Chengpu Zhang

Systematic Analyses of the Transcriptome, Translatome, and Proteome Provide a Global View and Potential Strategy for the C‑HPP

To estimate the potential of the state-of-the-art proteomics technologies on full coverage of the encoding gene products, the Chinese Human Chromosome Proteome Consortium (CCPC) applied a multiomics strategy to systematically analyze the transciptome, translatome, and proteome of the same cultured hepatoma cells with varied metastatic potential qualitatively and quantitatively. The results provide a global view of gene expression profiles. The 9064 identified high confident proteins covered 50.2% of all gene products in the translatome. Those proteins with function of adhesion, development, reproduction, and so on are low abundant in transcriptome and translatome but absent in proteome. Taking the translatome as the background of protein expression, we found that the protein abundance plays a decisive role and hydrophobicity has a greater influence than molecular weight and isoelectric point on protein detectability. Thus, the enrichment strategy used for low-abundant transcription factors helped to identify missing proteins. In addition, those peptides with single amino acid polymorphisms played a significant role for the disease research, although they might negligibly contribute to new protein identification. The proteome raw and metadata of proteome were collected using the iProX submission system and submitted to ProteomeXchange (PXD000529, PXD000533, and PXD000535). All detailed information in this study can be accessed from the Chinese Chromosome-Centric Human Proteome Database.

First proteomic exploration of protein-encoding genes on chromosome 1 in human liver, stomach, and colon.

The launch of the Chromosome-Centric Human Proteome Project provides an opportunity to gain insight into the human proteome. The Chinese Human Chromosome Proteome Consortium has initiated proteomic exploration of protein-encoding genes on human chromosomes 1, 8, and 20. Collaboration within the consortium has generated a comprehensive proteome data set using normal and carcinomatous tissues from human liver, stomach, and colon and 13 cell lines originating in these organs. We identified 12,101 proteins (59.8% coverage against Swiss-Prot human entries) with a protein false discovery rate of less than 1%. On chromosome 1, 1,252 proteins mapping to 1,227 genes, representing 60.9% of Swiss-Prot entries, were identified; however, 805 proteins remain unidentified, suggesting that analysis of more diverse samples using more advanced proteomic technologies is required. Genes encoding the unidentified proteins were concentrated in seven blocks, located at p36, q12-21, and q42-44, partly consistent with correlation of these blocks with cancers of the liver, stomach, and colon. Combined transcriptome, proteome, and cofunctionality analyses confirmed 23 coexpression clusters containing 165 genes. Biological information, including chromosome structure, GC content, and protein coexpression pattern was analyzed using multilayered, circular visualization and tabular visualization. Details of data analysis and updates are available in the Chinese Chromosome-Centric Human Proteome Database ( http://proteomeview.hupo.org.cn/chromosome/ ).

Tissue-Based Proteogenomics Reveals that Human Testis Endows Plentiful Missing Proteins.

Investigations of missing proteins (MPs) are being endorsed by many bioanalytical strategies. We proposed that proteogenomics of testis tissue was a feasible approach to identify more MPs because testis tissues have higher gene expression levels. Here we combined proteomics and transcriptomics to survey gene expression in human testis tissues from three post-mortem individuals. Proteins were extracted and separated with glycine- and tricine-SDS-PAGE. A total of 9597 protein groups were identified; of these, 166 protein groups were listed as MPs, including 138 groups (83.1%) with transcriptional evidence. A total of 2948 proteins are designated as MPs, and 5.6% of these were identified in this study. The high incidence of MPs in testis tissue indicates that this is a rich resource for MPs. Functional category analysis revealed that the biological processes that testis MPs are mainly involved in are sexual reproduction and spermatogenesis. Some of the MPs are potentially involved in tumorgenesis in other tissues. Therefore, this proteogenomics analysis of individual testis tissues provides convincing evidence of the discovery of MPs. All mass spectrometry data from this study have been deposited in the ProteomeXchange (data set identifier PXD002179).

