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. Author manuscript; available in PMC: 2015 Jan 3.
Published in final edited form as: J Proteome Res. 2013 Dec 6;13(1):137–146. doi: 10.1021/pr400792p

Proteogenomic Analysis of Human Chromosome 9-Encoded Genes from Human Samples and Lung Cancer Tissues

Jung-Mo Ahn †, Min-Sik Kim ‡, Yong-In Kim †, Seul-Ki Jeong ||, Hyoung-Joo Lee ||, Sun Hee Lee ||, Young-Ki Paik ||, Akhilesh Pandey ‡, Je-Yoel Cho †,*
PMCID: PMC3918476  NIHMSID: NIHMS547529  PMID: 24274035

Abstract

The Chromosome-centric Human Proteome Project (C-HPP) was recently initiated as an international collaborative effort. Our team adopted chromosome 9 (Chr 9) and performed a bioinformatics and proteogenomic analysis to catalog Chr 9-encoded proteins from normal tissues, lung cancer cell lines and lung cancer tissues. Approximately 74.7% of the Chr 9 genes of the human genome were identified, which included approximately 28% of missing proteins (46 of 162) on Chr 9 compared with the list of missing proteins from the neXtProt master table (2013-09). In addition, we performed a comparative proteomics analysis between normal lung and lung cancer tissues. Based on the data analysis, 15 proteins from Chr 9 were detected only in lung cancer tissues. Finally, we conducted a proteogenomic analysis to discover Chr 9-residing single nucleotide polymorphisms (SNP) and mutations described in the COSMIC cancer mutation database. We identified 21 SNPs and 4 mutations containing peptides on Chr 9 from normal human cells/tissues and lung cancer cell lines, respectively. In summary, this study provides valuable information of the human proteome for the scientific community as part of C-HPP. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD.

Keywords: C-HPP, missing proteins, lung cancer, biomarker

INTRODUCTION

The Chromosome-centric Human Proteome Project (C-HPP) was initiated to annotate and characterize all proteins encoded by each human chromosome1. Approximately 30% of all chromosome-encoded proteins currently lack of any experimental proof at the protein level2. The primary goal of this project was to identify and characterize chromosome-encoded proteins that exist based on genetic evidence but currently lack mass spectrometry (MS) evidence or antibody (Ab) detection, termed “missing proteins”. The C-HPP initiative defined missing proteins as those missing from the three mass spectrometry databases (neXtProt, GPMdb and PeptideAtlas) and an antibody-based database (Human Protein Atlas)1. Twenty-four groups worldwide have joined the C-HPP initiative to share the tasks including the annotation of identified protein-coding genes and new protein isoforms, including new types of variants such as SNPs3. Human Chr 9, our focus in this study, is known to have approximately 141 million base pairs, representing approximately 4.5% of the total DNA. At the time of the preparation of this manuscript, the number of protein-coding genes on Chr 9 was 821 based on the C-HPP Master Table (Ensembl v72, 2013-09) at the Yokohama HUPO meeting.

Mutations of genes on Chr 9 have been associated with various types of cancer, including lung cancers4–6. Our previous studies have focused on lung cancer biomarkers, but there is still a lack of specific proteins or their variants, in particular, as biomarkers for lung cancer. Reports of chromosomal genetic changes serve as useful information to identify specific variant forms of proteins during carcinogenesis. Specific chromosomal aberrations have been associated with particular cancer types. The partial loss of the Chr 9p arm has been reported as one of the most frequent genetic aberrations in lung cancer patients, and lost chromosomal segments have proven to harbor candidate tumor suppressor genes7, 8. Several well-characterized tumor suppressor genes, such as CDKN2A, MTAP and P16β, have been mapped to 9p, and the DMRT1, DMRT3, and DOCK8 genes, located on 9p24, and TSC1 gene, mapped to 9q, are often involved in amplifications, translocations or deletions in lung cancers9–11. Some of the proteins encoded by genes on Chr 9 with a high frequency of genetic alterations, such as single nucleotide polymorphism (SNPs) and alternative splice variants, can be identified by proteogenomic approaches and provide useful information for diagnoses and mechanistic studies.

In this study, we performed Chr 9-based data analysis to catalog Chr 9-encoded missing proteins and to identify Chr 9-based lung cancer-specific proteins, SNPs and mutations. To do so, we collected high quality mass spectrometry datasets using tandem mass spectral data that were acquired on a high resolution mass analyzer in the high-high mode with the HCD fragmentation method. Our bioinformatics analysis catalogued a number of proteins encoded by 614 genes on Chr 9 that included 46 missing proteins. In addition, we performed lung cancer biomarker discovery from the proteins on Chr 9 identified by mass spectrometry-based proteomics on human lung cancer tissues. We also performed an extensive analysis to identify peptide evidence of single nucleotide polymorphisms (SNPs) and mutations from the normal lung and lung cancer cell lines/tissues datasets. Our results will provide information about tissue-/cell line-specific Chr 9-encoded proteins.

