during periods gENaC[112–122] sequence assayed in this study is of dietary sodium restriction (secondary aldosteronism) pre- located in the extracellular N-terminal domain of gENaC, vents hypotension, whereas prolonged aldosterone excess near a putative cleavage site. Cleavage and removal of the (primary aldosteronism) produces hypertension. inhibitory peptide by proteases, such as furin, prostasin, The urinary exosomal gENaC[112–122] biomarker could kallikrein, plasmin, elastase, or CAP2, maximally activate provide a useful estimate of ENaC activation in future clinical murine ENaC in vitro, although it is not clear from the pres- studies. The urinary sodium-to-potassium ratio has been pre- ent studies if cleavage directly affects gENaC[112–122] abun- viously used in clinical studies to estimate ENaC activity, but dance.16,20 Other potential explanations for the increased this measure is not specific for ENaC and can be affected by abundance of this gENaC[112–122] peptide include increased other variables, such as altered potassium intake. Nasal poten- absolute gENaC production and urinary excretion, or more tial difference has also been used to estimate ENaC activity, efficient digestion after gENaC is proteolytically although it has not been proven to reflect renal ENaC activ- 26 cleaved in vivo. Although this gENaC[112–122] biomarker ity. In animal studies, Western blot analysis of ENaC subu- may be useful in humans, this peptide region is variable nits in kidney tissue has been extensively used to measure across species, so an analogous peptide sequence would be expression, but obtaining adequate renal tissue for this mea- needed in rodents. Further studies under different treatment surement in clinical studies is impractical. Western blot anal- conditions are warranted to determine the source of the in- ysis for ENaC has been performed on urinary exosomal and crease in target peptide levels and to explore the clinical total urinary protein in prior clinical studies,27,28 but these utility of this measure. have reported low sensitivity and none have demonstrated a Multiple serine proteases known to cleave gENaC were significant response during RAAS activation. The gENaC[112–122] readily detectable within urinary exosomes by MudPIT anal- biomarker identified in the present study was detected using a ysis, including plasmin, , , furin, and more sensitive and specificMRMmethod,increasedmarkedly prostasin. Although prostasin is increased by aldosterone in during exogenous and endogenous RAAS activation, and in- rodents and has previously been identified as a marker of creased within 1 day to 1 week of stimulation. hyperaldosteronism in humans,21 we observed no significant Although unbiased MudPIT proteomic analysis has the increase during the LS diet. The proteases furin, neutrophil advantage of discovering previously unknown relationships, elastase, and kallikrein-10 were significantly increased by the this approach also requires validation to verify significant LS diet, even after correction for multiple testing. Among all relationships. We performed validation studies of the relevant

652 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 646–656, 2016 www.jasn.org CLINICAL RESEARCH sodium channels using MRM analysis and stable isotope- the study, as described previously.29 We provided participants with a labeled standards in a separate cohort of individuals who diet containing 10 mmol (LS) or 200 mmol (HS) sodium/day for 7 received aldosterone infusion or LS diet. Targeted MRM days prepared by the Vanderbilt clinical research center kitchen under analysis is less costly, less time-consuming, and more sensitive the guidance of a registered dietitian. After presenting to the clinical than MudPIT analysis. Analysis with stable isotope-labeled research center at 7 A.M., urine samples were collected immediately standards allows accurate identification and quantification of into a protease inhibitor cocktail as previously described,4,5 and sam- target peptides and also overcomes the difficulty encountered ples were stored at 280°C until processing. Fourteen paired samples with -based methods due to nonspecific binding. were available for analysis by MudPIT and four for MRM. Although it is not feasible to perform such targeted analysis of all proteins, this general approach can be followed in future Aldosterone Infusion studies to generate custom MS assays targeting pathways of Urine exosome samples were collected from healthy participants who interest, potentially in a single multiplex assay. Another were recruited and enrolled as previously reported.14 Participants limitation of the MudPIT analysis is the high inter-subject reported to the Vanderbilt Clinical Research Center on the fifth and variation for individual protein expression, which greatly seventh evenings of a 160-mmol/day sodium controlled diet. Partici- reduces the ability to detect meaningful changes. This limi- pants were administered aldosterone (0.7 mg/kg per hour in 5% dex- tation was overcome in the present study by using a cross-over trose; Professional Compounding Corporation of America) or vehicle design in which paired samples were used to eliminate the by intravenous infusion between 10:00 P.M. and 08:00 A.M. as previously between-subject variation. For future studies, a similar ap- described and returned for the second study day for the remaining proach may be used to identify significantly altered proteins infusion.14 The order of drug administration was randomized and dou- and to develop sensitive and specific MRM methods before ble-blinded by Vanderbilt Investigational Drug Services. BP was mon- widespread application in heterogeneous populations. itored hourly during drug infusion (Dinamap, GE Medical). Serum The present study provides a proof-of-principle concept potassium was monitored throughout the drug infusion, and oral po- that urinaryexosome protein expression is altered by the renin- tassium chloride was administered as needed to maintain serum potas- angiotensin-aldosterone system, with a significant increase in sium at a level of $3.8 mEq/L. We collected all urine after 3 hours of gENaC[112–122] peptide and urinary proteases. Thus, appro- drug infusion (between 1:00 A.M. and 7:00 A.M.) into a protease inhibitor priate selection of urinary exosomal protein biomarkers may cocktail as described previously and stored at 280°C until processing.2 provide insight into renal physiology. Selection of specificpep- Four paired urine samples were available for analysis. tide targets within individual proteins, such as peptides adja- cent to proteolytic cleavage sites or phosphopeptides may Laboratory Assays provide a more sensitive measure than overall protein expres- Screening electrolytes and lipid panels were performed in the sion alone. We propose that exosomal gENaC[112–122] is a bio- Vanderbilt clinical laboratory. Plasma aldosterone and plasma renin marker of proteolytically activated ENaC. Identification of activity were measured by radioimmunoassay as described pre- additional specific biomarkers may require identification of viously.25,29 Urine sodium and potassium concentrations were mea- modified peptides, such as phosphorylated forms, which is sured by flame photometry and creatinine by the sodium picrate technically more difficult. Our study also demonstrates the method.25,29 importance of controlling for dietary sodium intake when analyzing urinary biomarkers, which may prove difficult in Urine Exosome Isolation translating urinary biomarker discovery into broader clinical Urine samples were thawed and exosomes isolated as previously applications. described.1 Briefly, urine specimens were thawed with running tap water, separated into aliquots, and centrifuged at 17,000 g for 20 minutes at 4°C. The 17,000 g supernatant was ultracentrifuged at CONCISE METHODS 200,000 g for 1 hour at 24°C and the pellet was saved. The centrifu- gation steps were repeated on each aliquot until all of the urine was Clinical Study Protocols processed. The pellets from all aliquots of the same patient sample All studies were approved by the Vanderbilt University Institutional were pooled together and resuspended in isolation solution (10 mM Review Board and conducted in accordance with the Declaration of triethanolamine and 250 mM sucrose). To denature and remove ex- Helsinki. Informed consent was obtained, and participants cess uromodulin (Tamm-Horsfall protein),which can co-sediment underwent a screening history and physical before study enrollment. with exosomes in the 200,000 g centrifugation step, the resuspended Participants with a history of coronary artery disease, diabetes, renal pellet was mixed with 200 mg/ml dithiothreitol (DTT) and incubated insufficiency (eGFR,60 ml/min per 1.73 m2), anemia, major med- at 95°C for 2 minutes, then diluted 1:20 with isolation solution.15 The ical problems, or inability to comply with the protocol were excluded. sample was centrifuged at 17,000 g for 20 minutes at 4°C, and the resulting supernatant was ultracentrifuged at 200,000 g for 1 hour at fi Dietary Sodium Restriction 24°C. The nal pellet was suspended in 100 ml of HPLC-grade H2O Participants with mild-to-moderate hypertension were recruited and and frozen at 280°C. The protein concentration of exosome prepa- washed out from antihypertensive medications for $3 weeks before rations was measured with the BCA protein assay kit (Pierce).

J Am Soc Nephrol 27: 646–656, 2016 RAAS Alters Urinary Exosomes 653 CLINICAL RESEARCH www.jasn.org

Liquid Chromatography-Tandem Mass Spectometry precursor ion mass-to-charge ratio and all other spectra were pro- Analysis cessed to form .dta files for both doubly and triply charged precursor Exosome protein from each sample (25 mg) was dried using a Speed- ions. The .dta files were searched against a human subset of the Uni- Vac and reduced with 20 mM DTT in 50 mM NH4HCO3,50%tri- prot KB protein database (June 2012 release) with a total of 20,360 fluoroethanol (TFE) at 56°C for 45 minutes, followed by alkylation protein entries using the Sequest algorithm.31 The search was trypsin with 40 mM iodoacetamide in the dark for 1 hour at room temper- strict and a precursor mass tolerance of 62.5 Da was used. The ature. DTT, 50 mmol, was added to the sample for another hour in searches were performed allowing for the following differential mod- the dark to destroy excess iodoacetamide. The samples were diluted ifications: +57 on (for carboxyamidomethylation) and +16 5-fold with 50 mM NH4HCO3 to reduce the TFE concentration to on (oxidation). A concatenated database of forward and 5% and were subjected to enzymatic digestion for 12–16 hours at reverse sequences was constructed to allow determination of false 37°C using trypsin gold (1:25 trypsin-to-exosome protein ratio; discovery rates. The search results were imported into ProteoIQ soft- Promega, Madison, WI). The digestion was stopped by adding 2 ml ware (Premier Biosoft, Palo Alto, CA) for comparative proteomics of 100% formic acid. The trypsin-digested samples were centrifuged analysis. Results were filtered using minimum Xcorr scores of 1.5 for at 20,000 g for 20 minutes at 4°C, the supernatant was saved, and the +1 precursor ions, 2.5 for +2 precursor ions, and 3.5 for $+3 pre- pellets were extracted twice with 50 ml of 50% acetonitrile/0.1% for- cursor ions, respectively, a minimum of two peptides per protein, an mic acid. Extracts and the supernatant were pooled and dried by average of two spectral counts per protein, and an overall maximum SpeedVac. Dried samples were reconstituted with 100 mlofHPLC false discovery rate of ,0.05 in each sample. water with 0.1% formic acid (FA), centrifuged at 215,000 g for 20 minutes at 4°C, and the supernatant containing tryptic peptides MRM Analysis 2 was collected and stored at 80°C until liquid chromatography-mass For MRM analysis, 10 mg of exosomal protein, isolated from each spectometry analysis. patient urine sample, was placed in 20 mM Tris, 40% TFE, 18.75 mM The tryptic peptides were analyzed using an LTQ Velos mass tris(2-carboxyethyl)phosphine) for reduction at room temperature fi spectrometer (Thermo Fisher Scienti c) with a 12-step MudPIT for 1 hour, followed by alkylation using 75 mM iodoacetamide in the 5 fl analysis as previously described. Brie y, tryptic peptides were loaded dark for 45 minutes. Samples were diluted by adding 3 volumes of 3 onto a 4 cm 150 mm internal diameter (ID) microcapillary fused 100 mM Tris and digested with 1 mgoftrypsinat37°Covernight. silica pre-column packed in-house with C18 resin (Jupiter C18, 5-mm Tryptic peptides were dried by SpeedVac. Isotope-labeled peptide particle size, 300-Å pore size) followed by 6 cm of strong cation- standards and three other external control peptides were spiked into exchange resin (Luna SCX, 5-mm particle size, 100-Å pore size). The the samples in a final volume of 20 ml. The samples were diluted into fl 2D trap column was coupled to a nano ow capillary analytical col- 140 ml of 0.1% trifluoroacetic acid (TFA) and acidified to final pH#3 umn (100 mm ID) packed with 10 cm of 3-mm C18 reverse-phase using 50% TFA followed by a clean-up step using homemade mini- resin (Jupiter C18, 3-mm particle size, 300-Å pore size) constructed C18 cartridges. In brief, a disc of C18 filter membrane (2215-C18 solid with a laser-pulled electrospray emitter tip. Multidimensional sepa- phase extraction, Disk Empore; Chrom Tech Inc.) was cored with a rations were performed using 5-mlpulsesofammoniumacetate 16-gauge needle and the cored piece was fitted tightly into a 200-ml (0, 25, 50, 75, 100, 150, 200, 250, 300, 500, 750, and 1000 mM pipette tip. Three milligrams of C18 (Jupiter C18, 5-mm particle size, ammonium acetate) in 0.1% FA delivered by an autosampler. After 300-Å pore size) resin, suspended in 200 ml of methanol, was loaded each salt pulse, the peptides were eluted with a 105-minute reverse phase into the pipette tip and spun at 3600 g for 1 minute at room temper- solvent gradient from 2% acetonitrile (ACN)/0.1% FA to 45% ACN/ ature to form a mini-C18 cartridge. The mini-C18 cartridges were fi 0.1% FA for the rst 10 salt pulses and a reverse solvent gradient from equilibrated with 600 ml 0.1% TFA in HPLC water. Five micrograms 2% ACN/0.1% FA to 95% ACN/ 0.1% FA for 1 M salt pulse at a 0.5-ml of digested urine exosome protein was loaded on the cartridge, fl per minute ow rate. Gradient-eluted peptides were introduced into the centrifuged at 3600 g for 1.5 minutes at room temperature, and LTQ Velos instrument via a nano-electrospray ionization source. washed three times with 0.1% TFA. The sample was eluted twice The LTQ Velos mass spectrometer was operated in data-dependent with 300 ml of 80% ACN/0.1% TFA. All eluted peptides were pooled fi mode in which the rst an initial full MS scan recorded the mass-to- and dried by SpeedVac. The sample was solubilized in 20 ml of 0.1% – charge ratios of ions over the mass-to-charge range of 300 2000, and vol/vol formic acid before MRM analysis. fi the ve most abundant ions were automatically selected for subse- Proteotypic peptides detected in MudPIT analysis were selected quent collision-induced dissociation. Dynamic exclusion (repeat through bioinformatics analysis as guided by Kuzyk et al.32 C-terminal count 1, exclusion list size 150, and exclusion duration 60 seconds) [13C]/[15N] labeled heavy peptides (AQUA) were synthesized and was enabled to allow detection of less abundant ions. purified by Sigma-Aldrich. Upon receipt, the quantity and quality of each isotope labeled peptide were evaluated by matrix-assisted Data Analysis of MudPIT laser desorption/ionization and LC-MS/MS analysis. The following Liquid chromatography-tandem mass spectometry (LC-MS/MS) raw isotopically labeled internal control peptides were included: SGGTY- files were converted into .dta files by the ScanSifter algorithm.30 Spec- FLISR (NCC) at 50 fmol, HLLADLEQETR (gENaC) at 50 fmol, tra that contained ,25 peaks or that had ,2e1 measured total ion AEQNDFIPLLSTVTGAR (aENaC) at 250 fmol, and three other ex- current were not converted. The .dta files for singly charged precursor ternal control peptides at 10 fmol (SSAAPPPPPR, TASEFDSAIAQDK, ions were created if 90% of the total ion current occurred below the and LTILEELR). A list of MRM transitions for the selected target

