Proteasome complexes experience profound structural and functional rearrangements throughout mammalian spermatogenesis.

During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific α4s subunit replaces the α4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function, and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>82 to 92%) during differentiation, presumably due to the shift from α4 to α4s expression. We demonstrated that s20S, but not c20S, interacts with components of the meiotic synaptonemal complex, where it may localize via association with the PI31 adaptor protein. In vitro, s20S preferentially binds to 19S and displays higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between α4 and α4s. We propose that these differences induced by α4s incorporation result in significant changes in the way the s20S interacts with its partners and dictate its role in germ cell differentiation.

The 2023 Report on the Proteome from the HUPO Human Proteome Project.

Since 2010, the Human Proteome Project (HPP), the flagship initiative of the Human Proteome Organization (HUPO), has pursued two goals: (1) to credibly identify the protein parts list and (2) to make proteomics an integral part of multiomics studies of human health and disease. The HPP relies on international collaboration, data sharing, standardized reanalysis of MS data sets by PeptideAtlas and MassIVE-KB using HPP Guidelines for quality assurance, integration and curation of MS and non-MS protein data by neXtProt, plus extensive use of antibody profiling carried out by the Human Protein Atlas. According to the neXtProt release 2023-04-18, protein expression has now been credibly detected (PE1) for 18,397 of the 19,778 neXtProt predicted proteins coded in the human genome (93%). Of these PE1 proteins, 17,453 were detected with mass spectrometry (MS) in accordance with HPP Guidelines and 944 by a variety of non-MS methods. The number of neXtProt PE2, PE3, and PE4 missing proteins now stands at 1381. Achieving the unambiguous identification of 93% of predicted proteins encoded from across all chromosomes represents remarkable experimental progress on the Human Proteome parts list. Meanwhile, there are several categories of predicted proteins that have proved resistant to detection regardless of protein-based methods used. Additionally there are some PE1-4 proteins that probably should be reclassified to PE5, specifically 21 LINC entries and ∼30 HERV entries; these are being addressed in the present year. Applying proteomics in a wide array of biological and clinical studies ensures integration with other omics platforms as reported by the Biology and Disease-driven HPP teams and the antibody and pathology resource pillars. Current progress has positioned the HPP to transition to its Grand Challenge Project focused on determining the primary function(s) of every protein itself and in networks and pathways within the context of human health and disease.

Naive Pluripotent and Trophoblastic Stem Cell Lines as a Model for Detecting Missing Proteins in the Context of the Chromosome-Centric Human Proteome Project.

The Chromosome-centric Human Proteome Project (C-HPP) aims at identifying the proteins as gene products encoded by the human genome, characterizing their isoforms and functions. The existence of products has now been confirmed for 93.2% of the genes at the protein level. The remaining mostly correspond to proteins of low abundance or difficult to access. Over the past years, we have significantly contributed to the identification of missing proteins in the human spermatozoa. We pursue our search in the reproductive sphere with a focus on early human embryonic development. Pluripotent cells, developing into the fetus, and trophoblast cells, giving rise to the placenta, emerge during the first weeks. This emergence is a focus of scientists working in the field of reproduction, placentation and regenerative medicine. Most knowledge has been harnessed by transcriptomic analysis. Interestingly, some genes are uniquely expressed in those cells, giving the opportunity to uncover new proteins that might play a crucial role in setting up the molecular events underlying early embryonic development. Here, we analyzed naive pluripotent and trophoblastic stem cells and discovered 4 new missing proteins, thus contributing to the C-HPP. The mass spectrometry proteomics data was deposited on ProteomeXchange under the data set identifier PXD035768.

The 2022 Report on the Human Proteome from the HUPO Human Proteome Project.

The 2022 Metrics of the Human Proteome from the HUPO Human Proteome Project (HPP) show that protein expression has now been credibly detected (neXtProt PE1 level) for 18 407 (93.2%) of the 19 750 predicted proteins coded in the human genome, a net gain of 50 since 2021 from data sets generated around the world and reanalyzed by the HPP. Conversely, the number of neXtProt PE2, PE3, and PE4 missing proteins has been reduced by 78 from 1421 to 1343. This represents continuing experimental progress on the human proteome parts list across all the chromosomes, as well as significant reclassifications. Meanwhile, applying proteomics in a vast array of biological and clinical studies continues to yield significant findings and growing integration with other omics platforms. We present highlights from the Chromosome-Centric HPP, Biology and Disease-driven HPP, and HPP Resource Pillars, compare features of mass spectrometry and Olink and Somalogic platforms, note the emergence of translation products from ribosome profiling of small open reading frames, and discuss the launch of the initial HPP Grand Challenge Project, "A Function for Each Protein".