EXPERIMENTAL METHODS

Chromosome 9 based protein identification

The publicly available high quality proteomics datasets were collected (PMID: 23933261, PMID: 22278370), using both MS and MS/MS scans that were acquired on an Orbitrap mass analyzer at high-high mode. Briefly, each sample was prepared for 24 fractions (high pH RPLC or SDS-PAGE) that were analyzed on either LTQ-Orbitrap Velos or LTQ-Orbitrap Elite (Thermo Fisher, San Jose, CA) with Agilent’s 1200 nano-LC system to a trap column (2 cm × 75 μm, C18 material 5 μm, 120 Å) and an analytical column (10 cm × 75 μm, C18 material 5 μm, 120 Å). Peptide samples were loaded onto trap column in 3% solvent B (90% acetonitrile in 0.1% formic acid) and washed for 5 minutes. Peptides were eluted using a gradient of 3–35% solvent B for 60 minutes at a constant flow rate of 0.4 μl/min. All tandem spectra were generated by the Higher-energy collision dissociation (HCD) using 40% normalized collision energy. We reanalyzed these datasets (more than 2,000 raw files) using our pipeline with the Proteome Discoverer Platform and performed searches against the human RefSeq Protein Database (version 50) using the SEQUEST and Mascot database search algorithms. A list of non-redundant peptides was collected, and the resulting peptide sequences were searched against the human genome database. After identifying the proteins, spectral counts were normalized by total spectral counts for each MS data. These sums were then scaled so that they were equivalent. Next, the peptides uniquely mapped to human Chr 9 were selected for further analysis in this study.

Missing proteins on human Chr 9

In this study, we integrated the following databases to develop a list of missing proteins on human Chr 9. We used C-HPP wiki website databases (SIB Swiss Institute of Bioinformatics, rel. 2013-09-26), which comprised database information from Ensembl (version 72), neXtProt (rel. 2013-09, 3,844 missing genes), GPMdb (green, rel. 2013-08), PeptideAtlas (rel. 2013-08), and Human Protein Atlas (rel. 2012-12).

Protein identification of lung cancer tissue lysates

Lung cancer tissues and adjacent normal lung tissues (1 mg each of tissues pooled from 5 patients) were lysed and suspended in RIPA buffer (Thermo, USA) containing protease inhibitors (1:200) (Roche, Germany). The samples were sonicated, vortexed on ice and centrifuged at 14,000 rpm at 4 °C for 10 min to collect the protein supernatants, followed by the evaporation of water using a speed vacuum. Tryptic digestion was conducted based on the filter-aided sample preparation protocol (FASP)12. Briefly, 1 mg of the protein sample was solubilized with 8 M urea in a 10 kDa cut-off Amicon spin column (Millipore, MA, USA). The proteins were reduced with 10 mM dithiothreitol (DTT) at 60 °C for 30 min and then alkylated with 10 mM iodoacetamide (IAA) in the dark at room temperature for 30 min. Following alkylation, the samples were washed with 8 M urea and then with ammonium bicarbonate solution; the samples were finally subjected to tryptic digestion overnight. The peptides were eluted from the spin column by centrifugation at 3,000 rpm. The OFFGEL electrophoresis fractionation (Agilent, 12 fractions) and tandem mass spectrometric analysis of normal lung and lung cancer tissue lysate samples were performed. The LTQ-Orbitrap mass spectrometry was used for acquiring the mass spectra to identify proteins. Briefly, Nano high-performance liquid chromatography (nano-HPLC) analysis was performed using an Easy n-LC system (Thermo Fisher). The capillary column used (150 × 0.075 mm) for LC-MS/MS analysis was obtained from Phoenix S&T (Chester, PA, USA), and the slurry was packed in-house using a 5-μm, 100-Å pore size Magic C18 stationary phase resin (Michrom Bio Resources, Auburn, CA, USA). The mobile phase A for LC separation was 0.1% formic acid in deionized water, and the mobile phase B was 0.1% formic acid in acetonitrile. The chromatography gradient was designed for a linear increase from 0 to 8% B in 5 min, 5 to 25% B in 100 min, 25 to 45% B in 10 min, and 45 to 60% B in 10 min. Orbitrap full MS scans were acquired from m/z 350 to 1500 at a resolution of 15 000 (at m/z 400). The minimum threshold was set to 100 000 ion counts. Parent ions were fragmented using the LTQ (isolation width of 2 m/z units) with a maximum injection time of 100 ms combined with an AGC value of 1 ×104 using three fragmentation modes such as collision-induced dissociation (CID) alone, the reagent ion source emission current, reagent ion electron energy, and reagent ion source chemical ionization pressure were set to 35 mA, 70 V, and 26 psi, respectively. The tandem mass spectra were extracted, and searches were conducted against the UniProt database (rel. 2012-06, 86,875 entries) using MASCOT software (version 2.2.04). The search parameters used were the same as those previously reported13.

Customization of peptides database for SNPs and mutations

The dbSNP (version 138) and COSMIC (version 66) (Forbes et. al. 2011, PMID: 20952405, Sherry et. al, 2001 PMID: 11125122) databases were downloaded from their respective FTP servers. Every genomic coordinate was searched against the hg19 human reference genome for confirmation. A total of 3,052,321 non-synonymous SNPs and 1,100,191 mutations were used to create the databases. The non-synonymous SNPs and mutations were selected and incorporated one at a time into the protein sequences. The altered protein sequences were trypsinized in silico to create all possible fully tryptic peptides with lengths between 6 and 30 amino acids. All non-synonymous SNPs and mutations were considered if they caused alterations of fully tryptic peptide sequences that were observable by mass spectrometry. When a fully tryptic peptide was altered compared with the annotated protein database, it was deposited to a custom peptide database in Fasta format. This annotation tool was developed using the Java 2 Platform, Standard Edition. Finally, SNP-specific and mutation-specific peptide databases were built for the proteogenomic analysis. The SNP database comprised a total of 1,746,675 fully tryptic peptide sequences, and the COSMIC database contained 426,869 peptide sequences. Mapping of SNPs found in the protein coding regions of the gene (cSNPs) was automated by a tool developed in-house using shell scripts.