654 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 646–656, 2016 www.jasn.org CLINICAL RESEARCH peptides (Supplemental Table 1) was generated by Skyline software Core), UL1-RR024975 and -TR000445 from NCATS/NIH (Vander- based on product ions detected in MudPIT analyses.33 The identi- bilt Institute for Clinical and Translational Research), and the Van- ties of the target peptides were confirmed according to retention derbilt Mass Spectrometry Research Center. time equivalence and transition intensity equivalence to the isotope- This research was presented in abstract form at the American labeled peptide. Society of Nephrology Renal Week 2013. Target peptides were analyzed by a 60-minute scheduled MRM LC- MS/MS analysis. Peptides were loaded onto a 40 mm30.1 mm (Jupiter C18, 5-mm particle size, 300-Å pore size) kasil fritted trap DISCLOSURES 3 column that was connected inline to a 200 mm 0.1 mm (Jupiter None. C18, 3-mm particle size, 300-Å pore size), self-packed analytical col- umn using a NanoAcuity HPLC system (Waters). After trapping and fl equilibration, peptides were gradient-eluted at a ow rate of 400 nl/ REFERENCES min using a 60-minute gradient from 1% ACN/0.1% FA to 45% ACN/0.1% FA followed by a 5-minute ramp to 90% ACN/0.1% FA. 1. Gonzales PA, Pisitkun T, Hoffert JD, Tchapyjnikov D, Star RA, Kleta R, The column effluent was delivered directly to a triple quadrupole Wang NS, Knepper MA: Large-scale proteomics and phosphoproteo- mass spectrometer (TSQ-Vantage; Thermo Fisher Scientific) via a mics of urinary exosomes. J Am Soc Nephrol 20: 363–379, 2009 fi fi nano-electrospray source. A scheduled MRM method was developed 2. Pisitkun T, Shen RF, Knepper MA: Identi cation and proteomic pro ling of exosomes in human urine. Proc Natl Acad Sci U S A 101: 13368– according to a series of unscheduled scouting experiments. The 13373, 2004 scheduled method used 8-minute windows around the measured re- 3. Gonzalez-Calero L, Martin-Lorenzo M, Alvarez-Llamas G: Exosomes: A tention time for each peptide. Q1 peak width resolution was set to 0.7, potential key target in cardio-renal syndrome. Front Immunol 5: 465, collision gas pressure was 1 mTorr, and an EZmethod cycle time of 2014 3 seconds was used. The resulting raw data files were imported into 4. Stamer WD, Hoffman EA, Luther JM, Hachey DL, Schey KL: Protein profile of exosomes from trabecular meshwork cells. J Proteomics 74: Skyline Software, version 1.1, and processed for peak integration and 796–804, 2011 quantitation. All data were manually inspected to ensure correct peak 5. Wang Z, Hill S, Luther JM, Hachey DL, Schey KL: Proteomic analysis of detection and accurate integration. Target peptide peak areas were urine exosomes by multidimensional protein identification technology normalized to their isotopically labeled analogs. (MudPIT). Proteomics 12: 329–338, 2012 6. Zhou H, Yuen PS, Pisitkun T, Gonzales PA, Yasuda H, Dear JW, Gross P, Knepper MA, Star RA: Collection, storage, preservation, and normali- Statistical Analyses zation of human urinary exosomes for biomarker discovery. Kidney Int Spectral count, or the total number of MS/MS spectra taken on 69: 1471–1476, 2006 7. Borges FT, Melo SA, Özdemir BC, Kato N, Revuelta I, Miller CA, peptides from a given protein in a given LC2MS/MS analysis, was b fi Gattone VH 2nd, LeBleu VS, Kalluri R: TGF- 1-containing exosomes used as the basis for protein quanti cation in MUDPITexperiments. from injured epithelial cells activate fibroblasts to initiate tissue re- Spectral count is linearly correlated with the protein abundance generative responses and fibrosis. J Am Soc Nephrol 24: 385–392, over a large dynamic range,34 and this simple but effective quantifi- 2013 cation method has found broad application in detecting differential 8. Hiemstra TF, Charles PD, Gracia T, Hester SS, Gatto L, Al-Lamki R, Floto 35–39 RA, Su Y, Skepper JN, Lilley KS, Karet Frankl FE: Human urinary exosomes or correlated protein expression. We used the global normaliza- – fi as innate immune effectors. J Am Soc Nephrol 25: 2017 2027, 2014 tion method to make the total numbers of identi ed spectra compa- 9. Hogan MC, Manganelli L, Woollard JR, Masyuk AI, Masyuk TV, rable across all samples.40 For statistical comparison between the LS Tammachote R, Huang BQ, Leontovich AA, Beito TG, Madden BJ, group and the HS group, we modified a previously published quasi- Charlesworth MC, Torres VE, LaRusso NF, Harris PC, Ward CJ: Char- likelihood generalized linear model in order to handle paired data.38 acterization of PKD protein-positive exosome-like vesicles. JAmSoc Nephrol 20: 278–288, 2009 The analysis was limited to proteins with at least 10 total spectral fi 10. Dear JW, Street JM, Bailey MA: Urinary exosomes: A reservoir for counts across the 28 samples to avoid quanti cation uncertainty as- biomarker discovery and potential mediators of intrarenal signalling. sociated with low-abundance proteins. Calculated P values based on Proteomics 13: 1572–1580, 2013 the quasi-likelihood model were further adjusted by the Benjamini- 11. van Balkom BW, Pisitkun T, Verhaar MC, Knepper MA: Exosomes and Hochberg procedure to correct for multiple testing.41 GO enrich- the kidney: Prospects for diagnosis and therapy of renal diseases. Kidney Int 80: 1138–1145, 2011 ment analysis was performed in WebGestalt by searching the 316 12. Lifton RP, Gharavi AG, Geller DS: Molecular mechanisms of human altered proteins (113 upregulated and 203 downregulated, searched hypertension. Cell 104: 545–556, 2001 separately) against the 2775 identified proteins in this study as the 13. Rozansky DJ, Cornwall T, Subramanya AR, Rogers S, Yang YF, David LL, reference set.42,43 Zhu X, Yang CL, Ellison DH: Aldosterone mediates activation of the thiazide-sensitive Na-Cl cotransporter through an SGK1 and WNK4 signaling pathway. JClinInvest119: 2601–2612, 2009 14. Luther JM, Byrne LM, Yu C, Wang TJ, Brown NJ: Dietary sodium re- ACKNOWLEDGMENTS striction decreases insulin secretion without affecting insulin sensitivity in humans. JClinEndocrinolMetab99: E1895–E1902, 2014 15. Fernández-Llama P, Khositseth S, Gonzales PA, Star RA, Pisitkun T, This work was supported by National Institutes of Health grants Knepper MA: Tamm-Horsfall protein and urinary exosome isolation. HL100016, DK081662, DK38226, DK20593 (DRTC Hormone Assay Kidney Int 77: 736–742, 2010

J Am Soc Nephrol 27: 646–656, 2016 RAAS Alters Urinary Exosomes 655 CLINICAL RESEARCH www.jasn.org

16. Soundararajan R, Pearce D, Hughey RP, Kleyman TR: Role of epithelial GR, Liebler DC: Supporting tool suite for production proteomics. Bio- sodium channels and their regulators in hypertension. J Biol Chem 285: informatics 27: 3214–3215, 2011 30363–30369, 2010 31. Eng JK, McCormack AL, Yates JR: An approach to correlate tandem 17. Masilamani S, Kim GH, Mitchell C, Wade JB, Knepper MA: Aldoste- mass spectral data of peptides with amino acid sequences in a protein rone-mediated regulation of ENaC alpha, beta, and gamma subunit database. J Am Soc Mass Spectrom 5: 976–989, 1994 proteins in rat kidney. J Clin Invest 104: R19–R23, 1999 32. Kuzyk MA, Smith D, Yang J, Cross TJ, Jackson AM, Hardie DB, 18. Uchimura K, Kakizoe Y, Onoue T, Hayata M, Morinaga J, Yamazoe R, Anderson NL, Borchers CH: Multiple reaction monitoring-based, mul- Ueda M, Mizumoto T, Adachi M, Miyoshi T, Shiraishi N, Sakai Y, Tomita tiplexed, absolute quantitation of 45 proteins in human plasma. Mol K, Kitamura K: In vivo contribution of serine proteases to the proteolytic Cell Proteomics 8: 1860–1877, 2009 activation of gENaC in aldosterone-infused rats. Am J Physiol Renal 33. MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, Physiol 303: F939–F943, 2012 Frewen B, Kern R, Tabb DL, Liebler DC, MacCoss MJ: Skyline: An open 19. Frindt G, Palmer LG: Acute effects of aldosterone on the epithelial Na source document editor for creating and analyzing targeted proteo- channel in rat kidney. Am J Physiol Renal Physiol 308: F572–F578, 2015 mics experiments. Bioinformatics 26: 966–968, 2010 20. Svenningsen P, Friis UG, Bistrup C, Buhl KB, Jensen BL, Skøtt O: 34. Liu H, Sadygov RG, Yates JR 3rd: A model for random sampling and Physiological regulation of epithelial sodium channel by proteolysis. estimation of relative protein abundance in shotgun proteomics. Anal Curr Opin Nephrol Hypertens 20: 529–533, 2011 Chem 76: 4193–4201, 2004 21. van der Lubbe N, Jansen PM, Salih M, Fenton RA, van den Meiracker 35. Halvey PJ, Zhang B, Coffey R, Liebler DC, Slebos RJ: Proteomic con- AH, Danser AH, Zietse R, Hoorn EJ: The phosphorylated sodium sequences of a single gene in a colorectal model. J chloride cotransporter in urinary exosomes is superior to prostasin as a Proteome Res 11: 1184–1195, 2012 marker for aldosteronism. Hypertension 60: 741–748, 2012 36. Kislinger T, Cox B, Kannan A, Chung C, Hu P, Ignatchenko A, Scott MS, 22. Navarrete M, Ho J, Krokhin O, Ezzati P, Rigatto C, Reslerova M, Rush Gramolini AO, Morris Q, Hallett MT, Rossant J, Hughes TR, Frey B, Emili DN, Nickerson P, Wilkins JA: Proteomic characterization of serine hydro- A: Global survey of organ and organelle protein expression in mouse: lase activity and composition in normal urine. Clin Proteomics 10: 17, 2013 Combined proteomic and transcriptomic profiling. Cell 125: 173–186, 23. Stengaard-Pedersen K, Thiel S, Gadjeva M, Møller-Kristensen M, 2006 Sørensen R, Jensen LT, Sjøholm AG, Fugger L, Jensenius JC: Inherited 37. Zhang B, VerBerkmoes NC, Langston MA, Uberbacher E, Hettich RL, deficiency of mannan-binding lectin-associated serine protease 2. Samatova NF: Detecting differential and correlated protein expres- NEnglJMed349: 554–560, 2003 sion in label-free shotgun proteomics. J Proteome Res 5: 2909–2918, 24. Thiel S, Vorup-Jensen T, Stover CM, Schwaeble W, Laursen SB, Poulsen 2006 K, Willis AC, Eggleton P, Hansen S, Holmskov U, Reid KB, Jensenius JC: 38. Li M, Gray W, Zhang H, Chung CH, Billheimer D, Yarbrough WG, Liebler A second serine protease associated with mannan-binding lectin that DC, Shyr Y, Slebos RJ: Comparative shotgun proteomics using spectral activates complement. Nature 386: 506–510, 1997 count data and quasi-likelihood modeling. J Proteome Res 9: 4295– 25. Luther JM, Gainer JV, Murphey LJ, Yu C, Vaughan DE, Morrow JD, 4305, 2010 Brown NJ: Angiotensin II induces interleukin-6 in humans through a 39. Ning K, Fermin D, Nesvizhskii AI: Comparative analysis of different la- mineralocorticoid receptor-dependent mechanism. Hypertension 48: bel-free mass spectrometry based protein abundance estimates and 1050–1057, 2006 their correlation with RNA-Seq data. JProteomeRes 26. Rowe SM, Liu B, Hill A, Hathorne H, Cohen M, Beamer JR, Accurso FJ, 11: 2261–2271, 2012 Dong Q, Ordoñez CL, Stone AJ, Olson ER, Clancy JP; VX06-770-101 40. Liu Q, Halvey PJ, Shyr Y, Slebos RJ, Liebler DC, Zhang B: Integrative Study Group: Optimizing nasal potential difference analysis for CFTR omics analysis reveals the importance and scope of translational re- modulator development: Assessment of ivacaftor in CF subjects with pression in microRNA-mediated regulation. Mol Cell Proteomics 12: the G551D-CFTR mutation. PLoS ONE 8: e66955, 2013 1900–1911, 2013 27. Esteva-Font C, Wang X, Ars E, Guillén-Gómez E, Sans L, González 41. Benjamini Y, Hochberg Y: Controlling the false discovery rate: A prac- Saavedra I, Torres F, Torra R, Masilamani S, Ballarín JA, Fernández- tical and powerful approach to multiple testing. J R Stat Soc B 57(1): Llama P: Are sodium transporters in urinary exosomes reliable markers 289–300, 1995 of tubular sodium reabsorption in hypertensive patients? Nephron, 42. Wang J, Duncan D, Shi Z, Zhang B: WEB-based GEne SeT AnaLysis Physiol 114: 25–34, 2010 Toolkit (WebGestalt): Update 2013. Nucleic Acids Res 41: W77-83, 28. Rennings AJ, Russel FG, Li Y, Deen PM, Masereeuw R, Tack CJ, Smits P: 2013 Preserved response to diuretics in rosiglitazone-treated subjects with 43. Zhang B, Kirov S, Snoddy J: WebGestalt: An integrated system for insulin resistance: A randomized double-blind placebo-controlled exploring gene sets in various biological contexts. Nucleic Acids Res crossover study. Clin Pharmacol Ther 89: 587–594, 2011 33: W741– W748, 2005 29. Gilbert K, Nian H, Yu C, Luther JM, Brown NJ: Fenofibrate lowers blood pressure in salt-sensitive but not salt-resistant hypertension. JHyper- tens 31: 820–829, 2013 30. Ma ZQ, Tabb DL, Burden J, Chambers MC, Cox MB, Cantrell MJ, Ham This article contains supplemental material online at http://jasn.asnjournals. AJ, Litton MD, Oreto MR, Schultz WC, Sobecki SM, Tsui TY, Wernke org/lookup/suppl/doi:10.1681/ASN.2014111137/-/DCSupplemental.

