ARID3C Acts as a Regulator of Monocyte-to-Macrophage Differentiation Interacting with NPM1.

ARID3C is a protein located on human chromosome 9 and expressed at low levels in various organs, yet its biological function has not been elucidated. In this study, we investigated both the cellular localization and function of ARID3C. Employing a combination of LC-MS/MS and deep learning techniques, we identified NPM1 as a binding partner for ARID3C's nuclear shuttling. ARID3C was found to predominantly localize with the nucleus, where it functioned as a transcription factor for genes STAT3, STAT1, and JUNB, thereby facilitating monocyte-to-macrophage differentiation. The precise binding sites between ARID3C and NPM1 were predicted by AlphaFold2. Mutating this binding site prevented ARID3C from interacting with NPM1, resulting in its retention in the cytoplasm instead of translocation to the nucleus. Consequently, ARID3C lost its ability to bind to the promoters of target genes, leading to a loss of monocyte-to-macrophage differentiation. Collectively, our findings indicate that ARID3C forms a complex with NPM1 to translocate to the nucleus, acting as a transcription factor that promotes the expression of the genes involved in monocyte-to-macrophage differentiation.

Tissue Proteogenomic Landscape Reveals the Role of Uncharacterized SEL1L3 in Progression and Immunotherapy Response in Lung Adenocarcinoma.

The fundamental pursuit to complete the human proteome atlas and the unmet clinical needs in lung adenocarcinoma have prompted us to study the functional role of uncharacterized proteins and explore their implications in cancer biology. In this study, we characterized SEL1L3, a previously uncharacterized protein encoded from chromosome 4 as a dysregulated protein in lung adenocarcinoma from the large-scale tissue proteogenomics data set established using the cohort of Taiwan Cancer Moonshot. SEL1L3 was expressed in abundance in the tumor parts compared with paired adjacent normal tissues in 90% of the lung adenocarcinoma patients in our cohorts. Moreover, survival analysis revealed the association of SEL1L3 with better clinical outcomes. Intriguingly, silencing of SEL1L3 imposed a reduction in cell viability and activation of ER stress response pathways, indicating a role of SEL1L3 in the regulation of cell stress. Furthermore, the immune profiles of patients with higher SEL1L3 expression were corroborated with its active role in immunophenotype and favorable clinical outcomes in lung adenocarcinoma. Taken together, our study revealed that SEL1L3 might play a vital role in the regulation of cell stress, interaction with cancer cells and the immune microenvironment. Our research findings provide promising insights for further investigation of its molecular signaling network and also suggest SEL1L3 as a potential emerging adjuvant for immunotherapy in lung adenocarcinoma.

Flashlight into the Function of Unannotated C11orf52 using Affinity Purification Mass Spectrometry.

For an enhanced understanding of the biological mechanisms of human disease, it is essential to investigate protein functions. In a previous study, we developed a prediction method of gene ontology (GO) terms by the I-TASSER/COFACTOR result, and we applied this to uPE1 in chromosome 11. Here, to validate the bioinformatics prediction of C11orf52, we utilized affinity purification and mass spectrometry to identify interacting partners of C11orf52. Using immunoprecipitation methods with three different peptide tags (Myc, Flag, and 2B8) in HEK 293T cell lines, we identified 79 candidate proteins that are expected to interact with C11orf52. The results of a pathway analysis of the GO and STRING database with candidate proteins showed that C11orf52 could be related to signaling receptor binding, cell-cell adhesion, and ribosome biogenesis. Then, we selected three partner candidates of DSG1, JUP, and PTPN11 for verification of the interaction with C11orf52 and confirmed them by colocalization at the cell-cell junctions by coimmunofluorescence experiments. On the basis of this study, we expect that C11orf52 is related to the Wnt signaling pathway via DSG1 from the protein-protein interactions, given the results of a comprehensive analysis of the bioinformatic predictions. The data set is available at the ProteomeXchange consortium via PRIDE repository (PXD026986).

Open-pFind Verified Four Missing Proteins from Multi-Tissues.

The Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2012 to perfect the annotation of human protein existence by identifying stronger evidence of the expression of missing proteins (MPs) at the protein level. After an 8 year effort all over the world, the number of MPs in the neXtProt database significantly decreased from 5511 (2012-02-24) to 1899 (2020-01-17). It is now more difficult to provide confident evidence of the remaining MPs because of their specific characteristics, including low abundance, low molecular weight, unexpected modifications, transmembrane structure, tissue-expression specificity, and so on. A higher resolution mass spectrometry (MS) interpretation engine might provide an opportunity to identify these buried MPs in complex samples by the combination with multi-tissue large-scale proteomics. In this study, open-pFind was used to dig MPs from 20 pairs of healthy human tissues by Wang et al. ( Mol. Syst. Biol. 2019, 15 (2), e8503) combined with our large-scale testis data set digested by three enzymes (Glu-C, Lys-C, and trypsin) with specificity for different amino acid residues ( J. Proteme Res. 2019, 18 (12), 4189-4196). A total of 1 535 536 peptides with 17 283 477 peptide-spectrum matches (PSMs) were mapped to 14 279 protein entries at a false discovery rate of <1% at the PSM, peptide, and protein levels. A total of 103 MP candidates were identified, among which 86 candidates had more unique peptide numbers compared with our single testis tissue. After rigorous screening, manual checks, peptide synthesis, and matching with documented peptides from PeptideAtlas, we validated four MPs, P0C7T8 (duodenum and small intestine), Q8WWZ4 (stomach and rectum), Q8IV35 (fallopian tube), and O14921 (tonsil), at the protein level. All MS raw files have been deposited to the ProteomeXchange with identifier PXD021391.

Human Proteome Project and Human Pluripotent Stem Cells: Odd Bedfellows or a Perfect Match?

The Chromosome-Centric Human Proteome Project (C-HPP) aims at the identification of missing proteins (MPs) and the functional characterization of functionally unannotated PE1 (uPE1) proteins. A major challenge in addressing this goal is that many human proteins and MPs are silent in adult cells. A promising approach to overcome such challenge is to exploit the advantage of novel tools such as pluripotent stem cells (PSCs), which are capable of differentiation into three embryonic germ layers, namely, the endoderm, mesoderm, and ectoderm. Here we present several examples of how the Human Y Chromosome Proteome Project (Y-HPP) benefited from this approach to meet C-HPP goals. Furthermore, we discuss how integrating CRISPR engineering, human-induced pluripotent stem cell (hiPSC)-derived disease modeling systems, and organoid technologies provides a unique platform for Y-HPP and C-HPP for MP identification and the functional characterization of human proteins, especially uPE1s.

Bioinformatic Prediction of Gene Ontology Terms of Uncharacterized Proteins from Chromosome 11.

In chromosome 11, 71 out of its 1254 proteins remain functionally uncharacterized on the basis of their existence evidence (uPE1s) following the latest version of neXtProt (release 2020-01-17). Because in vivo and in vitro experimental strategies are often time-consuming and labor-intensive, there is a need for a bioinformatics tool to predict the function annotation. Here, we used I-TASSER/COFACTOR provided on the neXtProt web site, which predicts gene ontology (GO) terms based on the 3D structure of the protein. I-TASSER/COFACTOR predicted 2413 GO terms with a benchmark dataset of the 22 proteins belonging to PE1 of chromosome 11. In this study, we developed a filtering algorithm in order to select specific GO terms using the GO map generated by I-TASSER/COFACTOR. As a result, 187 specific GO terms showed a higher average precision-recall score at the least cellular component term compared to 2413 predicted GO terms. Next, we applied 65 proteins belonging to uPE1s of chromosome 11, and then 409 out of 6684 GO terms survived, where 103 and 142 GO terms of molecular function and biological process, respectively, were included. Representatively, the cellular component GO terms of CCDC90B, C11orf52, and the SMAP were predicted and validated using the overexpression system into 293T cells and immunofluorescence staining. We will further study their biological and molecular functions toward the goal of the neXt-CP50 project as a part of C-HPP. We shared all results and programs in Github (https://github.com/heeyounh/I-TASSER-COFACTOR-filtering.git).

Open-pFind Enhances the Identification of Missing Proteins from Human Testis Tissue.