Identification of SNPs and mutation on Chr 9

All the unassigned tandem mass spectra were collected. In short, a tandem spectrum was set aside when it was not matched to any peptide at a 1% false discovery rate (FDR) in the SEQUEST and MASCOT searches. All unassigned spectra were then merged into peaklist files that were used to search for SNPs and mutations by SEQUEST. At a 1% FDR, peptides with SNPs or mutations derived from Chr 9 were collected for further analysis.

RESULTS AND DICUSSION

Catalog of Chr 9 proteome

The overall workflow is illustrated in Figure 1. Chr 9-encoded proteins were cataloged by using publicly available datasets. We analyzed Chr 9-encoded proteins using large human proteome profiling data of normal human samples (18 adult tissues, 6 primary hematopoietic cells and 6 fetal tissues) from Pandey lab (Supplementary Table 1). Based on these normal proteome datasets, the protein products of a total of 614 Chr 9 genes were detected (including peptides mapped to multiple protein entries), which were identified from 11,065 peptides. These identified proteins covered approximately 74.7% of the Chr 9 protein-coding genes compared with the neXtProt database. We also focused on Chr 9-centric analysis using lung cancer datasets, which resulted in the identification of a number of proteins derived from a total of 6,824 protein-coding genes, including 255 proteins located on Chr 9.

Figure 1.

Figure 1

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Schematic diagram of the overall workflow for the analysis of human chromosome 9-encoded missing proteins, single nucleotide polymorphisms, lung cancer-selective comparative analysis and mutational analysis.

The list of missing proteins on Chr 9 was generated by in silico database analysis, as described in the Experimental Methods. A total of 162 missing proteins that are awaiting validation are listed as Chr 9-encoded proteins, which have no proteomics evidence and have only protein level existence as neXtProt uncertain proteins. The entire chromosome 9-encoded missing protein data lists identified from normal human samples as well as normal lung and lung cancer cell lines and tissues were found from the missing protein list by manually searching by gene symbols and descriptions. When using all three datasets, missing proteins from 46 genes were detected, including Ankyrin repeat domain 20 families and olfactory receptor families (Table 1 and Supplementary Table 2). Interestingly, we have found FAM157B for Chr 9 in the adult colon, OR1J1 for Chr 9 in the fetal liver, and OR1L1 in the placenta. Some of the missing proteins were detected with a high number of unique peptides. For example, Actin-like 7B (ACTL7B) and Calicin (CCIN) were detected with more than 10 unique peptides. The missing protein list was compared with the GPMDB and HPA databases (Supplementary Table 2). Most of the missing proteins had no or low levels detected by MS or antibody, except ACTL7B (green level in GPMdb and high level in HPA).

Table 1.

The list of newly detected missing proteins on chromosome 9. The detailed information on the peptide sequences is available in Supplementary Table 2.

NCBI Accession No. Gene Symbol Description
NP_006677.1 ACTL7B actin-like 7B
NP_671728.2 ANKRD18A ankyrin repeat domain 18A
NP_115626.2 ANKRD20A1 ankyrin repeat domain 20 family, member A1
NP_001012421.1 ANKRD20A2 ankyrin repeat domain 20 family, member A2
NP_001012419.1 ANKRD20A3 ankyrin repeat domain 20 family, member A3
NP_001092275.1 ANKRD20A4 ankyrin repeat domain 20 family, member A4
NP_054729.3 ASTN2 astrotactin 2
NP_001012520.2 C9orf117 chromosome 9 open reading frame 117
NP_997394.1 C9orf139 chromosome 9 open reading frame 139
NP_955382.3 C9orf50 chromosome 9 open reading frame 50
NP_001122090.1 C9orf57 chromosome 9 open reading frame 57
NP_689782.2 C9orf66 chromosome 9 open reading frame 66
NP_001074020.1 C9orf84 chromosome 9 open reading frame 84
NP_775821.2 CCDC171 coiled-coil domain containing 171
NP_005884.2 CCIN calicin
NP_001124337.1 DMRT2 doublesex and mab-3 related transcription factor 2
NP_001138721.1 FAM157B family with sequence similarity 157, member B
NP_001001710.1 FAM166A family with sequence similarity 166, member A
NP_001157782.1 FAM166B family with sequence similarity 166, member B
NP_001092749.1 FOXD4L2 forkhead box D4-like 2
NP_954586.4 FOXD4L3 forkhead box D4-like 3
NP_954714.2 FOXD4L4 forkhead box D4-like 4
NP_001119806.1 FOXD4L5 forkhead box D4-like 5
NP_001078945.1 FOXD4L6 forkhead box D4-like 6
NP_002164.1 IFNA16 interferon, alpha 16
NP_009110.2 INSL6 insulin-like 6
NP_001017969.2 KIAA2026 Uncharacterized protein KIAA2026
NP_997393.3 LCNL1 lipocalin-like 1
NP_055396.1 OBP2B odorant binding protein 2B
NP_001001919.1 OR13C4 olfactory receptor, family 13, subfamily C, member 4
NP_001004483.1 OR13C8 olfactory receptor, family 13, subfamily C, member 8
NP_001004450.1 OR1B1 olfactory receptor, family 1, subfamily B, member 1
NP_001004451.1 OR1J1 olfactory receptor, family 1, subfamily J, member 1
NP_001005236.3 OR1L1 olfactory receptor, family 1, subfamily L, member 1
NP_001005234.1 OR1L3 olfactory receptor, family 1, subfamily L, member 3
NP_001005235.1 OR1L4 olfactory receptor, family 1, subfamily L, member 4
NP_001004453.2 OR1L6 olfactory receptor, family 1, subfamily L, member 6
NP_001001923.1 OR5C1 olfactory receptor, family 5, subfamily C, member 1
NP_001128691.1 PIP5KL1 phosphatidylinositol-4-phosphate 5-kinase-like 1
NP_659488.2 RNF183 ring finger protein 183
NP_001034484.3 SPATA6L spermatogenesis associated 6-like
NP_001007472.2 TRPM3 transient receptor potential cation channel, subfamily M, member 3
NP_001012361.1 WDR31 WD repeat domain 31
NP_001038941.1 WDR38 WD repeat domain 38
NP_003399.1 ZFP37 ZFP37 zinc finger protein
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Tissue-wise expression pattern of the Chr 9 proteome

