Combination of Multiple Spectral Libraries Improves the Current Search Methods Used to Identify Missing Proteins in the Chromosome-Centric Human Proteome Project

Cho, Jin-Young,Lee, Hyoung-Joo,Jeong, Seul-Ki,Kim, Kwang-Youl,Kwon, Kyung-Hoon,Yoo, Jong Shin,Omenn, Gilbert S.,Baker, Mark S.,Hancock, William S.,Paik, Young-Ki American Chemical Society 2015 JOURNAL OF PROTEOME RESEARCH Vol.14 No.12

<P>Approximately 2.9 billion long base-pair human reference genome sequences are known to encode some 20 000 representative proteins. However, 3000 proteins, that is, ∼15% of all proteins, have no or very weak proteomic evidence and are still missing. Missing proteins may be present in rare samples in very low abundance or be only temporarily expressed, causing problems in their detection and protein profiling. In particular, some technical limitations cause missing proteins to remain unassigned. For example, current mass spectrometry techniques have high limits and error rates for the detection of complex biological samples. An insufficient proteome coverage in a reference sequence database and spectral library also raises major issues. Thus, the development of a better strategy that results in greater sensitivity and accuracy in the search for missing proteins is necessary. To this end, we used a new strategy, which combines a reference spectral library search and a simulated spectral library search, to identify missing proteins. We built the human iRefSPL, which contains the original human reference spectral library and additional peptide sequence-spectrum match entries from other species. We also constructed the human simSPL, which contains the simulated spectra of 173 907 human tryptic peptides determined by MassAnalyzer (version 2.3.1). To prove the enhanced analytical performance of the combination of the human iRefSPL and simSPL methods for the identification of missing proteins, we attempted to reanalyze the placental tissue data set (PXD000754). The data from each experiment were analyzed using PeptideProphet, and the results were combined using iProphet. For the quality control, we applied the class-specific false-discovery rate filtering method. All of the results were filtered at a false-discovery rate of <1% at the peptide and protein levels. The quality-controlled results were then cross-checked with the neXtProt DB (2014-09-19 release). The two spectral libraries, iRefSPL and simSPL, were designed to ensure no overlap of the proteome coverage. They were shown to be complementary to spectral library searching and significantly increased the number of matches. From this trial, 12 new missing proteins were identified that passed the following criterion: at least 2 peptides of 7 or more amino acids in length or one of 9 or more amino acids in length with one or more unique sequences. Thus, the iRefSPL and simSPL combination can be used to help identify peptides that have not been detected by conventional sequence database searches with improved sensitivity and a low error rate.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2015/jprobs.2015.14.issue-12/acs.jproteome.5b00578/production/images/medium/pr-2015-00578f_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr5b00578'>ACS Electronic Supporting Info</A></P>

GenomewidePDB 2.0: A Newly Upgraded Versatile Proteogenomic Database for the Chromosome-Centric Human Proteome Project

Jeong, Seul-Ki,Hancock, William S.,Paik, Young-Ki American Chemical Society 2015 Journal of proteome research Vol.14 No.9

<P>Since the launch of the Chromosome-centric Human Proteome Project (C-HPP) in 2012, the number of “missing” proteins has fallen to 2932, down from ∼5932 since the number was first counted in 2011. We compared the characteristics of missing proteins with those of already annotated proteins with respect to transcriptional expression pattern and the time periods in which newly identified proteins were annotated. We learned that missing proteins commonly exhibit lower levels of transcriptional expression and less tissue-specific expression compared with already annotated proteins. This makes it more difficult to identify missing proteins as time goes on. One of the C-HPP goals is to identify alternative spliced product of proteins (ASPs), which are usually difficult to find by shot-gun proteomic methods due to their sequence similarities with the representative proteins. To resolve this problem, it may be necessary to use a targeted proteomics approach (e.g., selected and multiple reaction monitoring [S/MRM] assays) and an innovative bioinformatics platform that enables the selection of target peptides for rarely expressed missing proteins or ASPs. Given that the success of efforts to identify missing proteins may rely on more informative public databases, it was necessary to upgrade the available integrative databases. To this end, we attempted to improve the features and utility of GenomewidePDB by integrating transcriptomic information (e.g., alternatively spliced transcripts), annotated peptide information, and an advanced search interface that can find proteins of interest when applying a targeted proteomics strategy. This upgraded version of the database, GenomewidePDB 2.0, may not only expedite identification of the remaining missing proteins but also enhance the exchange of information among the proteome community. GenomewidePDB 2.0 is available publicly at <uri xlink:href='http://genomewidepdb.proteomix.org/' xlink:type='simple'>http://genomewidepdb.proteomix.org/</uri>.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2015/jprobs.2015.14.issue-9/acs.jproteome.5b00541/production/images/medium/pr-2015-00541m_0009.gif'></P>

