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Chromosome-8-Coded Proteome of Chinese Chromosome Proteome Data Set (CCPD) 2.0 with Partial Immunohistochemical Verifications Yang Liu,†,‡,◆ Wantao Ying,⊥,◆ Zhe Ren,▽,◆ Wei Gu,○,◆ Yang Zhang,† Guoquan Yan,† Pengyuan Yang,†,§ Yinkun Liu,†,∥ Xuefei Yin,†,§ Cheng Chang,⊥ Jing Jiang,⊥ Fengxu Fan,⊥ Chengpu Zhang,⊥ Ping Xu,⊥ Quanhui Wang,#,▽ Bo Wen,▽ Liang Lin,▽ Tingyou Wang,▽ Chaoqin Du,▽ Jiayong Zhong,○ Tong Wang,○ Qing-Yu He,○ Xiaohong Qian,*,⊥ Xiaomin Lou,*,#,▽ Gong Zhang,*,○ and Fan Zhong*,† †

Institutes of Biomedical Sciences, Fudan University, Mingdao Bldg. 815, 138 Yixueyuan Road, Shanghai 200032, China School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China § Department of Chemistry, Fudan University, 220 Handan Road, Shanghai 200433, China ∥ Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, China ⊥ State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, No. 33, Life Science Park Road, Beijing 102206, China # Beijing Institute of Genomics, Chinese Academy of Sciences, No. 1 Beichen West Road, Beijing 100101, China ▽ BGI-Shenzhen, Beishan Industrial Zone, Shenzhen 518083, China ○ Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China ‡

S Supporting Information *

ABSTRACT: We upgraded the preliminary CCPD 1.0 to CCPD 2.0 using the latest deep-profiling proteome (CCPD 2013) of three hepatocellular carcinoma (HCC) cell lines, namely, Hep3B, MHCC97H, and HCCLM3 (ProteomeXchange identifiers: PXD000529, PXD000533, and PXD000535). CCPD 2.0 totally covered 63.6% (438/689) of Chr. 8-coded proteins and 62.6% (439/701) of Chr. 8-coded protein-coding genes. Interestingly, we found that the missing proteins exhibited a tendency to form a cluster region in chromosomes, such as two β-defensins clusters in Chr. 8, caused perhaps by their inflammation-related features. For the 41 Chr. 8coded proteins being weakly or barely identified previously, we have performed an immunohistochemical (IHC) verification in 30 pairs of carcinoma/para-carcinoma HCC and 20 noncancerous liver tissues and confirmed their expressional evidence and occurrence proportions in tissue samples. We also verified 13 Chr. 8-coded HCC tumorigenesis-associated depleting or deficient proteins reported in CCPD 1.0 using IHC and screened 16 positive and 24 negative HCC metastatic potential-correlated proteins from large-scale label-free proteome quantitation data of CCPD 2013. Our results suggest that the selection of proper samples and the methodology to look for targeted missing proteins should be carefully considered in further verifications for the remaining Chr. 8-coded proteins. KEYWORDS: chromosome 8, proteome, hepatocellular carcinoma, metastasis, missing protein, immunohistochemistry, neXtProt

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INTRODUCTION During the initial phase of the Chromosome-Centric Human Proteome Project (C-HPP),1 the Chinese Human Chromosome Proteome Consortium (CCPC) has produced the Chinese Chromosome Proteome Data set (CCPD) based on three digestive organs, namely, stomach, colon, and liver, as well as their corresponding carcinoma tissues/cell lines.2 The analyses corresponding to, but not limited to, chromosomes 1,2 8,3 and 204 were carried out. Among these chromosomes, the Chr. 8 team was also curious on multiple 8p deficiencies in the tumorigenesis of the three digestive organs.3 CCPC has profiled proteomes of three human hepatocellular carcinoma (HCC) © 2013 American Chemical Society

cell lines in 2013 and contributed deeply covered proteome data in CCPD. These deeply covered proteomes with transcriptome comparative scale (8000+ proteins) can support more comprehensive and intensive analyses. In addition to the previous mass-spectrometry (MS)-based profiling, antibody (Ab) as one of the three pillars of the HPP is the other important complement to the MS pillar.5 In the Special Issue: Chromosome-centric Human Proteome Project Received: September 2, 2013 Published: November 29, 2013 126