656 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 646–656, 2016 Supplemental Table 1. MRM transitions for targeted peptides.

Precursor m/z Transitions Peptide Sequence (charge) (ions, m/z)

(2+) y9 y9 y8 y7 y6 y5 y4 NCC SGGTYFLISR 550.8 (2+) 1013.5 507.3 956.5 899.5 798.5 635.4 488.3

y12 y11 y10 y8 y7 y6 αENaC AEQNDFIPLLSTVTGAR 916.5 (2+) 1274.7 1127.7 1014.6 804.5 691.4 604.3

y10 y9 y8 y7 y6 y4 γENaC HLLADLEQETR 662.8 (2+) 1187.6 1074.5 961.5 890.4 775.4 533.3

Supplemental Table 2. Significantly altered proteins Ratio Adjusted Protein Annotation Protein Name/Description LS/HS p -value 1 sp|Q7L3S4|ZN771_HUMAN Zinc finger protein 771 OS=Homo sapiens GN=ZNF771 PE=1 SV=1 2.56 0.00000 2 sp|Q5SSJ5|HP1B3_HUMAN Heterochromatin protein 1-binding protein 3 OS=Homo sapiens GN=HP1BP3 PE=1 SV=1 0.37 0.00000 3 sp|O00187|MASP2_HUMAN Mannan-binding lectin serine protease 2 OS=Homo sapiens GN=MASP2 PE=1 SV=4 5.44 0.00005 4 sp|Q6NT55|CP4FN_HUMAN Cytochrome P450 4F22 OS=Homo sapiens GN=CYP4F22 PE=2 SV=1 0.43 0.00005 5 sp|Q92556|ELMO1_HUMAN Engulfment and cell motility protein 1 OS=Homo sapiens GN=ELMO1 PE=1 SV=2 0.51 0.00005 6 sp|P40394|ADH7_HUMAN Alcohol dehydrogenase class 4 mu/sigma chain OS=Homo sapiens GN=ADH7 PE=1 SV=2 0.45 0.00005 7 sp|Q9C075|K1C23_HUMAN , type I cytoskeletal 23 OS=Homo sapiens GN=KRT23 PE=1 SV=2 0.16 0.00005 8 sp|Q9BXJ7|AMNLS_HUMAN Protein amnionless OS=Homo sapiens GN=AMN PE=1 SV=2 1.29 0.00008 9 sp|Q92619|HMHA1_HUMAN Minor histocompatibility protein HA-1 OS=Homo sapiens GN=HMHA1 PE=1 SV=2 0.43 0.00008 10 sp|P49411|EFTU_HUMAN Elongation factor Tu, mitochondrial OS=Homo sapiens GN=TUFM PE=1 SV=2 0.37 0.00014 11 sp|Q9ULG6|CCPG1_HUMAN Cell cycle progression protein 1 OS=Homo sapiens GN=CCPG1 PE=1 SV=3 0.08 0.00021 12 sp|P52272|HNRPM_HUMAN Heterogeneous nuclear ribonucleoprotein M OS=Homo sapiens GN=HNRNPM PE=1 SV=3 0.23 0.00030 13 sp|K1HA_HUMAN| sp|K1HA_HUMAN| 0.19 0.00030 14 sp|Q15274|NADC_HUMAN Nicotinate- pyrophosphorylase [carboxylating] OS=Homo sapiens GN=QPRT PE=1 SV=3 3.06 0.00031 15 sp|P13639|EF2_HUMAN Elongation factor 2 OS=Homo sapiens GN=EEF2 PE=1 SV=4 0.47 0.00039 16 sp|Q9H2M9|RBGPR_HUMAN Rab3 GTPase-activating protein non-catalytic subunit OS=Homo sapiens GN=RAB3GAP2 PE=1 SV=1 0.48 0.00041 17 sp|Q8NI99|ANGL6_HUMAN Angiopoietin-related protein 6 OS=Homo sapiens GN=ANGPTL6 PE=1 SV=1 4.42 0.00043 18 sp|P19878|NCF2_HUMAN Neutrophil cytosol factor 2 OS=Homo sapiens GN=NCF2 PE=1 SV=2 0.38 0.00047 19 sp|Q9BRT3|MIEN1_HUMAN Migration and invasion enhancer 1 OS=Homo sapiens GN=MIEN1 PE=1 SV=1 2.15 0.00050 20 sp|Q5VVQ6|OTU1_HUMAN Ubiquitin thioesterase OTU1 OS=Homo sapiens GN=YOD1 PE=1 SV=1 0.35 0.00050 21 sp|Q9H0P0|5NT3_HUMAN Cytosolic 5'- 3 OS=Homo sapiens GN=NT5C3 PE=1 SV=3 0.50 0.00050 22 sp|Q13508|NAR3_HUMAN Ecto-ADP-ribosyltransferase 3 OS=Homo sapiens GN=ART3 PE=1 SV=2 0.52 0.00050 23 sp|P15880|RS2_HUMAN 40S ribosomal protein S2 OS=Homo sapiens GN=RPS2 PE=1 SV=2 0.30 0.00055 24 sp|P30519|HMOX2_HUMAN oxygenase 2 OS=Homo sapiens GN=HMOX2 PE=1 SV=2 0.49 0.00055 25 sp|O95171|SCEL_HUMAN Sciellin OS=Homo sapiens GN=SCEL PE=1 SV=2 0.26 0.00061 26 sp|O43149|ZZEF1_HUMAN Zinc finger ZZ-type and EF-hand domain-containing protein 1 OS=Homo sapiens GN=ZZEF1 PE=1 SV=6 0.19 0.00061 27 sp|Q9Y446|PKP3_HUMAN -3 OS=Homo sapiens GN=PKP3 PE=1 SV=1 0.29 0.00087 28 sp|P02545|LMNA_HUMAN Prelamin-A/C OS=Homo sapiens GN=LMNA PE=1 SV=1 0.31 0.00088 29 sp|O75594|PGRP1_HUMAN Peptidoglycan recognition protein 1 OS=Homo sapiens GN=PGLYRP1 PE=1 SV=1 3.33 0.00101 30 sp|P49327|FAS_HUMAN Fatty acid synthase OS=Homo sapiens GN=FASN PE=1 SV=3 0.44 0.00130 31 sp|P35232|PHB_HUMAN Prohibitin OS=Homo sapiens GN=PHB PE=1 SV=1 0.41 0.00130 32 sp|Q14515|SPRL1_HUMAN SPARC-like protein 1 OS=Homo sapiens GN=SPARCL1 PE=1 SV=2 3.28 0.00134 33 sp|Q14116|IL18_HUMAN Interleukin-18 OS=Homo sapiens GN=IL18 PE=1 SV=1 0.37 0.00134 34 sp|O75487|GPC4_HUMAN Glypican-4 OS=Homo sapiens GN=GPC4 PE=1 SV=4 4.41 0.00143 35 sp|O43175|SERA_HUMAN D-3-phosphoglycerate dehydrogenase OS=Homo sapiens GN=PHGDH PE=1 SV=4 0.61 0.00144 36 sp|P00749|UROK_HUMAN Urokinase-type plasminogen activator OS=Homo sapiens GN=PLAU PE=1 SV=2 2.78 0.00148 37 sp|P01033|TIMP1_HUMAN Metalloproteinase inhibitor 1 OS=Homo sapiens GN=TIMP1 PE=1 SV=1 1.12 0.00148 38 sp|P24158|PRTN3_HUMAN OS=Homo sapiens GN=PRTN3 PE=1 SV=3 2.91 0.00156 39 sp|Q86XP0|PA24D_HUMAN Cytosolic phospholipase A2 delta OS=Homo sapiens GN=PLA2G4D PE=2 SV=2 0.37 0.00156 40 sp|Q9H159|CAD19_HUMAN Cadherin-19 OS=Homo sapiens GN=CDH19 PE=2 SV=1 1.96 0.00171 41 sp|Q9HAT2|SIAE_HUMAN Sialate O-acetylesterase OS=Homo sapiens GN=SIAE PE=1 SV=1 2.79 0.00171 42 sp|Q96FV2|SCRN2_HUMAN Secernin-2 OS=Homo sapiens GN=SCRN2 PE=2 SV=3 1.60 0.00178 43 sp|P14923|PLAK_HUMAN Junction OS=Homo sapiens GN=JUP PE=1 SV=3 0.33 0.00180 44 sp|P20700|LMNB1_HUMAN -B1 OS=Homo sapiens GN=LMNB1 PE=1 SV=2 0.54 0.00205 45 sp|P18827|SDC1_HUMAN Syndecan-1 OS=Homo sapiens GN=SDC1 PE=1 SV=3 2.80 0.00223 46 sp|Q8N1N4|K2C78_HUMAN Keratin, type II cytoskeletal 78 OS=Homo sapiens GN=KRT78 PE=2 SV=2 0.49 0.00223 47 sp|P51572|BAP31_HUMAN B-cell receptor-associated protein 31 OS=Homo sapiens GN=BCAP31 PE=1 SV=3 0.46 0.00223 48 sp|P61254|RL26_HUMAN 60S ribosomal protein L26 OS=Homo sapiens GN=RPL26 PE=1 SV=1 0.35 0.00258 49 sp|P55083|MFAP4_HUMAN Microfibril-associated glycoprotein 4 OS=Homo sapiens GN=MFAP4 PE=1 SV=2 2.07 0.00316 50 sp|Q6W4X9|MUC6_HUMAN Mucin-6 OS=Homo sapiens GN=MUC6 PE=1 SV=3 9.31 0.00330 51 sp|Q92817|EVPL_HUMAN OS=Homo sapiens GN=EVPL PE=1 SV=3 0.42 0.00336 52 sp|P55290|CAD13_HUMAN Cadherin-13 OS=Homo sapiens GN=CDH13 PE=1 SV=1 1.46 0.00348 53 sp|Q7Z406|MYH14_HUMAN -14 OS=Homo sapiens GN=MYH14 PE=1 SV=2 0.39 0.00348 54 sp|A1L0T0|ILVBL_HUMAN Acetolactate synthase-like protein OS=Homo sapiens GN=ILVBL PE=1 SV=2 0.45 0.00348 55 sp|Q6B0K9|HBM_HUMAN Hemoglobin subunit mu OS=Homo sapiens GN=HBM PE=2 SV=1 1.72 0.00351 56 sp|P33121|ACSL1_HUMAN Long-chain-fatty-acid--CoA 1 OS=Homo sapiens GN=ACSL1 PE=1 SV=1 0.37 0.00351 57 sp|P62280|RS11_HUMAN 40S ribosomal protein S11 OS=Homo sapiens GN=RPS11 PE=1 SV=3 0.25 0.00351 58 sp|O15372|EIF3H_HUMAN Eukaryotic translation initiation factor 3 subunit H OS=Homo sapiens GN=EIF3H PE=1 SV=1 0.45 0.00351 59 sp|P07305|H10_HUMAN Histone H1.0 OS=Homo sapiens GN=H1F0 PE=1 SV=3 0.36 0.00351 60 sp|Q96NR8|RDH12_HUMAN Retinol dehydrogenase 12 OS=Homo sapiens GN=RDH12 PE=1 SV=3 0.64 0.00400 61 sp|Q6ZUI0|TPRG1_HUMAN Tumor protein p63-regulated gene 1 protein OS=Homo sapiens GN=TPRG1 PE=2 SV=1 0.44 0.00400 62 sp|P30101|PDIA3_HUMAN Protein - A3 OS=Homo sapiens GN=PDIA3 PE=1 SV=4 0.30 0.00406 63 sp|Q02083|NAAA_HUMAN N-acylethanolamine-hydrolyzing acid amidase OS=Homo sapiens GN=NAAA PE=1 SV=3 2.27 0.00426 64 sp|P30046|DOPD_HUMAN D-dopachrome decarboxylase OS=Homo sapiens GN=DDT PE=1 SV=3 1.63 0.00459 65 sp|Q9BTV4|TMM43_HUMAN Transmembrane protein 43 OS=Homo sapiens GN=TMEM43 PE=1 SV=1 0.43 0.00459 66 sp|P01611|KV119_HUMAN Ig kappa chain V-I region Wes OS=Homo sapiens PE=1 SV=1 2.12 0.00462 67 sp|Q96DA0|ZG16B_HUMAN Zymogen granule protein 16 homolog B OS=Homo sapiens GN=ZG16B PE=1 SV=3 4.13 0.00462 68 sp|P01833|PIGR_HUMAN Polymeric immunoglobulin receptor OS=Homo sapiens GN=PIGR PE=1 SV=4 2.57 0.00462 69 sp|P00367|DHE3_HUMAN Glutamate dehydrogenase 1, mitochondrial OS=Homo sapiens GN=GLUD1 PE=1 SV=2 0.39 0.00462 70 sp|P06576|ATPB_HUMAN ATP synthase subunit beta, mitochondrial OS=Homo sapiens GN=ATP5B PE=1 SV=3 0.29 0.00462 71 sp|Q3ZCV2|CA177_HUMAN Uncharacterized protein C1orf177 OS=Homo sapiens GN=C1orf177 PE=2 SV=3 0.40 0.00462 72 sp|Q07065|CKAP4_HUMAN -associated protein 4 OS=Homo sapiens GN=CKAP4 PE=1 SV=2 0.42 0.00473 73 sp|Q9UMR5|PPT2_HUMAN Lysosomal thioesterase PPT2 OS=Homo sapiens GN=PPT2 PE=1 SV=4 0.31 0.00473 74 sp|Q13835|PKP1_HUMAN Plakophilin-1 OS=Homo sapiens GN=PKP1 PE=1 SV=2 0.35 0.00518 75 sp|Q13332|PTPRS_HUMAN Receptor-type tyrosine-protein phosphatase S OS=Homo sapiens GN=PTPRS PE=1 SV=3 2.08 0.00519 76 sp|P15924|DESP_HUMAN OS=Homo sapiens GN=DSP PE=1 SV=3 0.36 0.00519 77 sp|P46776|RL27A_HUMAN 60S ribosomal protein L27a OS=Homo sapiens GN=RPL27A PE=1 SV=2 0.40 0.00531 78 sp|P12273|PIP_HUMAN