Progress on Identifying and Characterizing the Human Proteome: 2018 Metrics from the HUPO Human Proteome Project

Omenn, Gilbert S.,Lane, Lydie,Overall, Christopher M.,Corrales, Fernando J.,Schwenk, Jochen M.,Paik, Young-Ki,Van Eyk, Jennifer E.,Liu, Siqi,Snyder, Michael,Baker, Mark S.,Deutsch, Eric W. American Chemical Society 2018 Journal of proteome research Vol.17 No.12

<P>The Human Proteome Project (HPP) annually reports on progress throughout the field in credibly identifying and characterizing the human protein parts list and making proteomics an integral part of multiomics studies in medicine and the life sciences. NeXtProt release 2018-01-17, the baseline for this sixth annual HPP special issue of the <I>Journal of Proteome Research</I>, contains 17 470 PE1 proteins, 89% of all neXtProt predicted PE1-4 proteins, up from 17 008 in release 2017-01-23 and 13 975 in release 2012-02-24. Conversely, the number of neXtProt PE2,3,4 missing proteins has been reduced from 2949 to 2579 to 2186 over the past two years. Of the PE1 proteins, 16 092 are based on mass spectrometry results, and 1378 on other kinds of protein studies, notably protein-protein interaction findings. PeptideAtlas has 15 798 canonical proteins, up 625 over the past year, including 269 from SUMOylation studies. The largest reason for missing proteins is low abundance. Meanwhile, the Human Protein Atlas has released its Cell Atlas, Pathology Atlas, and updated Tissue Atlas, and is applying recommendations from the International Working Group on Antibody Validation. Finally, there is progress using the quantitative multiplex organ-specific popular proteins targeted proteomics approach in various disease categories.</P> [FIG OMISSION]</BR>

Launching the C-HPP neXt-CP50 Pilot Project for Functional Characterization of Identified Proteins with No Known Function

Paik, Young-Ki,Lane, Lydie,Kawamura, Takeshi,Chen, Yu-Ju,Cho, Je-Yoel,LaBaer, Joshua,Yoo, Jong Shin,Domont, Gilberto,Corrales, Fernando,Omenn, Gilbert S.,Archakov, Alexander,Encarnació,n-Guevara American Chemical Society 2018 JOURNAL OF PROTEOME RESEARCH Vol.17 No.12