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Figure 1. Identification and overlapping status of Chr. 8-coded proteome in CCPD. (A) Identification scales of CCPD 2.0 (blue), CCPD 2013 (red), PeptideAtlas (purple), and GPMDB “green” (green) to the baselines (orange) of Ensembl Gene, Swiss-Prot, and neXtProt “gold”, respectively. Venn diagrams comparing Chr. 8-coded proteins from CCPD 2.0, PeptideAtlas, and GPMDB “green” in gene (B) and protein (C) levels.

proteomecentral.proteomexchange.org) with the following identifiers: PXD000529, PXD000533, and PXD000535. We upgraded CCPD into 2.0 by adding the newly produced CCPD 2013 and remapping the newest Swiss-Prot (UniProt) AC of CCPD 1.0. The protein abundance of CCPD 2.0 was quantified by extracting ion current (XIC)-based label-free quantification method SLIVER.8 The iBAQ value of every peptide was then determined using the area of peptide XIC.9 Furthermore, we normalized the iBAQ values for comparative analysis of all samples by dividing their medians. The transcriptomes and translatomes of the three cell lines were profiled by RNA-seq and quantified by reads per kilo bases per million reads (RPKM) method.10 All sequencing data are available in Gene Expression Omnibus (http://www.ncbi. nlm.nih.gov/geo/) with the accession number GSE49994. Details of the identification, remapping, and quantification of CCPD 2.0, transcriptomes, and translatomes are described in a related article entitled “Systematic Analyses of Transcriptome, Translatome, and Proteome Provide a Global View and Potential Strategy for C-HPP” (Chang et al. J. Proteome Res. 2013, 10.1021/pr4009018).

present study, we utilized the Ab pillar to verify the targeting proteins in our C-HPP, especially in (1) missing protein finding and (2) pathological/clinical issues. Both issues have been enhanced by large-scale IHC assay.

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MATERIALS AND METHODS

Proteomes, Transcriptomes, Translatomes, and Quantification

The newly profiled proteomes, namely, CCPD 2013, were obtained from three human HCC cell lines: Hep3B,6 MHCC97H (97H), and HCCLM3 (LM3).7 97H and LM3 had 100% lung metastasis upon orthotopic inoculation, whereas Hep3B was nonmetastatic. All cell lines had an HBV infective background with HBAg+ and were also profiled in CCPD 1.0.2 A total of 12 batches of intact cell line samples were profiled by shotgun proteome strategy: Hep3B (2 batches), 97H (4 batches), and LM3 (2 batches) were profiled by Orbitrap QExactive (Thermo Fisher Scientific, San Jose, CA), whereas Hep3B (2 batches) and 97H (2 batches) were profiled by Triple TOF 5600 (AB SCIEX, Concord, ON). All proteomic data were deposited to ProteomeXchange (http:// 127

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Figure 2. Chromosome 8 proteome atlas with data on the identification (red box) and quantification (blue box) of CCPD, PeptideAtlas, and GPMDB “green”. The blue depth denotes the abundance of the protein expression data. The median normalized protein data were transformed by log10 and rescaled into a [−8, 0.5] region. The color legend is provided at the bottom left. Three distinct MS missing blocks in 8p23.1 and 8q24.3 are marked by yellow frames.

Database and Data Set Versions

nonmetastasis groups. The metastasis group contained six batches of 97H profiling and two batches of LM3 profiling, whereas the nonmetastasis group contained four batches of Hep3B profiling. Statistic signal-to-noise ratio (SNR), defined as the reciprocal of the coefficient of variation, that is, the ratio of mean to standard deviation (μA − μB)/(σA + σB), where μ is the mean and σ is the standard deviation of the noise, was applied to measure the difference in protein abundances between the two groups. We utilized the Java GSEA package13 to compute SNR with 1000 phenotype permutations and set the upper and lower SNR cut-point of ±4 to yield 16 positive and 24 negative HCC metastatic potential-correlated proteins.