Chr 9-encoded proteins in the human normal proteome dataset are presented in Table 2 and Supplementary Table 1. Among all the samples, the highest number of Chr 9-encoded proteins was identified in the adult testis (372), and the smallest number was identified in platelets (174). We observed four Chr 9-encoded proteins that were detected most abundantly in all sample types: Heat shock 70 kDa protein 5 (HSPA5), Spectrin alpha non-erythrocytic 1 (SPTAN1), Talin 1 (TLN1), and Valosin-containing protein (VCP). HSPA5, a glucose-related heat shock protein, plays a role in facilitating the assembly of multimeric protein complexes inside the endoplasmic reticulum14. SPTAN1, similar to erythrocyte spectrin, is involved in secretion and also interacts with calmodulin in the calcium-dependent movement of the cytoskeleton at the membrane15. TLN1, a cell junction protein, is involved in connections of major cytoskeletal structures to the plasma membrane16. VCP is necessary for the fragmentation of Golgi stacks during mitosis and is involved in the formation of the transitional ER17. Moreover, tissue or blood cell line selective abundant proteins (dominantly detected by more than 95% in one type of tissue or cell) were revealed in our large data set analysis, including Paired box 5 (PAX5) in B cells, Calicin (CCIN) in the adult testis, and Src homology 2 domain containing transforming protein 3 (SHC3) in the adult frontal cortex. PAX5 is known to be involved in B-cell differentiation, neural development and spermatogenesis18. CCIN is a cytoskeletal element involved in spermiogenic differentiation19. SHC3 is a signaling adaptor that couples activated growth factor receptors to signaling pathways in neurons and is highly expressed in the cerebral cortex and frontal lobes20. Our data indicate that the multi-tissue proteome analysis strongly confirms the previously known protein-tissue relationship and biology.

Table 2.

The number of chromosome 9-encoded proteins identified and the sum of normalized spectra counts in the normal human samples.

Samples Protein numbers identified Sum of normalized specrta count
Adult testis 372 28439
Adult ovary 353 27908
Adult retina 346 29355
Adult pancreas 336 26923
Fetal heart 333 27438
CD8+ T cells 328 30468
Fetal testis 323 24545
Fetal ovary 317 35325
Fetal liver 315 28612
Fetal brain 305 33194
Adult prostate 301 21395
Adult frontal cortex 296 42961
Adult liver 293 32734
B cells 293 22692
Adult adrenal gland 291 34811
Fetal gut 288 23047
Adult colon 285 21742
CD4+ T cells 273 18485
NK cells 269 23882
Adult rectum 265 24815
Adult spinal cord 258 21308
Adult urinary bladder 257 18523
Adult gallbladder 249 23021
Monocytes 247 16888
Adult heart 230 15517
Adult kidney 228 28469
Adult lung 216 22252
Placenta 214 21349
Adult esophagus 181 16445
Platelets 174 34265
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For the identification of the tissue or blood cell line-selective abundant Chr 9 protein lists, we compared the data for 30 different samples. A list of the top 10 abundantly detected Chr 9 proteins in each of the 30 different samples is presented in Supplementary Table 3. Spectrin alpha non-erythrocytic 1 (SPTAN1) was the most abundantly expressed Chr 9 protein in the 5 fetal tissues, except the fetal liver (heat shock 70 kDa protein 5, HSPA5). SPTAN1 was the most abundantly expressed Chr 9 protein in the 10 adult tissues, followed by talin 1 (TLN1) (5 out of 18). TLN1 was the most abundant Chr 9 protein in 5 out of 6 immune cell lines analyzed. The cell-/tissue-specific protein lists are provided in Table 3 with normalized spectra counts. With the detection sensitivity of mass spectrometry used in this study, a total of 122 proteins were identified as cell-/tissue-specific Chr 9 proteins (i.e., detected in only one sample and not in any others). Among 108 cell-/tissue-specific Chr 9-encoded proteins from the normal human samples, the adult retina showed the highest number of proteins (12); conversely, no tissue-specific protein was detected in the adult lung, adult rectum, and adult prostate. Most of the tissue-specific Chr 9-encoded proteins were identified with normalized spectra counts of 2.2 to 10. Notably, the proteins detected with lower spectra counts would not show high selectivity for the tissue and might be detected in other tissues using more sensitive analytical approaches. However, some of the specific proteins were identified as being highly specific, including Chr 9 open reading frame 169 in adult esophagus and CD 72 molecule in B cells. These results show that important tissue-selective Chr 9 encoded proteins are differentially expressed in not only fetal and adult tissues but also immune cells.

Table 3.

The list of cell-/tissue-specific proteins detected in normal human samples.