In recent years, high-throughput technologies have contributed to the development of a more precise picture of the human proteome. However, 2129 proteins remain listed as missing proteins (MPs) in the newest neXtProt release (2019-02). The main reasons for MPs are a low abundance, a low molecular weight, unexpected modifications, membrane characteristics, and so on. Moreover, >50% of the MS/MS data have not been successfully identified in shotgun proteomics. Open-pFind, an efficient open search engine, recently released by the pFind group in China, might provide an opportunity to identify these buried MPs in complex samples. In this study, proteins and potential MPs were identified using Open-pFind and three other search engines to compare their performance and efficiency with three large-scale data sets digested by three enzymes (Glu-C, Lys-C, and trypsin) with specificity on different amino acid (AA) residues. Our results demonstrated that Open-pFind identified 44.7-93.1% more peptide-spectrum matches and 21.3-61.6% more peptide sequences than the second-best search engine. As a result, Open-pFind detected 53.1% more MP candidates than MaxQuant and 8.8% more candidate MPs than Proteome Discoverer. In total, 5 (PE2) of the 124 MP candidates identified by Open-pFind were verified with 2 or 3 unique peptides containing more than 9 AAs by using a spectrum theoretical prediction with pDeep and synthesized peptide matching with pBuild after spectrum quality analysis, isobaric post-translational modification, and single amino acid variant filtering. These five verified MPs can be saved as PE1 proteins. In addition, three other MP candidates were verified with two unique peptides (one peptide containing more than 9 AAs and the other containing only 8 AAs), which was slightly lower than the criteria listed by C-HPP and required additional verification information. More importantly, unexpected modifications were detected in these MPs. All MS data sets have been deposited into ProteomeXchange with the identifier PXD015759.

Challenges of the Human Proteome Project: 10-Year Experience of the Russian Consortium.

This manuscript collects all the efforts of the Russian Consortium, bottlenecks revealed in the course of the C-HPP realization, and ways of their overcoming. One of the main bottlenecks in the C-HPP is the insufficient sensitivity of proteomic technologies, hampering the detection of low- and ultralow-copy number proteins forming the "dark part" of the human proteome. In the frame of MP-Challenge, to increase proteome coverage we suggest an experimental workflow based on a combination of shotgun technology and selected reaction monitoring with two-dimensional alkaline fractionation. Further, to detect proteins that cannot be identified by such technologies, nanotechnologies such as combined atomic force microscopy with molecular fishing and/or nanowire detection may be useful. These technologies provide a powerful tool for single molecule analysis, by analogy with nanopore sequencing during genome analysis. To systematically analyze the functional features of some proteins (CP50 Challenge), we created a mathematical model that predicts the number of proteins differing in amino acid sequence: proteoforms. According to our data, we should expect about 100 000 different proteoforms in the liver tissue and a little more in the HepG2 cell line. The variety of proteins forming the whole human proteome significantly exceeds these results due to post-translational modifications (PTMs). As PTMs determine the functional specificity of the protein, we propose using a combination of gene-centric transcriptome-proteomic analysis with preliminary fractionation by two-dimensional electrophoresis to identify chemically modified proteoforms. Despite the complexity of the proposed solutions, such integrative approaches could be fruitful for MP50 and CP50 Challenges in the framework of the C-HPP.

200+ Protein Concentrations in Healthy Human Blood Plasma: Targeted Quantitative SRM SIS Screening of Chromosomes 18, 13, Y, and the Mitochondrial Chromosome Encoded Proteome.

This work continues the series of the quantitative measurements of the proteins encoded by different chromosomes in the blood plasma of a healthy person. Selected Reaction Monitoring with Stable Isotope-labeled peptide Standards (SRM SIS) and a gene-centric approach, which is the basis for the implementation of the international Chromosome-centric Human Proteome Project (C-HPP), were applied for the quantitative measurement of proteins in human blood plasma. Analyses were carried out in the frame of C-HPP for each protein-coding gene of the four human chromosomes: 18, 13, Y, and mitochondrial. Concentrations of proteins encoded by 667 genes were measured in 54 blood plasma samples of the volunteers, whose health conditions were consistent with requirements for astronauts. The gene list included 276, 329, 47, and 15 genes of chromosomes 18, 13, Y, and the mitochondrial chromosome, respectively. This paper does not make claims about the detection of missing proteins. Only 205 proteins (30.7%) were detected in the samples. Of them, 84, 106, 10, and 5 belonged to chromosomes 18, 13, and Y and the mitochondrial chromosome, respectively. Each detected protein was found in at least one of the samples analyzed. The SRM SIS raw data are available in the ProteomeXchange repository (PXD004374, PASS01192).