The versions of the databases and data sets used as searching background, baseline, and external references in this work are as follows: UniProtKB/Swiss-Prot release 2013.6, neXtProt release 2013.9, Ensembl Gene v.72, PeptideAtlas release 2013.8, global proteome machine database (GPMDB) release 2013.8, and Human Protein Atlas (HPA) version 11.0. Isomap and Three-Dimensional Scatter

Isomap as a nonlinear principal component (PC) method can be used in measuring similar degree of samples.11 We utilized a set of isomap MATLAB script from Kevin Dawson12 to visualize the similarity of cell lines in 3D PC space. The 3D scatter plots and correlation coefficients (Pearson and Spearman) of the transcriptome, translatome, and proteome data were all realized by MATLAB.

Enrichment Analysis

The enrichment analysis of 251 missing Chr. 8-coded proteins was carried out using the web-accessible Database for Annotation, Visualization, and Integrated Discovery (DAVID).14 DAVID can recognize the UniProt AC from data sets. Medium classification stringency and default items were chosen for enrichment calculation.

Metastasis-Related Differential Protein Screen

Protein abundances from the 12 batches of intact label-free proteome quantification were divided into metastasis and 128

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Figure 3. Venn diagrams comparing total (A) and Chr. 8-coded (B) MS missing proteome from CCPD, PeptideAtlas, and GPMDB “green” according to Swiss-Prot baseline. (C) 41 Chr. 8-coded proteins with different MS identification statuses were selected to verify their occurrence proportion in liver by IHC. “Y” and “N” denote whether the protein was identified in CCPD 2.0 or PeptideAtlas, respectively. The existing protein evidence in GPMDB was given according to its four-level criterion. We used the highest staining levels from HPA as references. The yellow highlighted occurrence proportions in our IHC verification were ≤20%.

Tissue Array and Immunohistochemistry

Image-Pro Plus 6.0. Mean optical density (MOD) to represent staining depth was calculated as (IOD/S). Most IOD and MOD distributions were non-normal; thus, we constructed the Delta of paired samples as (IODcarcinoma − IODpara‑carcinoma) or (MODcarcinoma − MODpara‑carcinoma) and used the one-tailed Wilcoxon signed rank test to evaluate whether the Delta was significantly lower or higher than 0.

The tissue arrays used for IHC verification were supplied by Xi’an Alena Biotechnology. The arrays contained tumor tissues corresponding to adjacent liver tissues of the 30 HCC cases and noncancerous liver tissues of the 20 autopsies (Table S1 in the Supporting Information). All of the donors were Chinese. IHC verification was carried out for 41 proteins in the 30 pairs of carcinoma/para-carcinoma HCC and 20 noncancerous liver tissues, with three replications. Details of the 41 primary antibodies are shown in Table S2 in the Supporting Information. Tissue arrays were deparaffinized and rehydrated at 60 °C for 30 min. The antigen retrieval solution was 10 mM citrate buffer, pH 6.0. Sections were blocked for nonspecific binding with nonimmunized goat sera for 10 min at room temperature and then incubated with primary antibodies for 12−18 h at 4 °C. Wash buffer was used for rinsing two times, and biotinylated secondary antibody was then added for 10 min at room temperature. Finally, streptavidin-peroxidase of immunohistochemistry was used to detect the protein expressions. Sera from nonimmunized goats were used as negative controls.