<P>An important goal of the Human Proteome Organization (HUPO) Chromosome-centric Human Proteome Project (C-HPP) is to correctly define the number of canonical proteins encoded by their cognate open reading frames on each chromosome in the human genome. When identified with high confidence of protein evidence (PE), such proteins are termed PE1 proteins in the online database resource, neXtProt. However, proteins that have not been identified unequivocally at the protein level but that have other evidence suggestive of their existence (PE2-4) are termed missing proteins (MPs). The number of MPs has been reduced from 5511 in 2012 to 2186 in 2018 (neXtProt 2018-01-17 release). Although the annotation of the human proteome has made significant progress, the “parts list” alone does not inform function. Indeed, 1937 proteins representing ∼10% of the human proteome have no function either annotated from experimental characterization or predicted by homology to other proteins. Specifically, these 1937 “dark proteins” of the so-called dark proteome are composed of 1260 functionally uncharacterized but identified PE1 proteins, designated as uPE1, plus 677 MPs from categories PE2-PE4, which also have no known or predicted function and are termed uMPs. At the HUPO-2017 Annual Meeting, the C-HPP officially adopted the uPE1 pilot initiative, with 14 participating international teams later committing to demonstrate the feasibility of the functional <U>c</U>haracterization of large numbers of dark <U>p</U>roteins (CP), starting first with 50 uPE1 proteins, in a stepwise chromosome-centric organizational manner. The second aim of the feasibility phase to <U>c</U>haracterize protein (CP) functions of 50 uPE1 proteins, termed the neXt-CP50 initiative, is to utilize a variety of approaches and workflows according to individual team expertise, interest, and resources so as to enable the C-HPP to recommend experimentally proven workflows to the proteome community within 3 years. The results from this pilot will not only be the cornerstone of a larger characterization initiative but also enhance understanding of the human proteome and integrated cellular networks for the discovery of new mechanisms of pathology, mechanistically informative biomarkers, and rational drug targets.</P> [FIG OMISSION]</BR>

Systematic Proteogenomic Approach To Exploring a Novel Function for NHERF1 in Human Reproductive Disorder: Lessons for Exploring Missing Proteins

Na, Keun,Shin, Heon,Cho, Jin-Young,Jung, Sang Hee,Lim, Jaeseung,Lim, Jong-Sun,Kim, Eun Ah,Kim, Hye Sun,Kang, Ah Reum,Kim, Ji Hye,Shin, Jeong Min,Jeong, Seul-Ki,Kim, Chae-Yeon,Park, Jun Young,Chung, Hy American Chemical Society 2017 JOURNAL OF PROTEOME RESEARCH Vol.16 No.12

<P>One of the major goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to fill the knowledge gaps between human genomic information and the corresponding proteomic information. These gaps are due to “missing” proteins (MPs)predicted proteins with insufficient evidence from mass spectrometry (MS), biochemical, structural, or antibody analysesthat currently account for 2579 of the 19587 predicted human proteins (neXtProt, 2017-01). We address some of the lessons learned from the inconsistent annotations of missing proteins in databases (DB) and demonstrate a systematic proteogenomic approach designed to explore a potential new function of a known protein. To illustrate a cautious and strategic approach for characterization of novel function in vitro and in vivo, we present the case of Na(+)/H(+) exchange regulatory cofactor 1 (NHERF1/SLC9A3R1, located at chromosome 17q25.1; hereafter NHERF1), which was mistakenly labeled as an MP in one DB (Global Proteome Machine Database; GPMDB, 2011-09 release) but was well known in another public DB and in the literature. As a first step, NHERF1 was determined by MS and immunoblotting for its molecular identity. We next investigated the potential new function of NHERF1 by carrying out the quantitative MS profiling of placental trophoblasts (PXD004723) and functional study of cytotrophoblast JEG-3 cells. We found that NHERF1 was associated with trophoblast differentiation and motility. To validate this newly found cellular function of NHERF1, we used the <I>Caenorhabditis elegans</I> mutant of <I>nrfl-1</I> (a nematode ortholog of <I>NHERF1</I>), which exhibits a protruding vulva (Pvl) and egg-laying-defective phenotype, and performed genetic complementation work. The <I>nrfl-1</I> mutant was almost fully rescued by the transfection of the recombinant transgenic construct that contained human <I>NHERF1</I>. These results suggest that NHERF1 could have a previously unknown function in pregnancy and in the development of human embryos. Our study outlines a stepwise experimental platform to explore new functions of ambiguously denoted candidate proteins and scrutinizes the mandated DB search for the selection of MPs to study in the future.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2017/jprobs.2017.16.issue-12/acs.jproteome.7b00146/production/images/medium/pr-2017-00146s_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr7b00146'>ACS Electronic Supporting Info</A></P>