Prolactin-inducible protein OS=Homo sapiens GN=PIP PE=1 SV=1 5.85 0.00586 79 sp|Q92614|MY18A_HUMAN Unconventional myosin-XVIIIa OS=Homo sapiens GN=MYO18A PE=1 SV=3 0.49 0.00586 80 sp|P25705|ATPA_HUMAN ATP synthase subunit alpha, mitochondrial OS=Homo sapiens GN=ATP5A1 PE=1 SV=1 0.31 0.00616 Ratio Adjusted Protein Annotation Protein Name/Description LS/HS p -value 81 sp|P12956|XRCC6_HUMAN X-ray repair cross-complementing protein 6 OS=Homo sapiens GN=XRCC6 PE=1 SV=2 0.38 0.00633 82 sp|P05109|S10A8_HUMAN Protein S100-A8 OS=Homo sapiens GN=S100A8 PE=1 SV=1 2.27 0.00653 83 sp|Q00839|HNRPU_HUMAN Heterogeneous nuclear ribonucleoprotein U OS=Homo sapiens GN=HNRNPU PE=1 SV=6 0.28 0.00654 84 sp|P36639|8ODP_HUMAN 7,8-dihydro-8-oxoguanine triphosphatase OS=Homo sapiens GN=NUDT1 PE=1 SV=3 1.52 0.00666 85 sp|P04843|RPN1_HUMAN Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 OS=Homo sapiens GN=RPN1 PE=1 SV=1 0.39 0.00674 86 sp|P46781|RS9_HUMAN 40S ribosomal protein S9 OS=Homo sapiens GN=RPS9 PE=1 SV=3 0.49 0.00750 87 sp|Q02818|NUCB1_HUMAN Nucleobindin-1 OS=Homo sapiens GN=NUCB1 PE=1 SV=4 1.96 0.00756 88 sp|O14964|HGS_HUMAN Hepatocyte growth factor-regulated tyrosine kinase substrate OS=Homo sapiens GN=HGS PE=1 SV=1 0.48 0.00789 89 sp|Q07507|DERM_HUMAN Dermatopontin OS=Homo sapiens GN=DPT PE=2 SV=2 1.35 0.00794 90 sp|P49755|TMEDA_HUMAN Transmembrane emp24 domain-containing protein 10 OS=Homo sapiens GN=TMED10 PE=1 SV=2 0.44 0.00858 91 sp|P07942|LAMB1_HUMAN Laminin subunit beta-1 OS=Homo sapiens GN=LAMB1 PE=1 SV=2 1.43 0.00859 92 sp|O75015|FCG3B_HUMAN Low affinity immunoglobulin gamma Fc region receptor III-B OS=Homo sapiens GN=FCGR3B PE=1 SV=2 0.38 0.00859 93 sp|P09871|C1S_HUMAN Complement C1s subcomponent OS=Homo sapiens GN=C1S PE=1 SV=1 1.15 0.00893 94 sp|P45381|ACY2_HUMAN Aspartoacylase OS=Homo sapiens GN=ASPA PE=1 SV=1 1.36 0.00948 95 sp|P16615|AT2A2_HUMAN Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 OS=Homo sapiens GN=ATP2A2 PE=1 SV=1 0.46 0.00948 96 sp|P42766|RL35_HUMAN 60S ribosomal protein L35 OS=Homo sapiens GN=RPL35 PE=1 SV=2 0.46 0.00948 97 sp|P83731|RL24_HUMAN 60S ribosomal protein L24 OS=Homo sapiens GN=RPL24 PE=1 SV=1 0.33 0.00948 98 sp|P61970|NTF2_HUMAN Nuclear transport factor 2 OS=Homo sapiens GN=NUTF2 PE=1 SV=1 2.42 0.00974 99 sp|P60842|IF4A1_HUMAN Eukaryotic initiation factor 4A-I OS=Homo sapiens GN=EIF4A1 PE=1 SV=1 0.57 0.01009 100 sp|Q8WVV4|POF1B_HUMAN Protein POF1B OS=Homo sapiens GN=POF1B PE=1 SV=3 0.28 0.01025 101 sp|Q9H8H3|MET7A_HUMAN Methyltransferase-like protein 7A OS=Homo sapiens GN=METTL7A PE=1 SV=1 0.49 0.01035 102 sp|Q9NZ01|TECR_HUMAN Trans-2,3-enoyl-CoA reductase OS=Homo sapiens GN=TECR PE=1 SV=1 0.56 0.01035 103 sp|Q71DI3|H32_HUMAN Histone H3.2 OS=Homo sapiens GN=HIST2H3A PE=1 SV=3 0.34 0.01044 104 sp|O60613|SEP15_HUMAN 15 kDa selenoprotein OS=Homo sapiens GN=SEP15 PE=1 SV=3 1.43 0.01054 105 sp|Q9Y287|ITM2B_HUMAN Integral membrane protein 2B OS=Homo sapiens GN=ITM2B PE=1 SV=1 2.87 0.01054 106 sp|P18124|RL7_HUMAN 60S ribosomal protein L7 OS=Homo sapiens GN=RPL7 PE=1 SV=1 0.50 0.01054 107 sp|K1C15_SHEEP| sp|K1C15_SHEEP| 0.48 0.01054 108 sp|P40939|ECHA_HUMAN Trifunctional subunit alpha, mitochondrial OS=Homo sapiens GN=HADHA PE=1 SV=2 0.42 0.01054 109 sp|P61247|RS3A_HUMAN 40S ribosomal protein S3a OS=Homo sapiens GN=RPS3A PE=1 SV=2 0.41 0.01054 110 sp|Q13617|CUL2_HUMAN Cullin-2 OS=Homo sapiens GN=CUL2 PE=1 SV=2 0.52 0.01054 111 sp|Q9UGI8|TES_HUMAN Testin OS=Homo sapiens GN=TES PE=1 SV=1 0.45 0.01060 112 sp|Q4LDE5|SVEP1_HUMAN Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1 OS=Homo sapiens GN=SVEP1 PE=1 SV=3 1.14 0.01065 113 sp|Q9Y5Y2|NUBP2_HUMAN Cytosolic Fe-S cluster assembly factor NUBP2 OS=Homo sapiens GN=NUBP2 PE=1 SV=1 0.48 0.01071 114 sp|P01034|CYTC_HUMAN Cystatin-C OS=Homo sapiens GN=CST3 PE=1 SV=1 2.40 0.01110 115 sp|Q96GD0|PLPP_HUMAN Pyridoxal phosphate phosphatase OS=Homo sapiens GN=PDXP PE=1 SV=2 6.97 0.01167 116 sp|O60437|PEPL_HUMAN OS=Homo sapiens GN=PPL PE=1 SV=4 0.39 0.01172 117 sp|Q6ZMN7|PZRN4_HUMAN PDZ domain-containing RING finger protein 4 OS=Homo sapiens GN=PDZRN4 PE=1 SV=3 7.62 0.01180 118 sp|Q6P4A8|PLBL1_HUMAN Phospholipase B-like 1 OS=Homo sapiens GN=PLBD1 PE=1 SV=2 1.59 0.01180 119 sp|P09651|ROA1_HUMAN Heterogeneous nuclear ribonucleoprotein A1 OS=Homo sapiens GN=HNRNPA1 PE=1 SV=5 0.37 0.01180 120 sp|O00151|PDLI1_HUMAN PDZ and LIM domain protein 1 OS=Homo sapiens GN=PDLIM1 PE=1 SV=4 0.36 0.01180 121 sp|P20160|CAP7_HUMAN Azurocidin OS=Homo sapiens GN=AZU1 PE=1 SV=3 2.67 0.01219 122 sp|Q9H7P6|F125B_HUMAN Multivesicular body subunit 12B OS=Homo sapiens GN=FAM125B PE=1 SV=2 2.23 0.01243 123 sp|P52790|HXK3_HUMAN Hexokinase-3 OS=Homo sapiens GN=HK3 PE=1 SV=2 0.26 0.01243 124 sp|Q15904|VAS1_HUMAN V-type proton ATPase subunit S1 OS=Homo sapiens GN=ATP6AP1 PE=1 SV=2 1.94 0.01305 125 sp|P10619|PPGB_HUMAN Lysosomal protective protein OS=Homo sapiens GN=CTSA PE=1 SV=2 2.82 0.01331 126 sp|P04264|K2C1_HUMAN Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 PE=1 SV=6 0.56 0.01331 127 sp|Q9ULV0|MYO5B_HUMAN Unconventional myosin-Vb OS=Homo sapiens GN=MYO5B PE=1 SV=3 0.48 0.01331 128 sp|P57088|TMM33_HUMAN Transmembrane protein 33 OS=Homo sapiens GN=TMEM33 PE=1 SV=2 0.48 0.01331 129 sp|Q96S59|RANB9_HUMAN Ran-binding protein 9 OS=Homo sapiens GN=RANBP9 PE=1 SV=1 0.55 0.01331 130 sp|Q5K651|SAMD9_HUMAN Sterile alpha motif domain-containing protein 9 OS=Homo sapiens GN=SAMD9 PE=1 SV=1 0.38 0.01331 131 sp|Q9ULV4|COR1C_HUMAN Coronin-1C OS=Homo sapiens GN=CORO1C PE=1 SV=1 0.44 0.01331 132 sp|O95394|AGM1_HUMAN Phosphoacetylglucosamine mutase OS=Homo sapiens GN=PGM3 PE=1 SV=1 0.40 0.01331 133 sp|Q8WVN6|SCTM1_HUMAN Secreted and transmembrane protein 1 OS=Homo sapiens GN=SECTM1 PE=1 SV=2 3.36 0.01338 134 sp|P04196|HRG_HUMAN Histidine-rich glycoprotein OS=Homo sapiens GN=HRG PE=1 SV=1 2.10 0.01338 135 sp|P31947|1433S_HUMAN 14-3-3 protein sigma OS=Homo sapiens GN=SFN PE=1 SV=1 0.44 0.01338 136 sp|Q96SA4|SERC2_HUMAN Serine incorporator 2 OS=Homo sapiens GN=SERINC2 PE=2 SV=3 4.55 0.01343 137 sp|P00736|C1R_HUMAN Complement C1r subcomponent OS=Homo sapiens GN=C1R PE=1 SV=2 2.20 0.01379 138 sp|O95967|FBLN4_HUMAN EGF-containing fibulin-like extracellular matrix protein 2 OS=Homo sapiens GN=EFEMP2 PE=1 SV=3 1.80 0.01379 139 sp|Q15084|PDIA6_HUMAN Protein disulfide-isomerase A6 OS=Homo sapiens GN=PDIA6 PE=1 SV=1 0.36 0.01452 140 sp|Q16270|IBP7_HUMAN Insulin-like growth factor-binding protein 7 OS=Homo sapiens GN=IGFBP7 PE=1 SV=1 1.92 0.01458 141 sp|P63151|2ABA_HUMAN Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B alpha isoform OS=Homo sapiens GN=PPP2R2A PE=1 SV=1 0.47 0.01458 142 sp|O75390|CISY_HUMAN Citrate synthase, mitochondrial OS=Homo sapiens GN=CS PE=1 SV=2 0.35 0.01458 143 sp|Q8NCL4|GALT6_HUMAN Polypeptide N-acetylgalactosaminyltransferase 6 OS=Homo sapiens GN=GALNT6 PE=2 SV=2 1.35 0.01487 144 sp|P30085|KCY_HUMAN UMP-CMP kinase OS=Homo sapiens GN=CMPK1 PE=1 SV=3 0.52 0.01487 145 sp|P27824|CALX_HUMAN Calnexin OS=Homo sapiens GN=CANX PE=1 SV=2 0.45 0.01499 146 sp|P62826|RAN_HUMAN GTP-binding nuclear protein Ran OS=Homo sapiens GN=RAN PE=1 SV=3 0.40 0.01499 147 sp|Q15149|PLEC_HUMAN OS=Homo sapiens GN=PLEC PE=1 SV=3 0.42 0.01548 148 sp|O14773|TPP1_HUMAN Tripeptidyl-peptidase 1 OS=Homo sapiens GN=TPP1 PE=1 SV=2 2.00 0.01584 149 sp|P78527|PRKDC_HUMAN DNA-dependent protein kinase catalytic subunit OS=Homo sapiens GN=PRKDC PE=1 SV=3 0.42 0.01690 150 sp|Q92608|DOCK2_HUMAN Dedicator of cytokinesis protein 2 OS=Homo sapiens GN=DOCK2 PE=1 SV=2 0.51 0.01690 151 sp|P35052|GPC1_HUMAN Glypican-1 OS=Homo sapiens GN=GPC1 PE=1 SV=2 2.28 0.01707 152 sp|K2C1_HUMAN| sp|K2C1_HUMAN| 0.57 0.01758 153 sp|Q07020|RL18_HUMAN 60S ribosomal protein L18 OS=Homo sapiens GN=RPL18 PE=1 SV=2 0.44 0.01791 154 sp|O14786|NRP1_HUMAN Neuropilin-1 OS=Homo sapiens GN=NRP1 PE=1 SV=3 1.17 0.01833 155 sp|Q7Z5L0|VMO1_HUMAN Vitelline membrane outer layer protein 1 homolog OS=Homo sapiens GN=VMO1 PE=1 SV=1 2.30 0.01833 156 sp|Q9BS26|ERP44_HUMAN Endoplasmic reticulum resident protein 44 OS=Homo sapiens GN=ERP44 PE=1 SV=1 0.50 0.01833 157 sp|P00403|COX2_HUMAN Cytochrome c oxidase subunit 2 OS=Homo