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RESULTS

Status of CCPD on Chromosome 8

CCPD 2.0 totally covered 63.6% (438/689) of Chr. 8-coded proteins and 62.6% (439/701) of Chr. 8-coded protein-coding genes (Figure 1A). The coverage was similar to that of PeptideAtlas15 (68.5% in protein level and 67.6% in gene level) and GPMDB “green”16 (71.2% in protein level and 71.3% in gene level). The Chr. 8-coded proteome coverage of CCPD 2.0 (76.4%), PeptideAtlas (81.9%), and GPMDB “green” (85.8%) increased according to the neXtProt “gold” criterion.17 High degrees of overlap existed among CCPD 2.0, PeptideAtlas, and GPMDB (Figure 1B,C). The Jaccard index among the three data sets defined as the size of the intersection divided by the size of the union of the sample sets was 384/552 = 69.6% in the gene level and 383/550 = 69.6% in the protein level. The Chr. 8-coded proteome atlas of CCPD 2.0 and its detailed 12 batches of identification status are shown in Figure 2 and Figure S1 in the Supporting Information, respectively.

IHC Image Acquisition and Statistics

IHC images, with 11 333 × 8533 pixels, 600 dpi each, were captured with an Olympus DP72 camera attached to a CX31 microscope (Olympus Imaging, Tokyo, Japan). Integrated optical density (IOD) to represent the staining quantity and positive stained area S of the images was determined with 129

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Table 1. Sample Sources of the 17 CCPD Unique Identified Proteins with RNA Proofs protein ADAM18 BAALC CNGB3 CYP11B2 FABP9 KCNQ3 KCNU1 KIAA1875 NKX2−6 PIWIL2 POTEA SCRT1 SLC18A1 SNX31 UNC5D LRRC6 PPP1R42 a

UniProt Q9Y3Q7 Q8WXS3 Q9NQW8 P19099 Q0Z7S8 O43525 A8MYU2 A6NE52 A6NCS4 Q8TC59 Q6S8J7 Q9BWW7 P54219 Q8N9S9 Q6UXZ4 Q86X45 Q7Z4L9

RNA proofs form transcriptomesa

CCPD 2.0 liver liver liver liver liver liver liver liver liver liver liver liver liver liver liver liver, Hep3B, 97H 97H, LM3

b

HCC, Hep3B, stomach, GC, AGS, BGC823, CC,c HCT116, SW480 liver, HCC, Hep3B, 97H, SNU398, SNU475, stomach, GC, colon, CC, HCT116, SW480 liver, HCC, 97L, stomach, GC, AGS, colon, CC liver, HCC, stomach, GC, CC stomach, GC, CC liver, HCC, Hep3B, 97L, 97H, LM3, stomach, GC, BGC823, colon, CC, SW480 liver, HCC, stomach, GC, CC liver, HCC, Hep3B, 97H, LM3, LM6, stomach, GC, AGS, BGC823, CC liver, GC, CC liver, HCC, LM6, stomach, GC, AGS, BGC823, colon, CC liver, HCC, stomach, GC, colon stomach, GC, AGS, BGC823, CC liver, HCC, LM6, stomach, GC, colon, CC liver, HCC, stomach, GC, CC liver, HCC, stomach, GC, AGS, BGC823, CC liver, HCC, 97L, 97H, LM3, LM6, stomach, GC, AGS, BGC823, colon, CC, HCT116, SW480 liver, HCC, Hep3B, 97H, LM3, LM6, GC, CC

From this work and CCPD 1.0 (ref 1). bGastric cancer. cColorectal cancer.