Systematic analysis of missing proteins provides clues to help define all of the protein-coding genes on human chromosome 1

Our first proteomic exploration of human chromosome 1 began in 2012 (CCPD 1.0), and the genome-wide characterization of the human proteome through public resources revealed that 32-39% of proteins on chromosome 1 remain unidentified. To characterize all of the missing proteins, we applied an OMICS-integrated analysis of three human liver cell lines (Hep3B, MHCC97H, and HCCLM3) using mRNA and ribosome nascent-chain complex-bound mRNA deep sequencing and proteome profiling, contributing mass spectrometric evidence of 60 additional chromosome 1 gene products. Integration of the annotation information from public databases revealed that 84.6% of genes on chromosome 1 had high-confidence protein evidence. Hierarchical analysis demonstrated that the remaining 320 missing genes were either experimentally or biologically explainable; 128 genes were found to be tissue-specific or rarely expressed in some tissues, whereas 91 proteins were uncharacterized mainly due to database annotation diversity, 89 were genes with low mRNA abundance or unsuitable protein properties, and 12 genes were identifiable theoretically because of a high abundance of mRNAs/RNC-mRNAs and the existence of proteotypic peptides. The relatively large contribution made by the identification of enriched transcription factors suggested specific enrichment of low-abundance protein classes, and SRM/MRM could capture high-priority missing proteins. Detailed analyses of the differentially expressed genes indicated that several gene families located on chromosome 1 may play critical roles in mediating hepatocellular carcinoma invasion and metastasis. All mass spectrometry proteomics data corresponding to our study were deposited in the ProteomeXchange under the identifiers PXD000529, PXD000533, and PXD000535.

Discovery of novel genes and gene isoforms by integrating transcriptomic and proteomic profiling from mouse liver.

Comprehensively identifying gene expression in both transcriptomic and proteomic levels of one tissue is a prerequisite for a deeper understanding of its biological functions. Alternative splicing and RNA editing, two main forms of transcriptional processing, play important roles in transcriptome and proteome diversity and result in multiple isoforms for one gene, which are hard to identify by mass spectrometry (MS)-based proteomics approach due to the relative lack of isoform information in standard protein databases. In our study, we employed MS and RNA-Seq in parallel into mouse liver tissue and captured a considerable catalogue of both transcripts and proteins that, respectively, covered 60 and 34% of protein-coding genes in Ensembl. We then developed a bioinformatics workflow for building a customized protein database that for the first time included new splicing-derived peptides and RNA-editing-caused peptide variants, allowing us to more completely identify protein isoforms. Using this experimentally determined database, we totally identified 150 peptides not present in standard biological databases at false discovery rate of <1%, corresponding to 72 novel splicing isoforms, 43 new genetic regions, and 15 RNA-editing sites. Of these, 11 randomly selected novel events passed experimental verification by PCR and Sanger sequencing. New discoveries of gene products with high confidence in two omics levels demonstrated the robustness and effectiveness of our approach and its potential application into improve genome annotation. All the MS data have been deposited to the iProx ( http://ww.iprox.org ) with the identifier IPX00003601.

PepDistiller: A quality control tool to improve the sensitivity and accuracy of peptide identifications in shotgun proteomics

In this study, we presented a quality control tool named PepDistiller to facilitate the validation of MASCOT search results. By including the number of tryptic termini, and integrating a refined false discovery rate (FDR) calculation method, we demonstrated the improved sensitivity of peptide identifications obtained from semitryptic search results. Based on the analysis of a complex data set, approximately 7% more peptide identifications were obtained using PepDistiller than using MASCOT Percolator. Moreover, the refined method generated lower FDR estimations than the percentage of incorrect target (PIT) fixed method applied in Percolator. Using a standard data set, we further demonstrated the increased accuracy of the refined FDR estimations relative to the PIT‐fixed FDR estimations. PepDistiller is fast and convenient to use, and is freely available for academic access. The software can be downloaded from http://www.bprc.ac.cn/pepdistiller.

Omics evidence: single nucleotide variants transmissions on chromosome 20 in liver cancer cell lines.