Multiproteases Combined with High-pH Reverse-Phase Separation Strategy Verified Fourteen Missing Proteins in Human Testis Tissue.

Subsequent to conducting the Chromosome-Centric Human Proteome Project, we have focused on human testis-enriched missing proteins (MPs) since 2015. For protein coverage to be enhanced, a multiprotease strategy was used for separation of samples by 10% SDS-PAGE. For the separating efficiency to be improved, a high-pH reverse phase (RP) separation strategy was applied to fractionate complex samples in this study. A total of 11,558 proteins was identified, which is the largest proteome data set for single human tissue sample so far. On the basis of this large-scale data set, we verified 14 MPs (PE2) in neXtProt (2018-01) after spectrum quality analysis, isobaric post-translational modification, and single amino acid variant filtering, and synthesized peptide matching. Tissue expression analysis showed that 3 of 14 MPs were testis-specific proteins. Functional analysis showed that 10 of 14 MPs were closely related to liver tumor, liver carcinoma, and hepatocellular carcinoma. Another 100 MPs were listed as candidates but required additional verification information. All MS data sets have been deposited into the ProteomeXchange with the identifier PXD009737.

Digging for Missing Proteins Using Low-Molecular-Weight Protein Enrichment and a "Mirror Protease" Strategy.

In 2012, the Chromosome-centric Human Proteome Project (C-HPP) launched an investigation for missing proteins (MPs) to complete the Human Proteome Project (HPP). The majority of the MPs were distributed in low-molecular-weight (LMW) ranges, especially from 0 to 40 kDa. LMW protein identification is challenging, owing to their short length, low abundance, and hydrophobicity. Furthermore, many sequences from trypsin digestion are unlikely to yield detectable peptides or a reasonable quality of MS2 spectrum. Therefore, we focused on small MPs by combining LMW protein enrichment and a pair of complementary proteases strategy with trypsin and LysargiNase for human testis samples. In-depth testis LMW protein profiling resulted in the identification of 4063 proteins, of which 2565 were LMW proteins and 1130 had pairs of peptides generated from both trypsin and LysargiNase. This provided additional mass spectral evidence of further verification of small MPs. Finally, two MPs were verified from the seven MP candidates. One of them, Q8N688 , was verified with two series of continuous and complementary b/y-product ions from the pairs of spectra for tryptic and LysargiNase digested peptides after the "mirror spectrum" matching. This make the confident identification of the representative peptides for the target MPs. On the contrary, the two verified peptides for Q86WR6 were identified with the same strategy from the gel-separation and gel-elution samples, respectively. Although the other five MP candidates showed high-quality spectra, they could not be sufficiently distinguished as PE1s and require further verification. All MS data sets have been deposited in the ProteomeXchange with identifier PXD010093.

Bridging the Chromosome-centric and Biology/Disease-driven Human Proteome Projects: Accessible and Automated Tools for Interpreting the Biological and Pathological Impact of Protein Sequence Variants Detected via Proteogenomics.

The Chromosome-centric Human Proteome Project (C-HPP) seeks to comprehensively characterize all protein products coded by the genome, including those expressed sequence variants confirmed via proteogenomics methods. The closely related Biology/Disease-driven Human Proteome Project (B/D-HPP) seeks to understand the biological and pathological associations of expressed protein products, especially those carrying sequence variants that may be drivers of disease. To achieve these objectives, informatics tools are required that interpret potential functional or disease implications of variant protein sequence detected via proteogenomics. Toward this end, we have developed an automated workflow within the Galaxy for Proteomics (Galaxy-P) platform, which leverages the Cancer-Related Analysis of Variants Toolkit (CRAVAT) and makes it interoperable with proteogenomic results. Protein sequence variants confirmed by proteogenomics are assessed for potential structure-function effects as well as associations with cancer using CRAVAT's rich suite of functionalities, including visualization of results directly within the Galaxy user interface. We demonstrate the effectiveness of this workflow on proteogenomic results generated from an MCF7 breast cancer cell line. Our free and open software should enable improved interpretation of the functional and pathological effects of protein sequence variants detected via proteogenomics, acting as a bridge between the C-HPP and B/D-HPP.