MS Missing Protein

MBOAT4) in the liver that were not in HPA because of the adequate sample size of 78 to 80. As an initiative of Chinese CHPP Ab pillar construction, all IHC data with 9840 highresolution images (three replications for the 41 proteins screened in 80 tissue samples) will be integrated and obtained in Chinese Chromosome-Centric Human Proteome Database (http://proteomeview.bioso.org/chromosome.jsp). The 251 CCPD missing proteins of Chr. 8-coded proteome were found to be significantly enriched in the “defensin, antibiotic, and antimicrobial” cluster (enrichment score = 9.93, p = 4.6 × 10−16, Benjamini = 9.3 × 10−14); “Ly-6 antigen/uPA receptor-like and UPAR/Ly6 domain” cluster (enrichment score = 5.47, p = 1.4 × 10−10, Benjamini =2.3 × 10−8); and “extracellular region, secreted, and disulfide bond” cluster (enrichment score = 2.79, p = 3.5 × 10−6, Benjamini = 4.7 × 10−4) by DAVID.14 We found two insufficient protein-identified cytobands in CCPD 1.0, namely, 8p23.1 and 8q24.3.3 These two cytobands remained as three distinct vacuous blocks in CCPD 2.0 (Figure 2). In particular , two vacuous blocks in 8p23.1 that were mostly composed of β-defensin family members were also missed by PeptideAtlas, GPMDB, HPA, and even neXtProt “gold”, with the exception that only five PCGs DEFB4B, DEFB103B, DEFB103A, DEFB4A, and USP17L2, were found in HPA and neXtProt “gold” (Figure S2 in the Supporting Information). It means these two blocks have seldom been identified with protein proof. The missing block A in 8p23.1 contained 18 PCGs with approximate palindrome symbols: DEFB4B, DEFB103B, SPAG11B, DEFB104B, DEFB106B, DEFB105B, DEFB107B, RP11-1118M6.1, AC084121.16, DEFB107A, DEFB105A, DEFB106A, DEFB104A, SPAG11A, DEFB103A, DEFB4A, ZNF705B, and LRLE1. The missing block B in 8p23.1 contained seven PCGs: DEFB136, DEFB135, DEFB134, RP11-481A20.11, DEFB130, ZNF705D, and USP17L2. Both absences in transcriptome and translatome of the missing block A further suggest its inactive state in our samples (Figure S2 in the Supporting Information). The IHC detection of protein DEFB136 in 30 pairs of carcinoma/paracarcinoma HCC and 20 noncancerous liver tissues also shows its especially low emergence (25/80) in hepatic samples (Figure 3C). Aside from 8p23.1, β-defensin family members are

C-HPP and neXtProt defined the “missing protein” as lacking all three of the following types of evidence: (1) definitive MS identification (PeptideAtlas canonical or GPMDB “green”), (2) antibody-based tissue evidence high or medium and subcellular localization (HPA), and (3) structural or function information.1,18,19 A total of 4089 proteins were unidentified or missing in both PeptideAtlas and GPMDB, leaving them to be verified, and then wiped off from the “MS missing protein list”. Our latest CCPD 2.0 contributed to wipe off 451 (11.0%) of these proteins (Figure 3A). CCPD 2.0 contributed 17 unique identified proteins to wipe off 10.9% of the contents of Chr. 8 MS missing protein list (Figure 3B); 15 of 17 proteins were solely identified from normal liver (CCPD 1.0),2 with RNA proofs from transcriptomes (Table 1). Ab is one of the three pillars of the HPP and an important complement to the MS pillar in finding the missing proteins.5 The HPA with protein evidence of ∼12 000 protein coding genes (PCGs) provided a strong support to the Ab pillar.20,21 Thus, we validated 41 Chr. 8-coded proteins in 30 pairs of carcinoma/para-carcinoma HCC and 20 single noncancerous liver tissues by IHC (Figure 3C). These proteins were chosen from different MS identification statuses among the CCPD, PeptideAtlas, and GPMDB: (1) 14 proteins had identification in at least two data sets; (2) 3 proteins had CCPD unique; (3) 1 protein had GPMDB unique; and (4) 23 proteins were missing in all three data sets. All 41 proteins showed their presence in these 78−80 valid samples. Protein NKAIN3 exhibited the lowest overall occurrence proportion (11/79) among the hepatic samples. The 11 proteins exhibited ≤20% occurrence proportions in HCC: CA2 (0/30), LZTS1 (2/29), ADAM18 (3/29), SNX31 (2/29), EFCAB1 (4/29), NKAIN3 (6/30), NKX6-3 (2/30), ADAM2 (3/30), C8orf31 (3/30), C8orf44 (4/29), and DEFB136 (2/30). These low occurrence proportions can partially explain why they can be missed when an MS experiment was not designed well. This IHC evidence provided circumstantial evidence of its existence in samples as well as guidance to select samples for further missing protein finding by MS. Our IHC validation added evidence of seven proteins (IDO2, SLC26A7, TRHR, DEFB136, GPR20, KCNK9, and 130