sapiens GN=MT-CO2 PE=1 SV=1 0.52 0.01833 158 sp|Q00796|DHSO_HUMAN Sorbitol dehydrogenase OS=Homo sapiens GN=SORD PE=1 SV=4 2.07 0.01970 159 sp|P13646|K1C13_HUMAN Keratin, type I cytoskeletal 13 OS=Homo sapiens GN=KRT13 PE=1 SV=4 0.48 0.01994 160 sp|HBA_HUMAN| sp|HBA_HUMAN| 2.55 0.01995 161 sp|P53992|SC24C_HUMAN Protein transport protein Sec24C OS=Homo sapiens GN=SEC24C PE=1 SV=3 0.50 0.02014 Ratio Adjusted Protein Annotation Protein Name/Description LS/HS p -value 162 sp|P26599|PTBP1_HUMAN Polypyrimidine tract-binding protein 1 OS=Homo sapiens GN=PTBP1 PE=1 SV=1 0.41 0.02199 163 sp|P16435|NCPR_HUMAN NADPH--cytochrome P450 reductase OS=Homo sapiens GN=POR PE=1 SV=2 0.48 0.02217 164 sp|P41252|SYIC_HUMAN Isoleucine--tRNA ligase, cytoplasmic OS=Homo sapiens GN=IARS PE=1 SV=2 0.44 0.02217 165 sp|Q9BUF5|TBB6_HUMAN beta-6 chain OS=Homo sapiens GN=TUBB6 PE=1 SV=1 0.56 0.02221 166 sp|P13807|GYS1_HUMAN Glycogen [starch] synthase, muscle OS=Homo sapiens GN=GYS1 PE=1 SV=2 0.41 0.02221 167 sp|Q86UX7|URP2_HUMAN Fermitin family homolog 3 OS=Homo sapiens GN=FERMT3 PE=1 SV=1 0.47 0.02232 168 sp|P50914|RL14_HUMAN 60S ribosomal protein L14 OS=Homo sapiens GN=RPL14 PE=1 SV=4 0.35 0.02255 169 sp|Q93100|KPBB_HUMAN Phosphorylase b kinase regulatory subunit beta OS=Homo sapiens GN=PHKB PE=1 SV=3 1.29 0.02273 170 sp|P13727|PRG2_HUMAN Bone marrow proteoglycan OS=Homo sapiens GN=PRG2 PE=1 SV=2 1.49 0.02303 171 sp|P17900|SAP3_HUMAN Ganglioside GM2 activator OS=Homo sapiens GN=GM2A PE=1 SV=4 2.89 0.02358 172 sp|Q9Y5P6|GMPPB_HUMAN Mannose-1-phosphate guanyltransferase beta OS=Homo sapiens GN=GMPPB PE=1 SV=2 0.59 0.02368 173 sp|Q8WWI1|LMO7_HUMAN LIM domain only protein 7 OS=Homo sapiens GN=LMO7 PE=1 SV=3 0.44 0.02397 174 sp|Q8WUH2|TGFA1_HUMAN Transforming growth factor-beta receptor-associated protein 1 OS=Homo sapiens GN=TGFBRAP1 PE=1 SV=1 2.14 0.02404 175 sp|Q6UX73|CP089_HUMAN UPF0764 protein C16orf89 OS=Homo sapiens GN=C16orf89 PE=2 SV=2 2.00 0.02404 176 sp|P09958|FURIN_HUMAN Furin OS=Homo sapiens GN=FURIN PE=1 SV=2 1.45 0.02410 177 sp|Q8WZ75|ROBO4_HUMAN Roundabout homolog 4 OS=Homo sapiens GN=ROBO4 PE=1 SV=1 2.09 0.02410 178 sp|Q96TA1|NIBL1_HUMAN Niban-like protein 1 OS=Homo sapiens GN=FAM129B PE=1 SV=3 0.67 0.02410 179 sp|Q7Z589|EMSY_HUMAN Protein EMSY OS=Homo sapiens GN=EMSY PE=1 SV=2 1.79 0.02447 180 sp|P11940|PABP1_HUMAN Polyadenylate-binding protein 1 OS=Homo sapiens GN=PABPC1 PE=1 SV=2 0.50 0.02486 181 sp|Q15369|ELOC_HUMAN elongation factor B polypeptide 1 OS=Homo sapiens GN=TCEB1 PE=1 SV=1 0.46 0.02486 182 sp|P23142|FBLN1_HUMAN Fibulin-1 OS=Homo sapiens GN=FBLN1 PE=1 SV=4 2.24 0.02520 183 sp|P62753|RS6_HUMAN 40S ribosomal protein S6 OS=Homo sapiens GN=RPS6 PE=1 SV=1 0.42 0.02520 184 sp|O14817|TSN4_HUMAN Tetraspanin-4 OS=Homo sapiens GN=TSPAN4 PE=1 SV=1 3.32 0.02532 185 sp|Q15223|PVRL1_HUMAN Poliovirus receptor-related protein 1 OS=Homo sapiens GN=PVRL1 PE=1 SV=3 1.53 0.02544 186 sp|P55084|ECHB_HUMAN Trifunctional enzyme subunit beta, mitochondrial OS=Homo sapiens GN=HADHB PE=1 SV=3 0.33 0.02544 187 sp|Q24JP5|T132A_HUMAN Transmembrane protein 132A OS=Homo sapiens GN=TMEM132A PE=2 SV=1 0.34 0.02544 188 sp|Q9UFH2|DYH17_HUMAN heavy chain 17, axonemal OS=Homo sapiens GN=DNAH17 PE=2 SV=2 0.53 0.02560 189 sp|Q9UBR1|BUP1_HUMAN Beta-ureidopropionase OS=Homo sapiens GN=UPB1 PE=1 SV=1 1.13 0.02569 190 sp|Q9Y6R7|FCGBP_HUMAN IgGFc-binding protein OS=Homo sapiens GN=FCGBP PE=1 SV=3 2.16 0.02569 191 sp|P05155|IC1_HUMAN Plasma protease C1 inhibitor OS=Homo sapiens GN=SERPING1 PE=1 SV=2 1.95 0.02569 192 sp|Q13509|TBB3_HUMAN Tubulin beta-3 chain OS=Homo sapiens GN=TUBB3 PE=1 SV=2 0.65 0.02569 193 sp|P55786|PSA_HUMAN Puromycin-sensitive OS=Homo sapiens GN=NPEPPS PE=1 SV=2 0.51 0.02569 194 sp|Q6Q0C0|TRAF7_HUMAN E3 ubiquitin-protein ligase TRAF7 OS=Homo sapiens GN=TRAF7 PE=1 SV=1 0.49 0.02569 195 sp|Q13616|CUL1_HUMAN Cullin-1 OS=Homo sapiens GN=CUL1 PE=1 SV=2 0.47 0.02569 196 sp|O95758|PTBP3_HUMAN Polypyrimidine tract-binding protein 3 OS=Homo sapiens GN=PTBP3 PE=1 SV=2 0.53 0.02585 197 sp|P07437|TBB5_HUMAN Tubulin beta chain OS=Homo sapiens GN=TUBB PE=1 SV=2 0.72 0.02592 198 sp|P09601|HMOX1_HUMAN 1 OS=Homo sapiens GN=HMOX1 PE=1 SV=1 0.50 0.02670 199 sp|O60716|CTND1_HUMAN delta-1 OS=Homo sapiens GN=CTNND1 PE=1 SV=1 0.46 0.02677 200 sp|O15231|ZN185_HUMAN Zinc finger protein 185 OS=Homo sapiens GN=ZNF185 PE=1 SV=3 0.46 0.02687 201 sp|Q02809|PLOD1_HUMAN Procollagen-,2-oxoglutarate 5-dioxygenase 1 OS=Homo sapiens GN=PLOD1 PE=1 SV=2 2.05 0.02718 202 sp|B2MG_HUMAN| sp|B2MG_HUMAN| 2.23 0.02718 203 sp|Q7L2H7|EIF3M_HUMAN Eukaryotic translation initiation factor 3 subunit M OS=Homo sapiens GN=EIF3M PE=1 SV=1 0.58 0.02718 204 sp|Q7Z5L7|PODN_HUMAN Podocan OS=Homo sapiens GN=PODN PE=1 SV=2 0.49 0.02726 205 sp|Q13867|BLMH_HUMAN Bleomycin hydrolase OS=Homo sapiens GN=BLMH PE=1 SV=1 1.54 0.02756 206 sp|P16401|H15_HUMAN Histone H1.5 OS=Homo sapiens GN=HIST1H1B PE=1 SV=3 0.40 0.02846 207 sp|Q9BRK5|CAB45_HUMAN 45 kDa calcium-binding protein OS=Homo sapiens GN=SDF4 PE=1 SV=1 1.64 0.02904 208 sp|P20742|PZP_HUMAN Pregnancy zone protein OS=Homo sapiens GN=PZP PE=1 SV=4 2.90 0.02911 209 sp|Q53GQ0|DHB12_HUMAN Estradiol 17-beta-dehydrogenase 12 OS=Homo sapiens GN=HSD17B12 PE=1 SV=2 0.48 0.02911 210 sp|Q9Y6N5|SQRD_HUMAN Sulfide:quinone , mitochondrial OS=Homo sapiens GN=SQRDL PE=1 SV=1 0.41 0.03002 211 sp|P28331|NDUS1_HUMAN NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial OS=Homo sapiens GN=NDUFS1 PE=1 SV=3 0.46 0.03002 212 sp|P14550|AK1A1_HUMAN Alcohol dehydrogenase [NADP(+)] OS=Homo sapiens GN=AKR1A1 PE=1 SV=3 1.75 0.03075 213 sp|O43240|KLK10_HUMAN Kallikrein-10 OS=Homo sapiens GN=KLK10 PE=1 SV=3 1.70 0.03125 214 sp|P48047|ATPO_HUMAN ATP synthase subunit O, mitochondrial OS=Homo sapiens GN=ATP5O PE=1 SV=1 0.43 0.03125 215 sp|P39023|RL3_HUMAN 60S ribosomal protein L3 OS=Homo sapiens GN=RPL3 PE=1 SV=2 0.51 0.03125 216 sp|P83436|COG7_HUMAN Conserved oligomeric Golgi complex subunit 7 OS=Homo sapiens GN=COG7 PE=1 SV=1 0.38 0.03125 217 sp|P16403|H12_HUMAN Histone H1.2 OS=Homo sapiens GN=HIST1H1C PE=1 SV=2 0.44 0.03200 218 sp|O00571|DDX3X_HUMAN ATP-dependent RNA helicase DDX3X OS=Homo sapiens GN=DDX3X PE=1 SV=3 0.55 0.03231 219 sp|Q8N0X7|SPG20_HUMAN Spartin OS=Homo sapiens GN=SPG20 PE=1 SV=1 1.04 0.03266 220 sp|P62879|GBB2_HUMAN Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 OS=Homo sapiens GN=GNB2 PE=1 SV=3 2.10 0.03266 221 sp|P68371|TBB4B_HUMAN Tubulin beta-4B chain OS=Homo sapiens GN=TUBB4B PE=1 SV=1 0.70 0.03266 222 sp|P24752|THIL_HUMAN Acetyl-CoA acetyltransferase, mitochondrial OS=Homo sapiens GN=ACAT1 PE=1 SV=1 0.33 0.03275 223 sp|Q71U36|TBA1A_HUMAN Tubulin alpha-1A chain OS=Homo sapiens GN=TUBA1A PE=1 SV=1 0.73 0.03276 224 sp|Q9UPT5|EXOC7_HUMAN Exocyst complex component 7 OS=Homo sapiens GN=EXOC7 PE=1 SV=3 0.48 0.03276 225 sp|O95831|AIFM1_HUMAN Apoptosis-inducing factor 1, mitochondrial OS=Homo sapiens GN=AIFM1 PE=1 SV=1 0.40 0.03280 226 sp|ANXA5_HUMAN| sp|ANXA5_HUMAN| 1.53 0.03288 227 sp|P22735|TGM1_HUMAN Protein-glutamine gamma-glutamyltransferase K OS=Homo sapiens GN=TGM1 PE=1 SV=4 0.50 0.03288 228 sp|P06748|NPM_HUMAN Nucleophosmin OS=Homo sapiens GN=NPM1 PE=1 SV=2 0.43 0.03288 229 sp|P37088|SCNNA_HUMAN Amiloride-sensitive sodium channel subunit alpha OS=Homo sapiens GN=SCNN1A PE=1 SV=1 1.18 0.03297 230 sp|Q00325|MPCP_HUMAN Phosphate carrier protein, mitochondrial OS=Homo sapiens GN=SLC25A3 PE=1 SV=2 0.43 0.03308 231 sp|P01613|KV121_HUMAN Ig kappa chain V-I region Ni OS=Homo sapiens PE=1 SV=1 1.32 0.03342 232 sp|O75116|ROCK2_HUMAN Rho-associated protein kinase 2 OS=Homo sapiens GN=ROCK2 PE=1 SV=4 1.14 0.03342 233 sp|P08246|ELNE_HUMAN Neutrophil elastase OS=Homo sapiens GN=ELANE PE=1 SV=1 1.88 0.03342 234 sp|P53396|ACLY_HUMAN ATP-citrate synthase OS=Homo sapiens GN=ACLY PE=1 SV=3 0.55 0.03342 235 sp|P19012|K1C15_HUMAN Keratin, type I cytoskeletal 15 OS=Homo sapiens GN=KRT15 PE=1 SV=3 0.49 0.03342 236 sp|P05091|ALDH2_HUMAN Aldehyde dehydrogenase, mitochondrial OS=Homo sapiens GN=ALDH2 PE=1 SV=2 0.56 0.03342 237 sp|P52597|HNRPF_HUMAN Heterogeneous nuclear ribonucleoprotein F OS=Homo sapiens GN=HNRNPF PE=1 SV=3 0.61 0.03342 238 sp|P02533|K1C14_HUMAN