Tissue Gene Sum of normalized spectra count NCBI accession ID Protein existence Proteomics Antibody Description
Fetal heart AGTPBP1 4.8 NP_056054.2 protein level yes no ATP/GTP binding protein 1
C9orf66 4.7 NP_689782.2 transcript level no yes chromosome 9 open reading frame 66
IFNA14 4.7 NP_002163.2 protein level no no interferon, alpha 14
LCN6 2.9 NP_945184.1 protein level no no lipocalin 6
ZDHHC21 2.9 NP_848661.1 protein level yes no zinc finger, DHHC-type containing 21
CBWD1 2.8 NP_001138827.1 protein level yes no COBW domain containing 1
*Common Peptide A 2.8 - - - - -
ZNF510 2.8 NP_055745.1 transcript level yes yes zinc finger protein 510
Fetal liver FKTN 10.6 NP_001073270.1 protein level yes yes fukutin
OR5C1 5.5 NP_001001923.1 transcript level no no olfactory receptor, family 5, subfamily C, member 1
USP20 5.5 NP_006667.3 protein level yes yes ubiquitin specific peptidase 20
OR1J1 2.7 NP_001004451.1 transcript level no no olfactory receptor, family 1, subfamily J, member 1
IFNA21 2.3 NP_002166.2 protein level no no interferon, alpha 21
IFNK 2.3 NP_064509.2 protein level no no interferon, kappa
SLC35D2 2.3 NP_008932.2 protein level yes no solute carrier family 35, member D2
Fetal gut LCN15 2.2 NP_976222.1 protein level yes no lipocalin 15
*Common Peptide B 2.2 - - - - -
OBP2A 2.2 NP_055397.1 protein level no yes odorant binding protein 2A
Fetal ovary C9orf85 4.8 NP_872311.2 protein level yes no chromosome 9 open reading frame 85
GKAP1 3.6 NP_001129425.1 protein level yes yes G kinase anchoring protein 1
NTMT1 3.6 NP_054783.2 protein level yes yes N-terminal Xaa-Pro-Lys N-methyltransferase 1
Fetal testis SPATA6L 7.9 NP_001034484.3 transcript level no yes spermatogenesis associated 6-like
*Common Peptide C 2.4 - - - - -
Fetal brain NOTCH1 2.8 NP_060087.3 protein level yes yes notch 1
Adult frontal cortex LRSAM1 6.6 NP_001005374.1 protein level yes yes leucine rich repeat and sterile alpha motif containing 1
NTRK2 6.3 NP_001018074.1 protein level yes yes neurotrophic tyrosine kinase, receptor, type 2
PIP5K1B 5.2 NP_003549.1 protein level yes yes phosphatidylinositol-4-phosphate 5-kinase, type I, beta
*Common Peptide D 4.1 - - - - -
INSL4 3 NP_002186.1 protein level no no insulin-like 4 (placenta)
CAMSAP1 2.8 NP_056262.3 protein level yes yes calmodulin regulated spectrin-associated protein 1
PRUNE2 2.6 NP_056040.2 protein level yes yes prune homolog 2 (Drosophila)
*Common Peptide E 2.6 - - - - -
GPR21 2.6 NP_005285.1 transcript level yes yes G protein-coupled receptor 21
LRRC8A 2.6 NP_001120717.1 protein level yes yes leucine rich repeat containing 8 family, member A
SLC24A2 2.6 NP_001180217.1 protein level yes no solute carrier family 24 (sodium/potassium/calcium exchanger), member 2
Adult spinal cord CNTLN 3.3 NP_001107867.1 protein level yes yes centlein, centrosomal protein
FANCC 2.4 NP_000127.2 protein level yes yes Fanconi anemia, complementation group C
ZBTB34 2.4 NP_001092740.1 protein level yes yes zinc finger and BTB domain containing 34
Adult retina PDCL 10.1 NP_005379.3 protein level yes yes phosducin-like
FIBCD1 7.4 NP_116232.3 protein level yes no fibrinogen C domain containing 1
KIAA1958 5.9 NP_597722.1 protein level yes yes KIAA1958
ADAMTS13 5.5 NP_620595.1 protein level yes yes ADAM metallopeptidase with thrombospondin type 1 motif, 13
OLFM1 3.4 NP_055094.1 protein level yes no olfactomedin 1
ZMYND19 3.4 NP_612471.1 protein level yes yes zinc finger, MYND-type containing 19
LHX3 2.5 NP_055379.1 protein level no no LIM homeobox 3
LHX2 2.5 NP_004780.3 transcript level yes no LIM homeobox 2
MPDZ 2.5 NP_003820.2 protein level yes yes multiple PDZ domain protein
RASEF 2.5 NP_689786.2 protein level yes yes RAS and EF-hand domain containing
TMEM215 2.5 NP_997723.2 transcript level yes yes transmembrane protein 215
TRPM3 2.5 NP_079247.5 transcript level no no transient receptor potential cation channel, subfamily M, member 3
Adult heart INPP5E 4.7 NP_063945.2 protein level yes no inositol polyphosphate-5-phosphatase, 72 kDa
WDR31 4.7 NP_001012361.1 transcript level no yes WD repeat domain 31
TRUB2 3.8 NP_056494.1 protein level yes yes TruB pseudouridine (psi) synthase homolog 2 (E. coli)
CREB3 3.3 NP_006359.3 protein level yes no cAMP responsive element binding protein 3
MUSK 3.3 NP_005583.1 protein level no no muscle, skeletal, receptor tyrosine kinase
Adult liver ENTPD8 2.8 NP_001028285.1 protein level yes yes ectonucleoside triphosphate diphosphohydrolase 8
RGP1 2.8 NP_001073965.2 protein level yes yes RGP1 retrograde golgi transport homolog (S. cerevisiae)