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Figure 4. (A) Isomap of 12 batches of quantitative proteome data: four batches of Hep3B profiling (yellow and green balls), six batches of 97H profiling (red, pink, and light-blue balls), and two batches of LM3 profiling (blue balls). (B) 3D with 2D projected scatter plots of Hep3B, 97H, and LM3 quantitative proteome data. (C) Heat map of top differential proteins between the metastasis and nonmetastasis HCC cell lines. All 16 positive and 24 negative HCC metastatic potential-correlated proteins were with |SNR| ≥ 4. The red depth denotes the abundance of the protein expression data. The color legend is provided on the top.

Hep3B and 97H or LM3 (Pearson 0.69 and 0.74, Spearman 0.86 and 0.78) (Figure 4B). This phenomenon also appeared in the CCPD-coupled transcriptome and translatome data (Figure S4 in the Supporting Information). Thus, we focused on differentially expressed proteins for distinct metastasis versus nonmetastasis in this study. Sixteen positive and 24 negative HCC metastatic potential-correlated proteins were identified (Figure 4C). Seven of the 16 positively correlated candidates were reported as metastasis promotion proteins: AGR2 for HCC23 and prostate cancer; 24 S100A10 for papillary thyroid carcinoma;25 SNCG for ovary,26 mammary gland,26 colorectal,26−28 and pancreatic cancers;29 PTRF for prostate cancer;30 MYH9 for esophageal squamous31 and gastric cancers;32 G6PD for gastric cancer;33 and ANXA1 for gastric,34 lung,35,36 breast,37−39 esophageal, and esophagogastric junction adenocarcinomas.40 Among these proteins, AGR2, with the highest positive SNR, was fully demonstrated to have its HCC metastasis promotion ability by us.23 By contrast, only 2 of the 24 negatively correlated candidates have been reported as metastasis repression proteins: S100A14 for colorectal cancer41 and CDKN2A for HCC42,43 and penile carcinomas.44

also clustered in three other cytobands: 6p12.3, 20p13, and 20q11.21, totaling 30 PCGs. All of these clusters were missed in the identification by CCPD 2.0 (also absent in transcriptome and translatome), PeptideAtlas (with exception of DEFB129 and DEFB132), GPMDB, HPA (with exception of DEFB127), and neXtProt “gold” (with exception of DEFB127, DEFB129, and DEFB132) (Figure S3 in the Supporting Information). Notably, constitutive expression of β-defensin DEFB1 (also in 8p23.1) was highly detected in multiple data sets, whereas DEFB4A and DEFB4B, which were inducible by infectious and inflammatory stimuli, were not detected (Figure S2 in the Supporting Information).22 These results suggest that infectious and inflammatory stimuli-related β-defensins were seldom expressed in usual samples. According to the evidence from CCPD, these samples included noninflamed liver, stomach, and colon, their cancerous tissue, and derived cell lines. HCC Metastasis-Related Proteins

Two HCC cell lines, 97H and LM3, possessed metastatic potential,7 whereas the HCC cell line Hep3B was nonmetastatic.6 The nonlinear principal component isomap of the quantitative proteomics data reflected much closer biological characteristics between 97H and LM3 (Figure 4A). The correlation between 97H and LM3 (Pearson 0.93, Spearman 0.90) was also much higher than that between 131

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Table 2. IHC Data Statistics of 13 Verified and 20 Newly Discovered HCC Tumorigenesis-Associated Proteins Pa of decrease or increaseb in C vs PC

well-verified (11/13)

nonsignificant (2/13)

newly found lower in carcinoma tissue (16)

newly found higher in carcinoma tissue (4)

protein

UniProt

cytoband

IOD

MOD

CA1 CA2 DLC1 EPHX2 LZTS1 NRG1 PCM1 PROSC SCARA5 SORBS3 WRN SH2D4A ZNF703 ADAM2 C8orf31 C8orf34 C8orf44 FGF17 GPR20 GRINA IDO2 KCNK9 OPRK1 OR4F21 RGS22 TRHR WISP1 XKR6 ZNF596 BAALC C8orf12 MBOAT4 NKX6-3