DeepHistoClass: A Novel Strategy for Confident Classification of Immunohistochemistry Images Using Deep Learning.

A multitude of efforts worldwide aim to create a single-cell reference map of the human body, for fundamental understanding of human health, molecular medicine, and targeted treatment. Antibody-based proteomics using immunohistochemistry (IHC) has proven to be an excellent technology for integration with large-scale single-cell transcriptomics datasets. The golden standard for evaluation of IHC staining patterns is manual annotation, which is expensive and may lead to subjective errors. Artificial intelligence holds much promise for efficient and accurate pattern recognition, but confidence in prediction needs to be addressed. Here, the aim was to present a reliable and comprehensive framework for automated annotation of IHC images. We developed a multilabel classification of 7848 complex IHC images of human testis corresponding to 2794 unique proteins, generated as part of the Human Protein Atlas (HPA) project. Manual annotation data for eight different cell types was generated as a basis for training and testing a proposed Hybrid Bayesian Neural Network. By combining the deep learning model with a novel uncertainty metric, DeepHistoClass (DHC) Confidence Score, the average diagnostic performance improved from 86.9% to 96.3%. This metric not only reveals which images are reliably classified by the model, but can also be utilized for identification of manual annotation errors. The proposed streamlined workflow can be developed further for other tissue types in health and disease and has important implications for digital pathology initiatives or large-scale protein mapping efforts such as the HPA project.

Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock.

Ribosome biogenesis is a complex and multistep process that depends on various assembly factors. To understand this process and identify the ribosome assembly intermediates, most studies have set out to delete or deplete these assembly factors. Instead, we took advantage of the impact of heat stress (45 °C) on the late stages of the biogenesis of the 30S ribosomal subunit to explore authentic precursors. Under these conditions, reduced levels of the DnaK chaperone proteins devoted to ribosome assembly lead to the transient accumulation of 21S ribosomal particles, which are 30S precursors. We constructed strains with different affinity tags on one early and one late 30S ribosomal protein and purified the 21S particles that form under heat shock. A combination of relative quantification using mass spectrometry-based proteomics and cryo-electron microscopy (cryo-EM) was then used to determine their protein contents and structures.

The proteomic analysis of bovine embryos developed in vivo or in vitro reveals the contribution of the maternal environment to early embryo.

BACKGROUND: Despite many improvements with in vitro culture systems, the quality and developmental ability of mammalian embryos produced in vitro are still lower than their in vivo counterparts. Though previous studies have evidenced differences in gene expression between in vivo- and in vitro-derived bovine embryos, there is no comparison at the protein expression level. RESULTS: A total of 38 pools of grade-1 quality bovine embryos at the 4-6 cell, 8-12 cell, morula, compact morula, and blastocyst stages developed either in vivo or in vitro were analyzed by nano-liquid chromatography coupled with label-free quantitative mass spectrometry, allowing for the identification of 3,028 proteins. Multivariate analysis of quantified proteins showed a clear separation of embryo pools according to their in vivo or in vitro origin at all stages. Three clusters of differentially abundant proteins (DAPs) were evidenced according to embryo origin, including 463 proteins more abundant in vivo than in vitro across development and 314 and 222 proteins more abundant in vitro than in vivo before and after the morula stage, respectively. The functional analysis of proteins found more abundant in vivo showed an enrichment in carbohydrate metabolism and cytoplasmic cellular components. Proteins found more abundant in vitro before the morula stage were mostly localized in mitochondrial matrix and involved in ATP-dependent activity, while those overabundant after the morula stage were mostly localized in the ribonucleoprotein complex and involved in protein synthesis. Oviductin and other oviductal proteins, previously shown to interact with early embryos, were among the most overabundant proteins after in vivo development. CONCLUSIONS: The maternal environment led to higher degradation of mitochondrial proteins at early developmental stages, lower abundance of proteins involved in protein synthesis at the time of embryonic genome activation, and a global upregulation of carbohydrate metabolic pathways compared to in vitro production. Furthermore, embryos developed in vivo internalized large amounts of oviductin and other proteins probably originated in the oviduct as soon as the 4-6 cell stage. These data provide new insight into the molecular contribution of the mother to the developmental ability of early embryos and will help design better in vitro culture systems.

Knock out of specific maternal vitellogenins in zebrafish (Danio rerio) evokes vital changes in egg proteomic profiles that resemble the phenotype of poor quality eggs.