CNTRL 2.8 NP_008949.4 protein level yes yes centriolin
Adult ovary BNC2 2.6 NP_060107.3 protein level yes yes basonuclin 2
CCL21 2.6 NP_002980.1 protein level yes yes chemokine (C-C motif) ligand 21
KLF9 2.6 NP_001197.1 protein level yes yes Kruppel-like factor 9
NR5A1 2.6 NP_004950.2 protein level no no nuclear receptor subfamily 5, group A, member 1
PBX3 2.6 NP_001128250.1 protein level yes no pre-B-cell leukemia homeobox 3
ZNF658 2.6 NP_149350.3 transcript level yes yes zinc finger protein 658
Adult testis DCAF12 10.8 NP_056212.1 protein level yes yes DDB1 and CUL4 associated factor 12
C9orf91 5.4 NP_694590.2 transcript level yes yes chromosome 9 open reading frame 91
AK8 2.7 NP_689785.1 protein level no yes adenylate kinase 8
C9orf139 2.7 NP_997394.1 transcript level no yes chromosome 9 open reading frame 139
ZBTB5 2.7 NP_055687.1 protein level yes yes zinc finger and BTB domain containing 5
Adult lung - - - - - - -
Adult adrenal gland ABCA1 7.2 NP_005493.2 protein level yes no ATP-binding cassette, sub-family A (ABC1), member 1
WDR38 7.2 NP_001038941.1 transcript level no yes WD repeat domain 38
CDKN2A 2.8 NP_001182061.1 protein level yes yes cyclin-dependent kinase inhibitor 2A
Adult gallbladder ASS1 8.7 NP_446464.1 protein level yes yes argininosuccinate synthase 1
ACO1 2.9 NP_002188.1 protein level yes yes aconitase 1, soluble
ARID3C 2.9 NP_001017363.1 transcript level yes no AT rich interactive domain 3C (BRIGHT-like)
LCNL1 2.9 NP_997393.3 transcript level no no lipocalin-like 1
Adult pancreas MSMP 6.1 NP_001037729.1 protein level yes yes microseminoprotein, prostate associated
ADAMTSL2 2.9 NP_055509.2 protein level yes yes ADAMTS-like 2
C9orf50 2.9 NP_955382.3 transcript level no yes chromosome 9 open reading frame 50
LURAP1L 2.9 NP_981948.1 transcript level yes yes leucine rich adaptor protein 1-like
Adult kidney BARHL1 4.4 NP_064448.1 transcript level yes yes BarH-like homeobox 1
C9orf135 4.2 NP_001010940.1 protein level yes yes chromosome 9 open reading frame 135
Adult esophagus C9orf169 17.8 NP_945352.2 protein level yes yes chromosome 9 open reading frame 169
IFNA5 5.4 NP_002160.1 protein level no no interferon, alpha 5
Adult colon GALNT12 3.1 NP_078918.3 protein level yes yes N-acetylgalactosaminyltransferase 12
ABO 2.6 NP_065202.2 protein level yes no alpha 1–3-N-acetylgalactosaminyltransferase
FAM157B 2.6 NP_001138721.1 homology no no family with sequence similarity 157, member B
Adult rectum - - - - - - -
Adult urinary bladder GOLGA2 6.9 NP_004477.3 protein level yes yes golgin A2
OR1K1 2.5 NP_543135.1 protein level yes no olfactory receptor, family 1, subfamily K, member 1
Adult prostate - - - - - - -
Placenta OR1L1 5.2 NP_001005236.3 transcript level no no olfactory receptor, family 1, subfamily L, member 1
*Common Peptide F 3.4 - - - - -
B cells CD72 42.9 NP_001773.1 protein level yes yes CD72 molecule
ZNF79 9.5 NP_009066.2 protein level yes yes zinc finger protein 79
NAIF1 7.8 NP_931045.1 protein level yes no nuclear apoptosis inducing factor 1
CDK20 2.6 NP_848519.1 protein level yes yes cyclin-dependent kinase 20
ZBTB6 2.4 NP_006617.1 protein level yes yes zinc finger and BTB domain containing 6
CD4+ T cells MGC50722 12.2 NP_976223.1 - - - uncharacterized MGC50722
SPIN1 3.3 NP_006708.2 protein level yes yes spindlin 1
CD8+ T cells *Common Peptide G 5.3 - - - - -
IFNA16 2.6 NP_002164.1 transcript level no no interferon, alpha 16
NK cells LOC100287368 2.4 XP_002342958.2 - - - protein FAM27D1-like
Monocytes *Common Peptide H 14.7 - - - - -
KLF4 6 NP_004226.3 protein level yes yes Kruppel-like factor 4 (gut)
CBWD6 5.6 NP_001078926.1 protein level yes no COBW domain containing 6
Platelets CDC14B 2.2 NP_001070649.1 protein level yes yes cell division cycle 14B
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Eight peptides are shared between more than two Chr 9 proteins. Protein existence, Proteomics, and Antibody column information were used by the C-HPP wiki website neXtProt database. (*Common Peptide A~H): *Common Peptide A - ZNF658;ZNF883 (NP_149350.3;NP_001094808.1); *Common Peptide B - LOC100132859;LOC100505781 (XP_003960500.1;XP_003119217.1); *Common Peptide C - FOXD4;FOXD4L5;FOXD4L4;FOXD4L2;FOXD4L6;FOXD4L3 (NP_997188.2;NP_001119806.1;NP_954714.2;NP_001092749.1;NP_001078945.1;NP_954586.4); *Common Peptide D - GNAQ;GNA14 (NP_002063.2;NP_004288.1); *Common Peptide E - CNTNAP3B;CNTNAP3 (NP_001188309.1;NP_387504.2); *Common Peptide F - OR1L4;OR1L6 (NP_001005235.1;NP_001004453.2); *Common Peptide G - ZNF782;ZNF79 (NP_001001662.1;NP_009066.2); *Common Peptide H - FCN2;FCN1 (NP_004099.2;NP_001994.2).