P00915 P00918 Q96QB1 P34913 Q9Y250 Q02297 Q15154 O94903 Q6ZMJ2 O60504 Q14191 Q9H788 Q9H7S9 Q99965 Q8N9H6 Q49A92 Q96CB5 O60258 Q99678 Q7Z429 Q6ZQW0 Q9NPC2 P41145 O95013 Q8NE09 P34981 O95388 Q5GH73 Q8TC21 Q8WXS3 Q96KT0 Q96T53 A6NJ46

8q21.2 8q21.2 8p22 8p21.2 8p21.3 8p12 8p22 8p11.23 8p21.1 8p21.3 8p12 8p21.3 8p11.23 8p11.2 8q24.3 8q13 8q13.1 8p21.3 8q24.3 8q24.3 8p11.21 8q24.3 8q11.2 8p23.3 8q22.2 8q23.1 8q24.22 8p23.1 8p23.3 8q22.3 8p23.1 8p12 8p11.21

0.002 0.000 0.002 0.000 0.008 0.000 0.015 0.005 0.000 0.002 0.002 0.138b 0.125b 0.029 0.005 0.325 0.006 0.003 0.000 0.004 0.000 0.014 0.003 0.001 0.071 0.002 0.000 0.022 0.024 0.022b 0.000b 0.001b 0.015b

0.005 0.000 0.534 0.000 0.003 0.000 0.012 0.000 0.000 0.000 0.000 0.180b 0.155b 0.121 0.003 0.015 0.027 0.010 0.001 0.000 0.000 0.010 0.011 0.443b 0.030 0.009 0.074 0.084 0.005 0.069b 0.000b 0.002b 0.094b

One-tailed Wilcoxon signed rank test P value of Delta if it was lower (default) or higher than 0. “C” and “PC” represented carcinoma and paracarcinoma tissues, respectively. bOne-tailed Wilcoxon signed rank test P values of Delta if it was higher than 0.

a

IHC Validation of Chr. 8-Coded Protein Deficiencies in HCC Tumorigenesis

XKR6, and ZNF596, whereas four proteins were higher in the carcinoma tissue, BAALC, C8orf12, MBOAT4, and NKX6-3. Only three proteins that were lower in carcinoma tissue were reported as tumor or metastasis suppressor: ADAM2 for breast cancer45 and head and neck squamous cell carcinoma,46 KCNK9 for breast cancer,47 RGS22 for esophageal cancer,48 and WISP1 for lung cancer49 and melanoma.50 Only 1 protein that was higher in carcinoma tissue was reported as a cancer promoter: BAALC for acute promyelocytic leukemia51 and acute myeloid leukemia.52−55

We have found 8p21∼23 and some other Chr. 8 cytoband deficiencies during HCC tumorigenesis in a previous report of CCPD 1.0.3 On the basis of the label-free MS quantification, 13 proteins were found to be down-regulated or totally deficient in HCC cell lines. In this work, the 13 proteins were quantitatively validated in the 30 pairs of carcinoma/para-carcinoma HCC and 20 noncancerous liver tissues by IHC. All 13 proteins showed significant downregulation tendency in HCC, with the exception of two nonsignificant ones: H2D4A and ZNF703 (Table 2, Figure S5 in the Supporting Information). EPHX2 was 1 of the 11 well-verified proteins in HCC carcinoma/para-carcinoma comparison (Figure 5A,C,D) and also showed even higher expression level in noncancerous livers (Figure 5B,C). Aside from these 13 proteins, IHC discovered another 20 proteins with significantly differential expression between carcinoma and para-carcinoma tissues in HCC (Table 2, Figure S6 in the Supporting Information). A total of 16 proteins were lower in the carcinoma tissue, ADAM2, C8orf31, C8orf34, C8orf44, FGF17, GPR20, GRINA, IDO2, KCNK9, OPRK1, OR4F21, RGS22, TRHR, WISP1,