Keratin, type I cytoskeletal 14 OS=Homo sapiens GN=KRT14 PE=1 SV=4 0.46 0.03342 239 sp|Q15080|NCF4_HUMAN Neutrophil cytosol factor 4 OS=Homo sapiens GN=NCF4 PE=1 SV=2 0.46 0.03342 240 sp|Q8NBQ5|DHB11_HUMAN Estradiol 17-beta-dehydrogenase 11 OS=Homo sapiens GN=HSD17B11 PE=1 SV=3 0.43 0.03342 241 sp|P02649|APOE_HUMAN Apolipoprotein E OS=Homo sapiens GN=APOE PE=1 SV=1 1.88 0.03372 242 sp|CAH2_HUMAN| sp|CAH2_HUMAN| 2.03 0.03372 Ratio Adjusted Protein Annotation Protein Name/Description LS/HS p -value 243 sp|P13647|K2C5_HUMAN Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 PE=1 SV=3 0.47 0.03372 244 sp|P10412|H14_HUMAN Histone H1.4 OS=Homo sapiens GN=HIST1H1E PE=1 SV=2 0.45 0.03372 245 sp|P48444|COPD_HUMAN Coatomer subunit delta OS=Homo sapiens GN=ARCN1 PE=1 SV=1 0.49 0.03372 246 sp|P53618|COPB_HUMAN Coatomer subunit beta OS=Homo sapiens GN=COPB1 PE=1 SV=3 0.43 0.03372 247 sp|Q99798|ACON_HUMAN Aconitate hydratase, mitochondrial OS=Homo sapiens GN=ACO2 PE=1 SV=2 0.35 0.03372 248 sp|P84243|H33_HUMAN Histone H3.3 OS=Homo sapiens GN=H3F3A PE=1 SV=2 0.33 0.03372 249 sp|P08294|SODE_HUMAN Extracellular superoxide dismutase [Cu-Zn] OS=Homo sapiens GN=SOD3 PE=1 SV=2 2.11 0.03433 250 sp|Q03252|LMNB2_HUMAN Lamin-B2 OS=Homo sapiens GN=LMNB2 PE=1 SV=3 0.60 0.03433 251 sp|Q16836|HCDH_HUMAN Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial OS=Homo sapiens GN=HADH PE=1 SV=3 0.41 0.03433 252 sp|Q9BZ11|ADA33_HUMAN Disintegrin and metalloproteinase domain-containing protein 33 OS=Homo sapiens GN=ADAM33 PE=1 SV=2 0.43 0.03433 253 sp|P50552|VASP_HUMAN Vasodilator-stimulated phosphoprotein OS=Homo sapiens GN=VASP PE=1 SV=3 0.44 0.03448 254 sp|Q7Z2K6|ERMP1_HUMAN Endoplasmic reticulum metallopeptidase 1 OS=Homo sapiens GN=ERMP1 PE=1 SV=2 0.48 0.03448 255 sp|Q9Y5E6|PCDB3_HUMAN Protocadherin beta-3 OS=Homo sapiens GN=PCDHB3 PE=2 SV=1 1.73 0.03484 256 sp|A1KZ92|PXDNL_HUMAN Peroxidasin-like protein OS=Homo sapiens GN=PXDNL PE=2 SV=3 0.54 0.03544 257 sp|Q00610|CLH1_HUMAN Clathrin heavy chain 1 OS=Homo sapiens GN=CLTC PE=1 SV=5 0.66 0.03625 258 sp|P98095|FBLN2_HUMAN Fibulin-2 OS=Homo sapiens GN=FBLN2 PE=1 SV=2 1.37 0.03711 259 sp|P49447|CY561_HUMAN Cytochrome b561 OS=Homo sapiens GN=CYB561 PE=2 SV=2 1.01 0.03711 260 sp|P05787|K2C8_HUMAN Keratin, type II cytoskeletal 8 OS=Homo sapiens GN=KRT8 PE=1 SV=7 0.56 0.03711 261 sp|CASK_BOVIN| sp|CASK_BOVIN| 0.21 0.03711 262 sp|Q8WWA0|ITLN1_HUMAN Intelectin-1 OS=Homo sapiens GN=ITLN1 PE=1 SV=1 1.40 0.03721 263 sp|P02751|FINC_HUMAN OS=Homo sapiens GN=FN1 PE=1 SV=4 1.74 0.03721 264 sp|P05107|ITB2_HUMAN Integrin beta-2 OS=Homo sapiens GN=ITGB2 PE=1 SV=2 0.46 0.03774 265 sp|Q5TCZ1|SPD2A_HUMAN SH3 and PX domain-containing protein 2A OS=Homo sapiens GN=SH3PXD2A PE=1 SV=1 1.89 0.03799 266 sp|Q9ULT8|HECD1_HUMAN E3 ubiquitin-protein ligase HECTD1 OS=Homo sapiens GN=HECTD1 PE=1 SV=3 0.47 0.03799 267 sp|P68036|UB2L3_HUMAN Ubiquitin-conjugating enzyme E2 L3 OS=Homo sapiens GN=UBE2L3 PE=1 SV=1 2.01 0.03814 268 sp|Q58FF8|H90B2_HUMAN Putative heat shock protein HSP 90-beta 2 OS=Homo sapiens GN=HSP90AB2P PE=1 SV=2 0.53 0.03814 269 sp|P08779|K1C16_HUMAN Keratin, type I cytoskeletal 16 OS=Homo sapiens GN=KRT16 PE=1 SV=4 0.46 0.03814 270 sp|Q6ZRV2|FA83H_HUMAN Protein FAM83H OS=Homo sapiens GN=FAM83H PE=1 SV=3 0.45 0.03814 271 sp|P07814|SYEP_HUMAN Bifunctional glutamate/proline--tRNA ligase OS=Homo sapiens GN=EPRS PE=1 SV=5 0.41 0.03814 272 sp|Q7Z5N4|SDK1_HUMAN Protein sidekick-1 OS=Homo sapiens GN=SDK1 PE=1 SV=3 1.58 0.03820 273 sp|O43511|S26A4_HUMAN Pendrin OS=Homo sapiens GN=SLC26A4 PE=1 SV=1 2.86 0.03866 274 sp|Q14166|TTL12_HUMAN Tubulin--tyrosine ligase-like protein 12 OS=Homo sapiens GN=TTLL12 PE=1 SV=2 0.52 0.03873 275 sp|P02765|FETUA_HUMAN Alpha-2-HS-glycoprotein OS=Homo sapiens GN=AHSG PE=1 SV=1 2.63 0.04040 276 sp|P16278|BGAL_HUMAN Beta-galactosidase OS=Homo sapiens GN=GLB1 PE=1 SV=2 2.03 0.04040 277 sp|Q03013|GSTM4_HUMAN Glutathione S- Mu 4 OS=Homo sapiens GN=GSTM4 PE=1 SV=3 1.68 0.04040 278 sp|Q13885|TBB2A_HUMAN Tubulin beta-2A chain OS=Homo sapiens GN=TUBB2A PE=1 SV=1 0.70 0.04040 279 sp|P19013|K2C4_HUMAN Keratin, type II cytoskeletal 4 OS=Homo sapiens GN=KRT4 PE=1 SV=4 0.62 0.04040 280 sp|Q96LJ7|DHRS1_HUMAN Dehydrogenase/reductase SDR family member 1 OS=Homo sapiens GN=DHRS1 PE=1 SV=1 0.57 0.04040 281 sp|P31153|METK2_HUMAN S-adenosylmethionine synthase isoform type-2 OS=Homo sapiens GN=MAT2A PE=1 SV=1 0.50 0.04040 282 sp|P51659|DHB4_HUMAN Peroxisomal multifunctional enzyme type 2 OS=Homo sapiens GN=HSD17B4 PE=1 SV=3 0.43 0.04040 283 sp|P05388|RLA0_HUMAN 60S acidic ribosomal protein P0 OS=Homo sapiens GN=RPLP0 PE=1 SV=1 0.62 0.04051 284 sp|P48668|K2C6C_HUMAN Keratin, type II cytoskeletal 6C OS=Homo sapiens GN=KRT6C PE=1 SV=3 0.48 0.04084 285 sp|O75367|H2AY_HUMAN Core histone macro-H2A.1 OS=Homo sapiens GN=H2AFY PE=1 SV=4 0.44 0.04108 286 sp|P49368|TCPG_HUMAN T-complex protein 1 subunit gamma OS=Homo sapiens GN=CCT3 PE=1 SV=4 0.63 0.04134 287 sp|Q08211|DHX9_HUMAN ATP-dependent RNA helicase A OS=Homo sapiens GN=DHX9 PE=1 SV=4 0.46 0.04159 288 sp|P53621|COPA_HUMAN Coatomer subunit alpha OS=Homo sapiens GN=COPA PE=1 SV=2 0.50 0.04169 289 sp|Q15485|FCN2_HUMAN Ficolin-2 OS=Homo sapiens GN=FCN2 PE=1 SV=2 1.89 0.04216 290 sp|P02538|K2C6A_HUMAN Keratin, type II cytoskeletal 6A OS=Homo sapiens GN=KRT6A PE=1 SV=3 0.50 0.04216 291 sp|P50897|PPT1_HUMAN Palmitoyl-protein thioesterase 1 OS=Homo sapiens GN=PPT1 PE=1 SV=1 1.41 0.04244 292 sp|Q14134|TRI29_HUMAN Tripartite motif-containing protein 29 OS=Homo sapiens GN=TRIM29 PE=1 SV=2 0.44 0.04244 293 sp|Q02388|CO7A1_HUMAN Collagen alpha-1(VII) chain OS=Homo sapiens GN=COL7A1 PE=1 SV=2 1.16 0.04384 294 sp|P84095|RHOG_HUMAN Rho-related GTP-binding protein RhoG OS=Homo sapiens GN=RHOG PE=1 SV=1 1.89 0.04430 295 sp|O43390|HNRPR_HUMAN Heterogeneous nuclear ribonucleoprotein R OS=Homo sapiens GN=HNRNPR PE=1 SV=1 0.57 0.04432 296 sp|Q02413|DSG1_HUMAN Desmoglein-1 OS=Homo sapiens GN=DSG1 PE=1 SV=2 0.47 0.04463 297 sp|Q8IWE2|NXP20_HUMAN Protein NOXP20 OS=Homo sapiens GN=FAM114A1 PE=1 SV=2 0.56 0.04463 298 sp|Q8N0V5|GNT2A_HUMAN N-acetyllactosaminide beta-1,6-N-acetylglucosaminyl-transferase, isoform A OS=Homo sapiens GN=GCNT2 PE=2 SV=1 1.50 0.04505 299 sp|Q02383|SEMG2_HUMAN Semenogelin-2 OS=Homo sapiens GN=SEMG2 PE=1 SV=1 43.54 0.04546 300 sp|P04350|TBB4A_HUMAN Tubulin beta-4A chain OS=Homo sapiens GN=TUBB4A PE=1 SV=2 0.71 0.04546 301 sp|Q9H8L6|MMRN2_HUMAN Multimerin-2 OS=Homo sapiens GN=MMRN2 PE=1 SV=2 2.28 0.04588 302 sp|O00560|SDCB1_HUMAN Syntenin-1 OS=Homo sapiens GN=SDCBP PE=1 SV=1 2.11 0.04607 303 sp|Q09666|AHNK_HUMAN Neuroblast differentiation-associated protein AHNAK OS=Homo sapiens GN=AHNAK PE=1 SV=2 0.50 0.04607 304 sp|Q13423|NNTM_HUMAN NAD(P) transhydrogenase, mitochondrial OS=Homo sapiens GN=NNT PE=1 SV=3 0.40 0.04607 305 sp|Q14566|MCM6_HUMAN DNA replication licensing factor MCM6 OS=Homo sapiens GN=MCM6 PE=1 SV=1 2.60 0.04627 306 sp|P08865|RSSA_HUMAN 40S ribosomal protein SA OS=Homo sapiens GN=RPSA PE=1 SV=4 0.60 0.04627 307 sp|Q14203|DCTN1_HUMAN subunit 1 OS=Homo sapiens GN=DCTN1 PE=1 SV=3 0.51 0.04627 308 sp|P50453|SPB9_HUMAN Serpin B9 OS=Homo sapiens GN=SERPINB9 PE=1 SV=1 0.30 0.04651 309 sp|Q14CN4|K2C72_HUMAN Keratin, type II cytoskeletal 72 OS=Homo sapiens GN=KRT72 PE=1 SV=2 0.42 0.04767 310 sp|Q99988|GDF15_HUMAN Growth/differentiation factor 15 OS=Homo sapiens GN=GDF15 PE=1 SV=3 1.20 0.04791 311 sp|O95573|ACSL3_HUMAN Long-chain-fatty-acid--CoA ligase 3 OS=Homo sapiens GN=ACSL3 PE=1 SV=3 0.53 0.04791 312 sp|P62820|RAB1A_HUMAN Ras-related protein Rab-1A OS=Homo sapiens GN=RAB1A PE=1 SV=3 0.70 0.04821 313 sp|A8MVU1|NCF1C_HUMAN Putative neutrophil cytosol factor 1C OS=Homo sapiens GN=NCF1C PE=5 SV=1 0.48 0.04821 314 sp|Q9P2K2|TXD16_HUMAN Thioredoxin domain-containing protein 16 OS=Homo sapiens GN=TXNDC16 PE=2 SV=4 1.64 0.04871 315 sp|Q96G03|PGM2_HUMAN Phosphoglucomutase-2 OS=Homo sapiens GN=PGM2 PE=1 SV=4 0.46 0.04967 316 sp|P35268|RL22_HUMAN 60S ribosomal protein L22 OS=Homo sapiens GN=RPL22 PE=1 SV=2 0.44 0.04996 Count 0 500 1000 1500 2000 2500 −2 −1 Relative expression (mean subtracted) and Histogram Row Z−Score Color Key 0 1 2