Chromosome 17 Missing Proteins: Recent Progress and Future Directions as Part of the neXt-MP50 Challenge.

The Chromosome-centric Human Proteome Project (C-HPP), announced in September 2016, is an initiative to accelerate progress on the detection and characterization of neXtProt PE2,3,4 "missing proteins" (MPs) with a mandate to each chromosome team to find about 50 MPs over 2 years. Here we report major progress toward the neXt-MP50 challenge with 43 newly validated Chr 17 PE1 proteins, of which 25 were based on mass spectrometry, 12 on protein-protein interactions, 3 on a combination of MS and PPI, and 3 with other types of data. Notable among these new PE1 proteins were five keratin-associated proteins, a single olfactory receptor, and five additional membrane-embedded proteins. We evaluate the prospects of finding the remaining 105 MPs coded for on Chr 17, focusing on mass spectrometry and protein-protein interaction approaches. We present a list of 35 prioritized MPs with specific approaches that may be used in further MS and PPI experimental studies. Additionally, we demonstrate how in silico studies can be used to capture individual peptides from major data repositories, documenting one MP that appears to be a strong candidate for PE1. We are close to our goal of finding 50 MPs for Chr 17.

Multi-Protease Strategy Identifies Three PE2 Missing Proteins in Human Testis Tissue.

Although 5 years of the missing proteins (MPs) study have been completed, searching for MPs remains one of the core missions of the Chromosome-Centric Human Proteome Project (C-HPP). Following the next-50-MPs challenge of the C-HPP, we have focused on the testis-enriched MPs by various strategies since 2015. On the basis of the theoretical analysis of MPs (2017-01, neXtProt) using multiprotease digestion, we found that nonconventional proteases (e.g. LysargiNase, GluC) could improve the peptide diversity and sequence coverage compared with Trypsin. Therefore, a multiprotease strategy was used for searching more MPs in the same human testis tissues separated by 10% SDS-PAGE, followed by high resolution LC-MS/MS system (Q Exactive HF). A total of 7838 proteins were identified. Among them, three PE2 MPs in neXtProt 2017-01 have been identified: beta-defensin 123 ( Q8N688 , chr 20q), cancer/testis antigen family 45 member A10 ( P0DMU9 , chr Xq), and Histone H2A-Bbd type 2/3 ( P0C5Z0 , chr Xq). However, because only one unique peptide of ≥9 AA was identified in beta-defensin 123 and Histone H2A-Bbd type 2/3, respectively, further analysis indicates that each falls under the exceptions clause of the HPP Guidelines v2.1. After a spectrum quality check, isobaric PTM and single amino acid variant (SAAV) filtering, and verification with a synthesized peptide, and based on overlapping peptides from different proteases, these three MPs should be considered as exemplary examples of MPs found by exceptional criteria. Other MPs were considered as candidates but need further validation. All MS data sets have been deposited to the ProteomeXchange with identifier PXD006465.

Identification of Missing Proteins in the Phosphoproteome of Kidney Cancer.

Identifying missing proteins (MPs) has been one of the critical missions of the Chromosome-Centric Human Proteome Project (C-HPP). Since 2012, over 30 research teams from 17 countries have been trying to search adequate and accurate evidence of MPs through various biochemical strategies. MPs mainly fall into the following classes: (1) low-molecular-weight (LMW) proteins, (2) membrane proteins, (3) proteins that contained various post-translational modifications (PTMs), (4) nucleic acid-associated proteins, (5) low abundance, and (6) unexpressed genes. In this study, kidney cancer and adjacent tissues were used for phosphoproteomics research, and 8962 proteins were identified, including 6415 phosphoproteins, and 44 728 phosphosites, of which 10 266 were unreported previously. In total, 75 candidate detections were found, including 45 phoshoproteins. GO analysis for these 75 candidate detections revealed that these proteins mainly clustered as membrane proteins and took part in nephron and kidney development. After rigorous screening and manual check, 9 of them were verified with the synthesized peptides. Finally, only one missing protein was confirmed. All mass spectrometry data from this study have been deposited in the PRIDE with identifier PXD006482.