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DISCUSSION Sample selection determines the proteome top limit that we can obtain in accessible technological frame. Deeply covered proteome profiling of CCPD, especially in liver-related samples (normal liver and eight HCC cell lines of CCPD 2012, three HCC cell lines of CCPD 2013), allow us to approach a possible saturated proteome detection of human liver, relying on the current techniques. For the objective of missing protein finding in C-HPP, alternative samples other than the improvement of detecting techniques will be more efficient to reclaim the uncultivated land of missing proteins to date. For example, the 132

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Figure 5. IHC validation of protein EPHX2. EPHX2 IHC staining image thumbnails in 30 pairs of carcinoma/para-carcinoma HCC (A) and 20 noncancerous liver (B) tissues with three replications. The carcinoma tissues of patient P07 were invalid. Connected line plot of IOD (C) and MOD (D) shows significantly decreased level in carcinoma “C” versus para-carcinoma “PC” tissue (one-tailed Wilcoxon signed rank test PDelta = 0.000). The interval plot of IOD (right part of panel C) shows even higher EPHX2 level in noncancerous liver tissues “N”. 133

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infectious and inflammatory stimuli-inducible β-defensin family members may be detected under special conditions, such as in lesions of the tongue,56 inflammatory bowel disease,57,58 and H. pylori-induced gastritis.59 The organization of chromosomes during evolution clustered many functional associated genes together in physical location. Therefore, some chromosomes or cytobands are associated with physiological or pathological processes to a great extent, including the 6p21.3∼22.1 cytoband of major histocompatibility complex (MHC)60 and 8p deletion associated with HCC metastasis.61 Considering how proteomes of different human cells, tissues, and organs (CTOs) and physiopathological processes relate to one another and also whether some human chromosomes-coded proteomes show bias to any CTOs or physiopathological processes is interesting. Exploring the chromosome -categorized proteome with guidance from associated physiopathological issues should be beneficial.

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31170780, 91131009, 81322028, 81372135), State Key Project Specialized for Infectious Diseases (2012ZX10002012), Shenzhen Municipal Government of China (20101749), and Shenzhen Key Laboratory of Transomics Biotechnologies (CXB2011O8250096A).

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ASSOCIATED CONTENT

S Supporting Information *

Donor information of tissue array. Information of 41 primary antibodies. Chr. 8 proteome atlas with data on the identification and quantification of 12 batches proteome profiling. Chr. 8 proteome atlas with data on the identification and quantification of transcriptome, translatome profiling, detailed CCPD, and external proteomic references (HPA, PeptideAtlas, GPMDB “green”, neXtProt, and its “gold” data set). Heatmaps of 6p12.3, 20p13, and 20q11.2 with data on the identification and quantification of transcriptome, translatome profiling, detailed CCPD, and external proteomic references. Isomap and 3D with 2D projected scatter plots of Hep3B, 97H, and LM3 transcriptomes and translatomes. Connected line plots of IHC density in carcinoma vs para-carcinoma tissue and interval plots in noncancerous liver tissues of 13 chosen to validate and 20 newly discovered HCC tumorigenesis associated proteins, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Xiaohong Qian: Tel/Fax: + 86 10 80705055. E-mail: [email protected]. *Xiaomin Lou: Tel/Fax: +86 10 80485324. E-mail: louxm@ genomics.org.cn. *Gong Zhang: Tel: +86 20 85220431. Fax: +86 20 85222616. E-mail: [email protected]. *Fan Zhong: Tel/Fax: +86 21 54237158. E-mail: zonefan@ 163.com. Author Contributions ◆

Yang Liu, Wantao Ying, Zhe Ren, and Wei Gu contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The study was supported by MOST-S973/863 projects (2013CB910802, 2012AA020200, 2010CB912700, 2013CB910500, 2012CB910600, 2011CB910600, 2012CB910301, 2013CB911200, 2011AA02A114, 2013CB910502, 2011CB910700), National Natural Science Foundation of China projects (31000379, 31070673, 134

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