Subject 1

Subject 2

Subject 3

Subject 4

Subject 5

Subject 6

Subject 7

Subject 8

Subject 9

Subject 10

Subject 11

Subject 12

Subject 13

Subject 14 sp|Q14566|MCM6_HUMAN sp|Q16270|IBP7_HUMAN sp|P00749|UROK_HUMAN sp|CAH2_HUMAN| sp|O75487|GPC4_HUMAN sp|Q9Y287|ITM2B_HUMAN sp|Q02413|DSG1_HUMAN sp|Q8N1N4|K2C78_HUMAN sp|P04264|K2C1_HUMAN sp|K2C1_HUMAN| sp|K1C15_SHEEP| sp|P13646|K1C13_HUMAN sp|Q8WVV4|POF1B_HUMAN sp|P19013|K2C4_HUMAN sp|Q13835|PKP1_HUMAN sp|O95171|SCEL_HUMAN sp|P16615|AT2A2_HUMAN sp|P22735|TGM1_HUMAN sp|O60716|CTND1_HUMAN sp|Q8WWI1|LMO7_HUMAN sp|O15231|ZN185_HUMAN sp|P35268|RL22_HUMAN sp|P50914|RL14_HUMAN sp|P35232|PHB_HUMAN sp|Q96S59|RANB9_HUMAN sp|Q6ZUI0|TPRG1_HUMAN sp|Q9C075|K1C23_HUMAN sp|Q14116|IL18_HUMAN sp|Q9BTV4|TMM43_HUMAN sp|Q03252|LMNB2_HUMAN sp|P07305|H10_HUMAN sp|P15924|DESP_HUMAN sp|K1HA_HUMAN| sp|Q3ZCV2|CA177_HUMAN sp|Q8IWE2|NXP20_HUMAN sp|P20700|LMNB1_HUMAN sp|P84243|H33_HUMAN sp|P52597|HNRPF_HUMAN sp|Q92614|MY18A_HUMAN sp|Q96NR8|RDH12_HUMAN sp|P30519|HMOX2_HUMAN sp|P46781|RS9_HUMAN sp|P42766|RL35_HUMAN sp|P62280|RS11_HUMAN sp|P61254|RL26_HUMAN sp|O00151|PDLI1_HUMAN sp|O95758|PTBP3_HUMAN sp|Q6NT55|CP4FN_HUMAN sp|P62826|RAN_HUMAN sp|O75367|H2AY_HUMAN sp|P26599|PTBP1_HUMAN sp|P02545|LMNA_HUMAN sp|P30085|KCY_HUMAN sp|P40394|ADH7_HUMAN sp|Q86XP0|PA24D_HUMAN sp|Q9H0P0|5NT3_HUMAN sp|Q15084|PDIA6_HUMAN sp|P04843|RPN1_HUMAN sp|Q07065|CKAP4_HUMAN sp|Q5VVQ6|OTU1_HUMAN sp|O43175|SERA_HUMAN sp|Q96TA1|NIBL1_HUMAN sp|P49755|TMEDA_HUMAN sp|Q14134|TRI29_HUMAN sp|Q9ULT8|HECD1_HUMAN sp|Q00325|MPCP_HUMAN sp|P68371|TBB4B_HUMAN sp|Q13509|TBB3_HUMAN sp|Q92817|EVPL_HUMAN sp|Q13885|TBB2A_HUMAN sp|P04350|TBB4A_HUMAN sp|P07437|TBB5_HUMAN sp|O14964|HGS_HUMAN sp|P49327|FAS_HUMAN sp|Q9ULV0|MYO5B_HUMAN sp|P15880|RS2_HUMAN sp|P13639|EF2_HUMAN sp|P08865|RSSA_HUMAN sp|P06576|ATPB_HUMAN sp|P25705|ATPA_HUMAN sp|Q9H8H3|MET7A_HUMAN sp|O60437|PEPL_HUMAN sp|P14923|PLAK_HUMAN sp|Q7Z406|MYH14_HUMAN sp|Q9Y446|PKP3_HUMAN sp|O00571|DDX3X_HUMAN sp|Q58FF8|H90B2_HUMAN sp|Q9UGI8|TES_HUMAN sp|P62753|RS6_HUMAN sp|P31947|1433S_HUMAN sp|Q07020|RL18_HUMAN sp|Q15369|ELOC_HUMAN sp|O95573|ACSL3_HUMAN sp|Q13616|CUL1_HUMAN sp|P19012|K1C15_HUMAN sp|P02533|K1C14_HUMAN sp|P08779|K1C16_HUMAN sp|P09601|HMOX1_HUMAN sp|Q08211|DHX9_HUMAN sp|Q9NZ01|TECR_HUMAN sp|O43149|ZZEF1_HUMAN sp|Q9ULG6|CCPG1_HUMAN sp|Q96LJ7|DHRS1_HUMAN sp|Q9ULV4|COR1C_HUMAN sp|Q9Y5Y2|NUBP2_HUMAN sp|Q24JP5|T132A_HUMAN sp|Q16836|HCDH_HUMAN sp|O95831|AIFM1_HUMAN sp|Q9BZ11|ADA33_HUMAN sp|O75015|FCG3B_HUMAN sp|Q9UMR5|PPT2_HUMAN sp|Q92608|DOCK2_HUMAN sp|P50453|SPB9_HUMAN sp|Q14203|DCTN1_HUMAN sp|O95394|AGM1_HUMAN sp|Q9BUF5|TBB6_HUMAN sp|Q13423|NNTM_HUMAN sp|CASK_BOVIN| sp|Q7Z5L7|PODN_HUMAN sp|P62820|RAB1A_HUMAN sp|P48047|ATPO_HUMAN sp|Q71U36|TBA1A_HUMAN sp|P00367|DHE3_HUMAN sp|Q6Q0C0|TRAF7_HUMAN sp|P49368|TCPG_HUMAN sp|Q9Y5P6|GMPPB_HUMAN sp|P05107|ITB2_HUMAN sp|Q15080|NCF4_HUMAN sp|P19878|NCF2_HUMAN sp|Q92556|ELMO1_HUMAN sp|Q8NBQ5|DHB11_HUMAN sp|A8MVU1|NCF1C_HUMAN sp|Q13508|NAR3_HUMAN sp|P20160|CAP7_HUMAN sp|Q96G03|PGM2_HUMAN sp|Q9UPT5|EXOC7_HUMAN sp|P53396|ACLY_HUMAN sp|P55786|PSA_HUMAN sp|P31153|METK2_HUMAN sp|P83436|COG7_HUMAN sp|P52790|HXK3_HUMAN sp|Q99798|ACON_HUMAN sp|P55084|ECHB_HUMAN sp|O75390|CISY_HUMAN sp|Q5K651|SAMD9_HUMAN sp|P05787|K2C8_HUMAN sp|Q14CN4|K2C72_HUMAN sp|P13647|K2C5_HUMAN sp|P02538|K2C6A_HUMAN sp|P48668|K2C6C_HUMAN sp|Q09666|AHNK_HUMAN sp|P39023|RL3_HUMAN sp|P06748|NPM_HUMAN sp|Q7Z2K6|ERMP1_HUMAN sp|P07814|SYEP_HUMAN sp|P28331|NDUS1_HUMAN sp|P41252|SYIC_HUMAN sp|P51659|DHB4_HUMAN sp|Q9Y6N5|SQRD_HUMAN sp|A1KZ92|PXDNL_HUMAN sp|P13807|GYS1_HUMAN sp|P53618|COPB_HUMAN sp|Q53GQ0|DHB12_HUMAN sp|P16435|NCPR_HUMAN sp|P40939|ECHA_HUMAN sp|P53992|SC24C_HUMAN sp|P83731|RL24_HUMAN sp|P18124|RL7_HUMAN sp|P46776|RL27A_HUMAN sp|Q02383|SEMG2_HUMAN sp|P24752|THIL_HUMAN sp|Q5SSJ5|HP1B3_HUMAN sp|P78527|PRKDC_HUMAN sp|P11940|PABP1_HUMAN sp|Q6ZRV2|FA83H_HUMAN sp|P09651|ROA1_HUMAN sp|Q7L2H7|EIF3M_HUMAN sp|Q13617|CUL2_HUMAN sp|O43390|HNRPR_HUMAN sp|P57088|TMM33_HUMAN sp|P53621|COPA_HUMAN sp|P33121|ACSL1_HUMAN sp|Q9UFH2|DYH17_HUMAN sp|Q14166|TTL12_HUMAN sp|P61247|RS3A_HUMAN sp|P16401|H15_HUMAN sp|P50552|VASP_HUMAN sp|P00403|COX2_HUMAN sp|P51572|BAP31_HUMAN sp|Q71DI3|H32_HUMAN sp|Q9BS26|ERP44_HUMAN sp|Q86UX7|URP2_HUMAN sp|O15372|EIF3H_HUMAN sp|Q00610|CLH1_HUMAN sp|P05091|ALDH2_HUMAN sp|Q00839|HNRPU_HUMAN sp|A1L0T0|ILVBL_HUMAN sp|P60842|IF4A1_HUMAN sp|Q92619|HMHA1_H sp|P08246|ELNE_HUMAN sp|Q6P4A8|PLBL1_HUMAN sp|P24158|PRTN3_HUMAN sp|P37088|SCNNA_HUMAN sp|P01613|KV121_HUMAN sp|Q6UX73|CP089_HUMAN sp|Q96DA0|ZG16B_HUMAN sp|P23142|FBLN1_HUMAN sp|P04196|HRG_HUMAN sp|O60613|SEP15_HUMAN sp|O43240|KLK10_HUMAN sp|Q9Y6R7|FCGBP_HUMAN sp|P20742|PZP_HUMAN sp|P07942|LAMB1_HUMAN sp|Q15485|FCN2_HUMAN sp|P36639|8ODP_HUMAN sp|Q99988|GDF15_HUMAN sp|P17900|SAP3_HUMAN sp|Q96FV2|SCRN2_HUMAN sp|Q9H7P6|F125B_HUMAN sp|P02765|FETUA_HUMAN sp|O00187|MASP2_HUMAN sp|Q8WVN6|SCTM1_HUMAN sp|P08294|SODE_HUMAN sp|Q7Z5L0|VMO1_HUMAN sp|Q13867|BLMH_HUMAN sp|P01611|KV119_HUMAN sp|Q02809|PLOD1_HUMAN sp|O75116|ROCK2_HUMAN sp|Q9H8L6|MMRN2_HUMAN sp|P55290|CAD13_HUMAN sp|P68036|UB2L3_HUMAN sp|Q02818|NUCB1_HUMAN sp|P55083|MFAP4_HUMAN sp|Q9UBR1|BUP1_HUMAN sp|Q8N0X7|SPG20_HUMAN sp|O43511|S26A4_HUMAN sp|O14817|TSN4_HUMAN sp|P49447|CY561_HUMAN sp|P62879|GBB2_HUMAN sp|O00560|SDCB1_HUMAN sp|Q96SA4|SERC2_HUMAN sp|Q9HAT2|SIAE_HUMAN sp|P05109|S10A8_HUMAN sp|HBA_HUMAN| sp|Q9P2K2|TXD16_HUMAN sp|Q02388|CO7A1_HUMAN sp|Q7Z589|EMSY_HUMAN sp|P13727|PRG2_HUMAN sp|Q8WWA0|ITLN1_HUMAN sp|Q03013|GSTM4_HUMAN sp|Q9BRK5|CAB45_HUMAN sp|Q7L3S4|ZN771_HUMAN sp|O14786|NRP1_HUMAN sp|P50897|PPT1_HUMAN sp|P84095|RHOG_HUMAN sp|Q96GD0|PLPP_HUMAN sp|P02751|FINC_HUMAN sp|Q8N0V5|GNT2A_HUMAN sp|Q8NCL4|GALT6_HUMAN sp|Q9BXJ7|AMNLS_HUMAN sp|P12273|PIP_HUMAN sp|Q4LDE5|SVEP1_HUMAN sp|P35052|GPC1_HUMAN sp|P00736|C1R_HUMAN sp|Q8WZ75|ROBO4_HUMAN sp|P61970|NTF2_HUMAN sp|Q14515|SPRL1_HUMAN sp|Q9H159|CAD19_HUMAN sp|Q13332|PTPRS_HUMAN sp|O75594|PGRP1_HUMAN sp|P18827|SDC1_HUMAN sp|P01034|CYTC_HUMAN sp|P09871|C1S_HUMAN sp|O95967|FBLN4_HUMAN sp|Q15274|NADC_HUMAN sp|Q9BRT3|MIEN1_HUMAN sp|P01833|PIGR_HUMAN sp|Q7Z5N4|SDK1_HUMAN sp|Q02083|NAAA_HUMAN sp|P30046|DOPD_HUMAN sp|Q6B0K9|HBM_HUMAN sp|P45381|ACY2_HUMAN sp|Q6W4X9|MUC6_HUMAN sp|Q07507|DERM_HUMAN sp|ANXA5_HUMAN| sp|P09958|FURIN_HUMAN sp|P02649|APOE_HUMAN sp|P05155|IC1_HUMAN sp|P10619|PPGB_HUMAN sp|Q00796|DHSO_HUMAN sp|O14773|TPP1_HUMAN sp|P14550|AK1A1_HUMAN sp|Q6ZMN7|PZRN4_HUMAN sp|Q15223|PVRL1_HUMAN sp|Q15904|VAS1_HUMAN sp|Q8NI99|ANGL6_HUMAN sp|Q5TCZ1|SPD2A_HUMAN sp|P01033|TIMP1_HUMAN sp|Q8WUH2|TGFA1_HUMAN sp|P98095|FBLN2_HUMAN sp|P16278|BGAL_HUMAN sp|B2MG_HUMAN| sp|Q93100|KPBB_HUMAN sp|Q9Y5E6|PCDB3_HUMAN Subject 1