Chromosome-Centric Human Proteome Project Allies with Developmental Biology: A Case Study of the Role of Y Chromosome Genes in Organ Development.

One of the main goals of Chromosome-Centric Human Proteome Project is to identify protein evidence for missing proteins (MPs). Here, we present a case study of the role of Y chromosome genes in organ development and how to overcome the challenges facing MPs identification by employing human pluripotent stem cell differentiation into cells of different organs yielding unprecedented biological insight into adult silenced proteins. Y chromosome is a male-specific sex chromosome which escapes meiotic recombination. From an evolutionary perspective, Y chromosome has preserved 3% of ancestral genes compared to 98% preservation of the X chromosome based on Ohno's law. Male specific region of Y chromosome (MSY) contains genes that contribute to central dogma and govern the expression of various targets throughout the genome. One of the most well-known functions of MSY genes is to decide the male-specific characteristics including sex, testis formation, and spermatogenesis, which are majorly formed by ampliconic gene families. Beyond its role in sex-specific gonad development, MSY genes in coexpression with their X counterparts, as single copy and broadly expressed genes, inhibit haplolethality and play a key role in embryogenesis. The role of X-Y related gene mutations in the development of hereditary syndromes suggests an essential contribution of sex chromosome genes to development. MSY genes, solely and independent of their X counterparts and/or in association with sex hormones, have a considerable impact on organ development. In this Review, we present major recent findings on the contribution of MSY genes to gonad formation, spermatogenesis, and the brain, heart, and kidney development and discuss how Y chromosome proteome project may exploit developmental biology to find missing proteins.

Y Chromosome Missing Protein, TBL1Y, May Play an Important Role in Cardiac Differentiation.

Despite evidence for sex-specific cardiovascular physiology and pathophysiology, the biological basis for this dimorphism remains to be explored. Apart from hormonal factors, gender-related characteristics may reside in the function of sex chromosomes during cardiac development. In this study, we investigated the differential expression of the male-specific region of the Y chromosome (MSY) genes and their X counterparts during cardiac differentiation of human embryonic stem cells (hESC). We observed alterations in mRNA and protein levels of TBL1Y, PCDH11Y, ZFY, KDM5D, USP9Y, RPS4Y1, DDX3Y, PRY, XKRY, BCORP1, RBMY, HSFY, and UTY, which accompanied changes in intracellular localization. Of them, the abundance of a Y chromosome missing protein, TBL1Y, showed a significant increase during differentiation while the expression level of its X counterpart decreased. Consistently, reducing TBL1Y cellular level using siRNA approach influenced cardiac differentiation by reducing its efficacy as well as increasing the probability of impaired contractions. TBL1Y knockdown may have negatively impacted cardiogenesis by CtBP stabilization. Furthermore, we presented compelling experimental evidence to distinguish TBL1Y from TBL1X, its highly similar X chromosome homologue, and proposed reclassification of TBL1Y as "found missing protein" (PE1). Our results demonstrated that MSY proteins may play an important role in cardiac development.

Next Generation Proteomic Pipeline for Chromosome-Based Proteomic Research Using NeXtProt and GENCODE Databases.