Cancer-specific Chr 9 encoded proteins in lung cancer tissues

We also performed proteomic analysis and comparative data analysis using lung cancer tissues along with the adjacent normal lung tissue (Supplementary Table 4). The Chr 9-encoded proteins matched data analysis revealed that 38 and 47 proteins were allocated to Chr 9-encoded proteins in normal lung and lung cancer tissues, respectively. The portion of Chr 9 proteins was 5.3% (38 out of 723) in normal lung tissue and 4.8% (47 out of 987) in lung cancer tissue.

Compared with normal lung tissue, 15 lung cancer-selective Chr 9 proteins were identified (Table 4). Based on a literature search, we present herein the significance of those proteins with respect to lung cancer and their speculated functions in lung cancer. The UV excision repair protein RAD23 homolog B (RAD23B) was identified as a lung cancer-selective protein and is known to be involved in nucleotide excision repair. It is also capable of binding polyubiquitinated substrates and delivers ubiquitinated proteins to the proteasome21. Nucleotide excision repair proteins play a key role in reversing DNA damage from exposure to environmental carcinogens. RAD23B variants have been reported to be associated with primary lung cancer risk in different ethnic groups22. 40S ribosomal protein S6 (RPS6) is the major substrate of protein kinase in eukaryote ribosomes23. RPS6 plays a role in AKT signaling in drug resistance. Strong activation was observed in the drug-resistant clones for several key AKT substrates, including RPS6 in a non-small cell lung cancer cell line24. SET is a proto-oncogene involved in apoptosis, transcription, nucleosome assembly and histone chaperoning. The protein encoded by this gene inhibits the acetylation of nucleosomes, especially histone H4, by histone acetylases25. The nuclear exit of SET correlates with cell spreading, and SET cooperates with Rac1 to stimulate cell migration26. The acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) works as a cell cycle progression factor and a cell survival factor, with functions that are largely unknown27. ANP32B is known to contribute to the retinoic acid-induced differentiation of leukemic cells28. Far upstream element-binding protein 3 (FUBP3) is a single strand DNA binding protein that recognizes the far upstream element (FUSE; originally identified upstream of the c-myc promoter)29. FUBP 3, as an activator of c-myc, has an important function in the high proliferation rate of renal cell carcinoma30. Perilipin 2 (PLIN2) is involved in the formation of lipid droplets for the development and maintenance of adipose tissues31. PLIN2 protein levels were increased in the plasma samples from colorectal cancer patients32. Actin-related protein 2/3 (Arp2/3) complex subunit 5-link protein (ARPC5L) is a component of the Arp2/3 complex, which is involved in the regulation of actin polymerization and the branched actin network. Actin is rapidly formed in response to specific cellular signals that converge on the Arp2/3 complex to regulate its assembly33. The increased expression of ARPC5 is known to reduce the levels of tumor suppressor miR-133a in lung squamous cell carcinoma34. Histidine triad nucleotide-binding protein 2 (HINT2) is a nuclear-encoded mitochondrial hydrolase. HINT2 overexpression sensitizes hepatocellular carcinoma to apoptosis35. HINT2 has been demonstrated to have low expression in endometrial cancer cells compared with control cells36. Sialic acid synthase (NANS) produces N-acetylneuraminic acid. Glycan structures of glycoprotein change during carcinogenesis and affect various biological behaviors of tumor cells. Typical glycan changes have also been reported to occur at the level of fucose and sialic acid in cancer cells37. Constitutive coactivator of PPAR-gamma-like protein 1 (FAM120A) was found in an mRNA-protein complex and might participate in mRNA transport throughout the cytoplasm38. FAM120A is a critical component of the oxidative stress-induced survival signaling pathway and is highly expressed in gastric scirrhous carcinoma compared with normal gastric mucosa39. Dipeptidyl peptidase 2 (DPP2/DPP7) is a serine protease that prevents spontaneous cell cycle progression in quiescent cells40. DPP2 inhibition tends to induce apoptosis of 60% of chronic lymphocytic leukemia cells41, suggesting its oncogenic activity. The list of lung cancer–selective Chr 9 proteins shows that most of these proteins are not well known as being associated with lung cancer; however, their functions are considered to be related to lung cancer. The selective biomarkers described here need further verification.

Table 4.

The list of lung cancer tissue-selective proteins on chromosome 9 compared with adjacent normal lung tissues.

NCBI accession ID Gene symbol Description
NP_055037.1 NDUFA8 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8
NP_001231653.1 RAD23B UV excision repair protein RAD23 homolog B
NP_001001.2 RPS6 40S ribosomal protein S6
NP_001116293.1 SET Protein SET
NP_001139580.1 PTGR1 Prostaglandin reductase 1
NP_056073.1 FKBP15 FK506-binding protein 15
NP_006392.1 ANP32B Acidic leucine-rich nuclear phosphoprotein 32 family member B
NP_003925.1 FUBP3 Far upstream element-binding protein 3
NP_001113.2 PLIN2 Perilipin-2
NP_112240.1 ARPC5L Actin-related protein 2/3 complex subunit 5-like protein
NP_115982.1 HINT2 Histidine triad nucleotide-binding protein 2, mitochondrial
NP_061819.2 NANS Sialic acid synthase
NP_064530.1 SH3GLB2 Endophilin-B2
NP_055427.2 FAM120A Constitutive coactivator of PPAR-gamma-like protein 1
NP_037511.2 DPP7 Dipeptidyl peptidase 2
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Identification of SNP- and mutation-containing peptides derived from Chr 9-encoded proteins