Chromosome-Centric Human Proteome Study of Chromosome 11 Team

황희연,김진영,JONG SHIN YOO 사단법인 한국질량분석학회 2021 Mass spectrometry letters Vol.12 No.3

As a part of the Chromosome-centric Human Proteome Project (C-HPP), we have developed a few algorithms for accurate identification of missing proteins, alternative splicing variants, single amino acid variants, and characterization of func- tion unannotated proteins. We have found missing proteins, novel and known ASVs, and SAAVs using LC-MS/MS data from human brain and olfactory epithelial tissue, where we validated their existence using synthetic peptides. According to the neXtProt database, the number of missing proteins in chromosome 11 shows a decreasing pattern. The development of genomic and transcriptomic sequencing techniques make the number of protein variants in chromosome 11 tremendously increase. We developed a web solution named as SAAvpedia for identification and function annotation of SAAVs, and the SAAV information is automatically transformed into the neXtProt web page using REST API service. For the 73 uPE1 in chromosome 11, we have studied the function annotaion of CCDC90B (NX_Q9GZT6), SMAP (NX_O00193), and C11orf52 (NX_Q96A22).

Quest for Missing Proteins: Update 2015 on Chromosome-Centric Human Proteome Project

Horvatovich, Pé,ter,Lundberg, Emma K.,Chen, Yu-Ju,Sung, Ting-Yi,He, Fuchu,Nice, Edouard C.,Goode, Robert J.,Yu, Simon,Ranganathan, Shoba,Baker, Mark S.,Domont, Gilberto B.,Velasquez, Erika,Li, D American Chemical Society 2015 Journal of Proteome Research Vol.14 No.9

<P>This paper summarizes the recent activities of the Chromosome-Centric Human Proteome Project (C-HPP) consortium, which develops new technologies to identify yet-to-be annotated proteins (termed “missing proteins”) in biological samples that lack sufficient experimental evidence at the protein level for confident protein identification. The C-HPP also aims to identify new protein forms that may be caused by genetic variability, post-translational modifications, and alternative splicing. Proteogenomic data integration forms the basis of the C-HPP’s activities; therefore, we have summarized some of the key approaches and their roles in the project. We present new analytical technologies that improve the chemical space and lower detection limits coupled to bioinformatics tools and some publicly available resources that can be used to improve data analysis or support the development of analytical assays. Most of this paper’s content has been compiled from posters, slides, and discussions presented in the series of C-HPP workshops held during 2014. All data (posters, presentations) used are available at the C-HPP Wiki (<uri xlink:href='http://c-hpp.webhosting.rug.nl/' xlink:type='simple'>http://c-hpp.webhosting.rug.nl/</uri>) and in the Supporting Information.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2015/jprobs.2015.14.issue-9/pr5013009/production/images/medium/pr-2014-013009_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr5013009'>ACS Electronic Supporting Info</A></P>

Integrated Proteomic Pipeline Using Multiple Search Engines for a Proteogenomic Study with a Controlled Protein False Discovery Rate

Park, Gun Wook,Hwang, Heeyoun,Kim, Kwang Hoe,Lee, Ju Yeon,Lee, Hyun Kyoung,Park, Ji Yeong,Ji, Eun Sun,Park, Sung-Kyu Robin,Yates, John R.,Kwon, Kyung-Hoon,Park, Young Mok,Lee, Hyoung-Joo,Paik, Young-K AMERICAN CHEMICAL SOCIETY 2016 Journal of Proteome Research Vol.15 No.11