Human Proteome Project aims to map all human proteins including missing proteins as well as proteoforms with post translational modifications, alternative splicing variants (ASVs), and single amino acid variants (SAAVs). neXtProt and Ensemble databases are usually used to provide curated information on human coding genes. However, to find these proteoforms, we (Chr #11 team) first introduce a streamlined pipeline using customized and concatenated neXtProt and GENCODE originated from Ensemble, with controlled false discovery rate (FDR). Because of large sized databases used in this pipeline, we found more stringent FDR filtering (0.1% at the peptide level and 1% at the protein level) to claim novel findings, such as GENCODE ASVs and missing proteins, from human hippocampus data set (MSV000081385) and ProteomeXchange (PXD007166). Using our next generation proteomic pipeline (nextPP) with neXtProt and GENCODE databases, two missing proteins such as activity-regulated cytoskeleton-associated protein (ARC, Chr 8) and glutamate receptor ionotropic, kainite 5 (GRIK5, Chr 19) were additionally identified with two or more unique peptides from human brain tissues. Additionally, by applying the pipeline to human brain related data sets such as cortex (PXD000067 and PXD000561), spinal cord, and fetal brain (PXD000561), seven GENCODE ASVs such as ACTN4-012 (Chr.19), DPYSL2-005 (Chr.8), MPRIP-003 (Chr.17), NCAM1-013 (Chr.11), EPB41L1-017 (Chr.20), AGAP1-004 (Chr.2), and CPNE5-005 (Chr.6) were identified from two or more data sets. The identified peptides of GENCODE ASVs were mapped onto novel exon insertions, alternative translations at 5'-untranslated region, or novel protein coding sequence. Applying the pipeline to male reproductive organ related data sets, 52 GENCODE ASVs were identified from two testis (PXD000561 and PXD002179) and a spermatozoa (PXD003947) data sets. Four out of 52 GENCODE ASVs such as RAB11FIP5-008 (Chr. 2), RP13-347D8.7-001 (Chr. X), PRDX4-002 (Chr. X), and RP11-666A8.13-001 (Chr. 17) were identified in all of the three samples.

Advances in the Chromosome-Centric Human Proteome Project: looking to the future.

INTRODUCTION: The mission of the Chromosome-Centric Human Proteome Project (C-HPP), is to map and annotate the entire predicted human protein set (~20,000 proteins) encoded by each chromosome. The initial steps of the project are focused on 'missing proteins (MPs)', which lacked documented evidence for existence at protein level. In addition to remaining 2,579 MPs, we also target those annotated proteins having unknown functions, uPE1 proteins, alternative splice isoforms and post-translational modifications. We also consider how to investigate various protein functions involved in cis-regulatory phenomena, amplicons lncRNAs and smORFs. Areas covered: We will cover the scope, historic background, progress, challenges and future prospects of C-HPP. This review also addresses the question of how we can best improve the methodological approaches, select the optimal biological samples, and recommend stringent protocols for the identification and characterization of MPs. A new strategy for functional analysis of some of those annotated proteins having unknown function will also be discussed. Expert commentary: If the project moves well by reshaping the original goals, the current working modules and team work in the proposed extended planning period, it is anticipated that a progressively more detailed draft of an accurate chromosome-based proteome map will become available with functional information.

Deep Coverage Proteomics Identifies More Low-Abundance Missing Proteins in Human Testis Tissue with Q-Exactive HF Mass Spectrometer.

Since 2012, missing proteins (MPs) investigation has been one of the critical missions of Chromosome-Centric Human Proteome Project (C-HPP) through various biochemical strategies. On the basis of our previous testis MPs study, faster scanning and higher resolution mass-spectrometry-based proteomics might be conducive to MPs exploration, especially for low-abundance proteins. In this study, Q-Exactive HF (HF) was used to survey proteins from the same testis tissues separated by two separating methods (tricine- and glycine-SDS-PAGE), as previously described. A total of 8526 proteins were identified, of which more low-abundance proteins were uniquely detected in HF data but not in our previous LTQ Orbitrap Velos (Velos) reanalysis data. Further transcriptomics analysis showed that these uniquely identified proteins by HF also had lower expression at the mRNA level. Of the 81 total identified MPs, 74 and 39 proteins were listed as MPs in HF and Velos data sets, respectively. Among the above MPs, 47 proteins (43 neXtProt PE2 and 4 PE3) were ranked as confirmed MPs after verifying with the stringent spectra match and isobaric and single amino acid variants filtering. Functional investigation of these 47 MPs revealed that 11 MPs were testis-specific proteins and 7 MPs were involved in spermatogenesis process. Therefore, we concluded that higher scanning speed and resolution of HF might be factors for improving the low-abundance MP identification in future C-HPP studies. All mass-spectrometry data from this study have been deposited in the ProteomeXchange with identifier PXD004092.