Cancer genomics unveils many cancer-related mutations, including some chromosome 20 (Chr.20) genes. The mutated messages have been found in the corresponding mRNAs; however, whether they could be translated to proteins still requires more evidence. Herein, we proposed a transomics strategy to profile the expression status of human Chr.20 genes (555 in Ensembl v72). The data of transcriptome and translatome (the mRNAs bound with ribosome, translating mRNAs) revealed that ∼80% of the coding genes on Chr.20 were detected with mRNA signals in three liver cancer cell lines, whereas of the proteome identified, only ∼45% of the Chr.20 coding genes were detected. The high amount of overlapping of identified genes in mRNA and RNC-mRNA (ribosome nascent-chain complex-bound mRNAs, translating mRNAs) and the consistent distribution of the abundance averages of mRNA and RNC-mRNA along the Chr.20 subregions in three liver cancer cell lines indicate that the mRNA information is efficiently transmitted from transcriptional to translational stage, qualitatively and quantitatively. Of the 457 genes identified in mRNAs and RNC-mRNA, 136 were found to contain SNVs with 213 sites, and >40% of these SNVs existed only in metastatic cell lines, suggesting them as the metastasis-related SNVs. Proteomics analysis showed that 16 genes with 20 SNV sites were detected with reliable MS/MS signals, and some SNVs were further validated by the MRM approach. With the integration of the omics data at the three expression phases, therefore, we are able to achieve the overall view of the gene expression of Chr.20, which is constructive in understanding the potential trend of encoding genes in a cell line and exploration of a new type of markers related to cancers.

Qualitative and Quantitative Expression Status of the Human Chromosome 20 Genes in Cancer Tissues and the Representative Cell Lines

Under the guidance of the Chromosome-centric Human Proteome Project (C-HPP), (1, 2) we conducted a systematic survey of the expression status of genes located at human chromosome 20 (Chr.20) in three cancer tissues, gastric, colon, and liver carcinoma, and their representative cell lines. We have globally profiled proteomes in these samples with combined technology of LC-MS/MS and acquired the corresponding mRNA information upon RNA-seq and RNAchip. In total, 323 unique proteins were identified, covering 60% of the coding genes (323/547) in Chr.20. With regards to qualitative information of proteomics, we overall evaluated the correlation of the identified Chr.20 proteins with target genes of transcription factors or of microRNA, conserved genes and cancer-related genes. As for quantitative information, the expression abundances of Chr.20 genes were found to be almost consistent in both tissues and cell lines of mRNA in all individual chromosome regions, whereas those of Chr.20 proteins in cells are different from tissues, especially in the region of 20q13.33. Furthermore, the abundances of Chr.20 proteins were hierarchically evaluated according to tissue- or cancer-related distribution. The analysis revealed several cancer-related proteins in Chr.20 are tissue- or cell-type dependent. With integration of all the acquired data, for the first time we established a solid database of the Chr.20 proteome.

Proteome atlas of human chromosome 8 and its multiple 8p deficiencies in tumorigenesis of the stomach, colon, and liver

Chromosome 8, a medium-length euchromatic unit in humans that has an extraordinarily high mutation rate, can be detected not only in evolution but also in multiple mutant diseases, such as tumorigenesis, and further invasion/metastasis. The Chromosome-Centric Human Proteome Project of China systematically profiles the proteomes of three digestive organs (i.e., stomach, colon, and liver) and their corresponding carcinoma tissues/cell lines according to a chromosome organizational roadmap. By rigorous standards, we have identified 271 (38.7%), 330 (47.1%), and 325 (46.4%) of 701 chromosome 8-coded proteins from stomach, colon, and liver samples, respectively, in Swiss-Prot and observed a total coverage rate of up to 58.9% by 413 identified proteins. Using large-scale label-free proteome quantitation, we also found some 8p deficiencies, such as the presence of 8p21-p23 in tumorigenesis of the above-described digestive organs, which is in good agreement with previous reports. To our best knowledge, this is the first study to have verified these 8p deficiencies at the proteome level, complementing genome and transcriptome data.

Chromosome-8-Coded Proteome of Chinese Chromosome Proteome Data Set (CCPD) 2.0 with Partial Immunohistochemical Verifications

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. 8-coded 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.