We performed a proteogenomic analysis by searching against two customized peptides sequence databases: the SNP-specific and mutation-specific databases. To decrease the false discovery rate, we first generated peaklist files that contained only tandem mass spectra that did not match any peptides from the initial protein database searches. Unmatched spectra from two available lung cancer cell line datasets (A549 and NCI-H460) and our adult lung tissue dataset were used to search against the two databases through the Proteome Discover Platform (version 1.4) using SEQUEST. We identified 249 SNPs on Chr 9 genes from normal human cells/tissues, with 21 SNP-containing peptides being mapped to 19 genes that were identified from normal adult lung tissue. For example, we observed a peptide (GIQLVEEELDR) that differed by one amino acid from the sequence of the tropomyosin beta chain (TPM2) protein in the databases (glycine instead of arginine at position 91), likely due to a known SNP in the gene (rs104894127) (Figure 2a). In addition, we observed four peptides from the two lung cancer cell lines that contained amino acid substitutions, which could be explained on the basis of mutations in the COSMIC database. For example, a peptide derived from the gene ALDH1B1 (aldehyde dehydrogenase X, mitochondrial) (LLNRLADLVER) was identified from a mutant-specific database search that contained a single amino acid substitution (L107R) from the database sequence (Figure 2b). These results show that peptides with genetic alterations can be identified by such a proteogenomic analysis.

Figure 2.

Figure 2

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Two representative mass spectrometry peak profiles that show the identification of two mutations in peptide sequences. (a) A peptide (GIQLVEEELDR) in tropomyosin beta chain (TPM2) differed by one amino acid identified in normal adult lung tissue from the known sequence of the TPM2 protein in databases. Arginine at position 91 was changed to glycine, likely due to a known SNP in the gene (rs104894127). (b) Based on mutations in the COSMIC database, a peptide (LLNRLADLVER) derived from the gene aldehyde dehydrogenase X, mitochondrial (ALDH1B1) was identified by a mutant-specific database search. A single amino acid substitution, arginine at position 107 to leucine, was identified from the database sequence.

CONCLUSIONS

In this study, we have participated in the first year of C-HPP project implementation to annotate proteins encoded by genes located on Chr 9 using proteomics approaches. We have analyzed data pertaining to Chr 9-encoded proteins from a panel of normal human samples and lung cancer cell lines/tissues. We have obtained data regarding the distribution of proteins in normal human tissues that are encoded by genes located on Chr 9; this information could be further mined for the study of tissue-selective biomarkers. Proteomics and data comparison analyses for the human lung cancer tissues and adjacent normal lung tissues provided 15 lung cancer-specific Chr 9-encoded proteins, most of which are functionally associated with cancer. We have also described 46 Chr 9-encoded proteins that have not been detected previously and account for ~28% of the missing proteins located on Chr 9. These results demonstrate how a global proteomic analysis of a large number of samples using high-resolution MS instruments can increase the possibility of detecting missing proteins on each chromosome. In addition, we performed preliminary analyses to find peptide-based evidence of SNPs and mutations in the Chr 9-encoded proteins from the normal lung and lung cancer cell line/tissue datasets.

In the future, we will seek more Chr 9 protein information with the MS data, such as splicing variant proteins, expressed pseudogenes and post-translationally modified proteins. To detect additional missing proteins encoded by Chr 9, a deeper analysis of the proteins from multiple tissues after subcellular fractionation or the enrichment of certain classes of proteins should be performed. These data will also assist in the analysis of genomics data generated by next generation sequencing. Another strategy is to analyze the quantitative levels of missing proteins and lung cancer-associated Chr 9-encoded proteins in various samples using targeted MRM proteomics approaches.

Supplementary Material

1_si_002. Supplementary Table 1.

Total chromosome 9-encoded protein list from normal human samples

2_si_004. Supplementary Table 2.

The list of newly detected missing proteins on chromosome 9.

3_si_003. Supplementary Table 3.

The list of detected top 10 abundance chromosome 9-encoded proteins among the normal human sample data.

4_si_001. Supplementary Table 4.

The lists in the identification of chromosome 9 proteins from normal lung and lung cancer tissues.

Acknowledgments

We thank Gyoung-Beom Heo for his assistance in the data arrangement of the Excel files. We acknowledge Srikanth S. Manda for assistance with the proteogenomic analysis. This research was supported by the 21C Frontier Follow-On Support Research and Development Program (No. 2012M3C5A1053342), Proteogenomics Research Program grants through the National Research Foundation of Korea (NRF) grant and Converging Research Center Program (2012K001536) funded by Ministry of Science, ICT & Future Planning (MSIP). This work was supported by NCI’s Clinical Proteomic Tumor Analysis Consortium initiative (U24CA160036).

Footnotes

The authors declare no competing financial interest.

ASSOCIATED CONTENT

Supplementary Tables 1–4, as described in the main text, are provided as MS Excel files (*.xlsx). Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org. The table descriptions are as follows.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1_si_002. Supplementary Table 1.

Total chromosome 9-encoded protein list from normal human samples

NIHMS547529-supplement-1_si_002.xls (27.1KB, xls)
2_si_004. Supplementary Table 2.

The list of newly detected missing proteins on chromosome 9.

NIHMS547529-supplement-2_si_004.xls (723.1KB, xls)
3_si_003. Supplementary Table 3.

The list of detected top 10 abundance chromosome 9-encoded proteins among the normal human sample data.

NIHMS547529-supplement-3_si_003.xls (230.4KB, xls)
4_si_001. Supplementary Table 4.

The lists in the identification of chromosome 9 proteins from normal lung and lung cancer tissues.

NIHMS547529-supplement-4_si_001.xls (1.8MB, xls)

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