<P>In the Chromosome-Centric Human Proteome Project (C-HPP), false positive identification by peptide spectrum matches (PSMs) after database searches is a major issue for proteogenomic studies using liquid-chromatography and mass-spectrometry-based large proteomic profiling. Here we developed a simple strategy for protein identification, with a controlled false discovery rate (FDR) at the protein level, using an integrated proteomic pipeline (IPP) that consists of four engrailed steps as follows. First, using three different search engines, SEQUEST, MASCOT, and MS-GF +, individual proteomic searches were performed against the neXtProt database. Second, the search results from the PSMs were combined using statistical evaluation tools including DTASelect and Percolator. Third, the peptide search scores were converted into E-scores normalized using an in-house program. Last, Proteinlnferencer was used to filter the proteins containing two or more peptides with a controlled FDR of 1.0% at the protein level. Finally, we compared the performance of the IPP to a conventional proteomic pipeline (CPP) for protein identification using a controlled FDR of <1% at the protein level. Using the IPP, a total of 5756 proteins (vs 4453 using the CPP) including 477 alternative splicing variants (vs 182 using the CPP) were identified from human hippocampal tissue. In addition, a total of 10 missing proteins (vs 7 using the CPP) were identified with two or more unique peptides, and their tryptic peptides were validated using MS/MS spectral pattern from a repository database or their corresponding synthetic peptides. This study shows that the IPP effectively improved the identification of proteins, including alternative splicing variants and missing proteins, in human hippocampal tissues for the C-HPP. All RAW files used in this study were deposited in ProteomeXchange (PXD000395).</P>

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

Ahn, Jung-Mo,Kim, Min-Sik,Kim, Yong-In,Jeong, Seul-Ki,Lee, Hyoung-Joo,Lee, Sun Hee,Paik, Young-Ki,Pandey, Akhilesh,Cho, Je-Yoel American Chemical Society 2014 JOURNAL OF PROTEOME RESEARCH Vol.13 No.1

<P>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. On the basis of 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 four 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 PXD000603.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2014/jprobs.2014.13.issue-1/pr400792p/production/images/medium/pr-2013-00792p_0003.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr400792p'>ACS Electronic Supporting Info</A></P>

Proteogenomic Study beyond Chromosome 9: New Insight into Expressed Variant Proteome and Transcriptome in Human Lung Adenocarcinoma Tissues

Kim, Yong-In,Lee, Jongan,Choi, Young-Jin,Seo, Jawon,Park, Jisook,Lee, Soo-Youn,Cho, Je-Yoel American Chemical Society 2015 Journal of proteome research Vol.14 No.12

<P>This is a report of a human proteome project (HPP) related to chromosome 9 (Chr 9). To reveal missing proteins and undiscovered features in proteogenomes, both LC–MS/MS analysis and next-generation RNA sequencing (RNA-seq)-based identification and characterization were conducted on five pairs of lung adenocarcinoma tumors and adjacent nontumor tissues. Before our previous Chromosome-Centric Human Proteome Project (C-HPP) special issue, there were 170 remaining missing proteins on Chr 9 (neXtProt 2013.09.26 rel.); 133 remain at present (neXtProt 2015.04.28 rel.). In the proteomics study, we found two missing protein candidates that require follow-up work and one unrevealed protein across all chromosomes. RNA-seq analysis detected RNA expression for four nonsynonymous (NS) single nucleotide polymorphisms (SNPs) (in CDH17, HIST1H1T, SAPCD2, and ZNF695) and three synonymous SNPs (in CDH17, CST1, and HNF1A) in all five tumor tissues but not in any of the adjacent normal tissues. By constructing a cancer patient sample-specific protein database based on individual RNA-seq data and by searching the proteomics data from the same sample, we identified four missense mutations in four genes (LTF, HDLBP, TF, and HBD). Two of these mutations were found in tumor samples but not in paired normal tissues. In summary, our proteogenomic study of human primary lung tumor tissues detected additional and revealed novel missense mutations and synonymous SNP signatures, some of which are specific to lung cancers. Data from mass spectrometry have been deposited in the ProteomeXchange with the identifier PXD002523.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2015/jprobs.2015.14.issue-12/acs.jproteome.5b00544/production/images/medium/pr-2015-00544w_0006.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr5b00544'>ACS Electronic Supporting Info</A></P>