Subject 2

Subject 3

Subject 4

Subject 5

Subject 6

Subject 7

Subject 8

Subject 9

Subject 10

Subject 11

Subject 12

Subject 13

Subject 14 sp|Q13423|NNTM_HUMAN sp|CASK_BOVIN| sp|Q7Z5L7|PODN_HUMAN sp|P62820|RAB1A_HUMAN sp|P48047|ATPO_HUMAN sp|Q71U36|TBA1A_HUMAN sp|P00367|DHE3_HUMAN sp|Q6Q0C0|TRAF7_HUMAN sp|P49368|TCPG_HUMAN sp|Q9Y5P6|GMPPB_HUMAN sp|P05107|ITB2_HUMAN sp|Q15080|NCF4_HUMAN sp|P19878|NCF2_HUMAN sp|Q92556|ELMO1_HUMAN sp|Q8NBQ5|DHB11_HUMAN sp|A8MVU1|NCF1C_HUMAN sp|Q13508|NAR3_HUMAN sp|P20160|CAP7_HUMAN sp|Q96G03|PGM2_HUMAN sp|Q9UPT5|EXOC7_HUMAN sp|P53396|ACLY_HUMAN sp|P55786|PSA_HUMAN sp|P31153|METK2_HUMAN sp|P83436|COG7_HUMAN sp|P52790|HXK3_HUMAN sp|Q99798|ACON_HUMAN sp|P55084|ECHB_HUMAN sp|O75390|CISY_HUMAN sp|Q5K651|SAMD9_HUMAN sp|P05787|K2C8_HUMAN sp|Q14CN4|K2C72_HUMAN sp|P13647|K2C5_HUMAN sp|P02538|K2C6A_HUMAN sp|P48668|K2C6C_HUMAN sp|Q09666|AHNK_HUMAN sp|P39023|RL3_HUMAN sp|P06748|NPM_HUMAN sp|Q7Z2K6|ERMP1_HUMAN sp|P07814|SYEP_HUMAN sp|P28331|NDUS1_HUMAN sp|P41252|SYIC_HUMAN sp|P51659|DHB4_HUMAN sp|Q9Y6N5|SQRD_HUMAN sp|A1KZ92|PXDNL_HUMAN sp|P13807|GYS1_HUMAN sp|P53618|COPB_HUMAN sp|Q53GQ0|DHB12_HUMAN sp|P16435|NCPR_HUMAN sp|P40939|ECHA_HUMAN sp|P53992|SC24C_HUMAN sp|P83731|RL24_HUMAN sp|P18124|RL7_HUMAN sp|P46776|RL27A_HUMAN sp|Q02383|SEMG2_HUMAN sp|P24752|THIL_HUMAN sp|Q5SSJ5|HP1B3_HUMAN sp|P78527|PRKDC_HUMAN sp|P11940|PABP1_HUMAN sp|Q6ZRV2|FA83H_HUMAN sp|P09651|ROA1_HUMAN sp|Q7L2H7|EIF3M_HUMAN sp|Q13617|CUL2_HUMAN sp|O43390|HNRPR_HUMAN sp|P57088|TMM33_HUMAN sp|P53621|COPA_HUMAN sp|P33121|ACSL1_HUMAN sp|Q9UFH2|DYH17_HUMAN sp|Q14166|TTL12_HUMAN sp|P61247|RS3A_HUMAN sp|P16401|H15_HUMAN sp|P50552|VASP_HUMAN sp|P00403|COX2_HUMAN sp|P51572|BAP31_HUMAN sp|Q71DI3|H32_HUMAN sp|Q9BS26|ERP44_HUMAN sp|Q86UX7|URP2_HUMAN sp|O15372|EIF3H_HUMAN sp|Q00610|CLH1_HUMAN sp|P05091|ALDH2_HUMAN sp|Q00839|HNRPU_HUMAN sp|A1L0T0|ILVBL_HUMAN sp|P60842|IF4A1_HUMAN sp|Q92619|HMHA1_HUMAN sp|Q9H2M9|RBGPR_HUMAN sp|P10412|H14_HUMAN sp|P16403|H12_HUMAN sp|P63151|2ABA_HUMAN sp|P05388|RLA0_HUMAN sp|P48444|COPD_HUMAN sp|P27824|CALX_HUMAN sp|Q15149|PLEC_HUMAN sp|P12956|XRCC6_HUMAN sp|P30101|PDIA3_HUMAN sp|P52272|HNRPM_HUMAN sp|P49411|EFTU_HUMAN sp|P08246|ELNE_HUMAN sp|Q6P4A8|PLBL1_HUMAN sp|P24158|PRTN3_HUMAN sp|P37088|SCNNA_HUMAN sp|P01613|KV121_HUMAN sp|Q6UX73|CP089_HUMAN sp|Q96DA0|ZG16B_HUMAN sp|P23142|FBLN1_HUMAN sp|P04196|HRG_HUMAN sp|O60613|SEP15_HUMAN sp|O43240|KLK10_HUMAN sp|Q9Y6R7|FCGBP_HUMAN sp|P20742|PZP_HUMAN sp|P07942|LAMB1_HUMAN sp|Q15485|FCN2_HUMAN sp|P36639|8ODP_HUMAN sp|Q99988|GDF15_HUMAN sp|P17900|SAP3_HUMAN sp|Q96FV2|SCRN2_HUMAN sp|Q9H7P6|F125B_HUMAN sp|P02765|FETUA_HUMAN sp|O00187|MASP2_HUMAN sp|Q8WVN6|SCTM1_HUMAN sp|P08294|SODE_HUMAN sp|Q7Z5L0|VMO1_HUMAN sp|Q13867|BLMH_HUMAN sp|P01611|KV119_HUMAN sp|Q02809|PLOD1_HUMAN sp|O75116|ROCK2_HUMAN sp|Q9H8L6|MMRN2_HUMAN sp|P55290|CAD13_HUMAN sp|P68036|UB2L3_HUMAN sp|Q02818|NUCB1_HUMAN sp|P55083|MFAP4_HUMAN sp|Q9UBR1|BUP1_HUMAN sp|Q8N0X7|SPG20_HUMAN sp|O43511|S26A4_HUMAN sp|O14817|TSN4_HUMAN sp|P49447|CY561_HUMAN sp|P62879|GBB2_HUMAN sp|O00560|SDCB1_HUMAN sp|Q96SA4|SERC2_HUMAN sp|Q9HAT2|SIAE_HUMAN sp|P05109|S10A8_HUMAN sp|HBA_HUMAN| sp|Q9P2K2|TXD16_HUMAN sp|Q02388|CO7A1_HUMAN sp|Q7Z589|EMSY_HUMAN sp|P13727|PRG2_HUMAN sp|Q8WWA0|ITLN1_HUMAN sp|Q03013|GSTM4_HUMAN sp|Q9BRK5|CAB45_HUMAN sp|Q7L3S4|ZN771_HUMAN sp|O14786|NRP1_HUMAN sp|P50897|PPT1_HUMAN sp|P84095|RHOG_HUMAN sp|Q96GD0|PLPP_HUMAN sp|P02751|FINC_HUMAN sp|Q8N0V5|GNT2A_HUMAN sp|Q8NCL4|GALT6_HUMAN sp|Q9BXJ7|AMNLS_HUMAN sp|P12273|PIP_HUMAN sp|Q4LDE5|SVEP1_HUMAN sp|P35052|GPC1_HUMAN sp|P00736|C1R_HUMAN sp|Q8WZ75|ROBO4_HUMAN sp|P61970|NTF2_HUMAN sp|Q14515|SPRL1_HUMAN sp|Q9H159|CAD19_HUMAN sp|Q13332|PTPRS_HUMAN sp|O75594|PGRP1_HUMAN sp|P18827|SDC1_HUMAN sp|P01034|CYTC_HUMAN sp|P09871|C1S_HUMAN sp|O95967|FBLN4_HUMAN sp|Q15274|NADC_HUMAN sp|Q9BRT3|MIEN1_HUMAN sp|P01833|PIGR_HUMAN sp|Q7Z5N4|SDK1_HUMAN sp|Q02083|NAAA_HUMAN sp|P30046|DOPD_HUMAN sp|Q6B0K9|HBM_HUMAN sp|P45381|ACY2_HUMAN sp|Q6W4X9|MUC6_HUMAN sp|Q07507|DERM_HUMAN sp|ANXA5_HUMAN| sp|P09958|FURIN_HUMAN sp|P02649|APOE_HUMAN sp|P05155|IC1_HUMAN sp|P10619|PPGB_HUMAN sp|Q00796|DHSO_HUMAN sp|O14773|TPP1_HUMAN sp|P14550|AK1A1_HUMAN sp|Q6ZMN7|PZRN4_HUMAN sp|Q15223|PVRL1_HUMAN sp|Q15904|VAS1_HUMAN sp|Q8NI99|ANGL6_HUMAN sp|Q5TCZ1|SPD2A_HUMAN sp|P01033|TIMP1_HUMAN sp|Q8WUH2|TGFA1_HUMAN sp|P98095|FBLN2_HUMAN sp|P16278|BGAL_HUMAN sp|B2MG_HUMAN| sp|Q93100|KPBB_HUMAN sp|Q9Y5E6|PCDB3_HUMAN sp|Q14566|MCM6_HUMAN sp|Q16270|IBP7_HUMAN sp|P00749|UROK_HUMAN sp|CAH2_HUMAN| sp|O75487|GPC4_HUMAN sp|Q9Y287|ITM2B_HUMAN sp|Q02413|DSG1_HUMAN sp|Q8N1N4|K2C78_HUMAN sp|P04264|K2C1_HUMAN sp|K2C1_HUMAN| sp|K1C15_SHEEP| sp|P13646|K1C13_HUMAN sp|Q8WVV4|POF1B_HUMAN sp|P19013|K2C4_HUMAN sp|Q13835|PKP1_HUMAN sp|O95171|SCEL_HUMAN sp|P16615|AT2A2_HUMAN sp|P22735|TGM1_HUMAN sp|O60716|CTND1_HUMAN sp|Q8WWI1|LMO7_HUMAN sp|O15231|ZN185_HUMAN sp|P35268|RL22_HUMAN sp|P50914|RL14_HUMAN sp|P35232|PHB_HUMAN sp|Q96S59|RANB9_HUMAN sp|Q6ZUI0|TPRG1_HUMAN sp|Q9C075|K1C23_HUMAN sp|Q14116|IL18_HUMAN sp|Q9BTV4|TMM43_HUMAN sp|Q03252|LMNB2_HUMAN sp|P07305|H10_HUMAN sp|P15924|DESP_HUMAN sp|K1HA_HUMAN| sp|Q3ZCV2|CA177_HUMAN sp|Q8IWE2|NXP20_HUMAN sp|P20700|LMNB1_HUMAN sp|P84243|H33_HUMAN sp|P52597|HNRPF_HUMAN sp|Q92614|MY18A_HUMAN sp|Q96NR8|RDH12_HUMAN sp|P30519|HMOX2_HUMAN sp|P46781|RS9_HUMAN sp|P42766|RL35_HUMAN sp|P62280|RS11_HUMAN sp|P61254|RL26_HUMAN sp|O00151|PDLI1_HUMAN sp|O95758|PTBP3_HUMAN sp|Q6NT55|CP4FN_HUMAN sp|P62826|RAN_HUMAN sp|O75367|H2AY_HUMAN sp|P26599|PTBP1_HUMAN sp|P02545|LMNA_HUMAN sp|P30085|KCY_HUMAN sp|P40394|ADH7_HUMAN sp|Q86XP0|PA24D_HUMAN sp|Q9H0P0|5NT3_HUMAN sp|Q15084|PDIA6_HUMAN sp|P04843|RPN1_HUMAN sp|Q07065|CKAP4_HUMAN sp|Q5VVQ6|OTU1_HUMAN sp|O43175|SERA_HUMAN sp|Q96TA1|NIBL1_HUMAN sp|P49755|TMEDA_HUMAN sp|Q14134|TRI29_HUMAN sp|Q9ULT8|HECD1_HUMAN sp|Q00325|MPCP_HUMAN sp|P68371|TBB4B_HUMAN sp|Q13509|TBB3_HUMAN sp|Q92817|EVPL_HUMAN sp|Q13885|TBB2A_HUMAN sp|P04350|TBB4A_HUMAN sp|P07437|TBB5_HUMAN sp|O14964|HGS_HUMAN sp|P49327|FAS_HUMAN sp|Q9ULV0|MYO5B_HUMAN sp|P15880|RS2_HUMAN sp|P13639|EF2_HUMAN sp|P08865|RSSA_HUMAN sp|P06576|ATPB_HUMAN sp|P25705|ATPA_HUMAN sp|Q9H8H3|MET7A_HUMAN sp|O60437|PEPL_HUMAN sp|P14923|PLAK_HUMAN sp|Q7Z406|MYH14_HUMAN sp|Q9Y446|PKP3_HUMAN sp|O00571|DDX3X_HUMAN sp|Q58FF8|H90B2_HUMAN sp|Q9UGI8|TES_HUMAN sp|P62753|RS6_HUMAN sp|P31947|1433S_HUMAN sp|Q07020|RL18_HUMAN sp|Q15369|ELOC_HUMAN sp|O95573|ACSL3_HUMAN sp|Q13616|CUL1_HUMAN sp|P19012|K1C15_HUMAN sp|P02533|K1C14_HUMAN sp|P08779|K1C16_HUMAN sp|P09601|HMOX1_HUMAN sp|Q08211|DHX9_HUMAN sp|Q9NZ01|TECR_HUMAN sp|O43149|ZZEF1_HUMAN sp|Q9ULG6|CCPG1_HUMAN sp|Q96LJ7|DHRS1_HUMAN sp|Q9ULV4|COR1C_HUMAN sp|Q9Y5Y2|NUBP2_HUMAN sp|Q24JP5|T132A_HUMAN sp|Q16836|HCDH_HUMAN sp|O95831|AIFM1_HUMAN sp|Q9BZ11|ADA33_HUMAN sp|O75015|FCG3B_HUMAN sp|Q9UMR5|PPT2_HUMAN sp|Q92608|DOCK2_HUMAN sp|P50453|SPB9_HUMAN sp|Q14203|DCTN1_HUMAN sp|O95394|AGM1_HUMAN sp|Q9BUF5|TBB6_HUMAN