BACKGROUND: We previously reported the results of CRISPR/Cas9 knock-out (KO) of type-I and type-III vitellogenins (Vtgs) in zebrafish, which provided the first experimental evidence on essentiality and disparate functioning of Vtgs at different stages during early development. However, the specific contributions of different types of Vtg to major cellular processes remained to be investigated. The present study employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to meet this deficit. Proteomic profiles of zebrafish eggs lacking three type-I Vtgs simultaneously (vtg1-KO), or lacking only type III Vtg (vtg3-KO) were compared to those of wild type (Wt) eggs. Obtained spectra were searched against a zebrafish proteome database and identified proteins were quantified based on normalized spectral counts. RESULTS: The vtg-KO caused severe changes in the proteome of 1-cell stage zebrafish eggs. These changes were disclosed by molecular signatures that highly resembled the proteomic phenotype of poor quality zebrafish eggs reported in our prior studies. Proteomic profiles of vtg-KO eggs and perturbations in abundances of hundreds of proteins revealed unique, noncompensable contributions of multiple Vtgs to protein and in energy homeostasis. The lack of this contribution appears to have a significant impact on endoplasmic reticulum and mitochondrial functions, and thus embryonic development, even after zygotic genome activation. Increased endoplasmic reticulum stress, Redox/Detox activities, glycolysis/gluconeogenesis, enrichment in cellular proliferation and in human neurodegenerative disease related activities in both vtg1- and vtg3-KO eggs were found to be indicators of the aforementioned conditions. Distinctive increase in apoptosis and Parkinson disease pathways, as well as the decrease in lipid metabolism related activities in vtg3-KO eggs implies compelling roles of Vtg3, the least abundant form of Vtgs in vertebrate eggs, in mitochondrial activities. Several differentially abundant proteins representing the altered molecular mechanisms have been identified as strong candidate markers for studying the details of these mechanisms during early embryonic development in zebrafish and possibly other vertebrates. CONCLUSIONS: These findings indicate that the global egg proteome is subject to extensive modification depending on the presence or absence of specific Vtgs and that these modifications can have a major impact on developmental competence.

Study of the Distribution of Acetaminophen and Its Metabolites in Rats, from the Whole-Body to Isolated Organ Levels, by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging after On-Tissue Chemical Derivatization.

During drug development, detailed investigations of the pharmacokinetic profile of the drug are required to characterize its absorption, distribution, metabolism, and excretion properties. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an established technique for studies of the distribution of drugs and their metabolites. It has advantages over autoradiography, which is conventionally used for distribution studies: it does not require the radiolabeling of drugs and can distinguish between the drug and its metabolites directly in the tissue. However, its lack of sensitivity in certain cases remains challenging. Novel procedures, such as on-tissue chemical derivatization (OTCD), could be developed to increase sensitivity. We used OTCD to enhance the sensitivity of MALDI-MSI for one of the most widely used drugs, acetaminophen, and to study its distribution in tissues. Without derivatization, this drug and some of its metabolites are undetectable by MALDI-MSI in the tissues of treated rats. We used 2-fluoro-1-methylpyridinium p-toluene sulfonate as a derivatization reagent, to increase the ionization yield of acetaminophen and some of its metabolites. The OTCD protocol made it possible to study the distribution of acetaminophen and its metabolites in whole-body sections at a spatial resolution of 400 µm and in complex anatomical structures, such as the testis and epididymis, at a spatial resolution <50 µm. The OTCD is also shown to be compatible with the quantification of acetaminophen by MALDI-MSI in whole-body tissues. This protocol could be applied to other molecules bearing phenol groups and presenting a low ionization efficiency.

The Functionally Unannotated Proteome of Human Male Tissues: A Shared Resource to Uncover New Protein Functions Associated with Reproductive Biology.

In the context of the Human Proteome Project, we built an inventory of 412 functionally unannotated human proteins for which experimental evidence at the protein level exists (uPE1) and which are highly expressed in tissues involved in human male reproduction. We implemented a strategy combining literature mining, bioinformatics tools to collate annotation and experimental information from specific molecular public resources, and efficient visualization tools to put these unknown proteins into their biological context (protein complexes, tissue and subcellular location, expression pattern). The gathered knowledge allowed pinpointing five uPE1 for which a function has recently been proposed and which should be updated in protein knowledge bases. Furthermore, this bioinformatics strategy allowed to build new functional hypotheses for five other uPE1s in link with phenotypic traits that are specific to male reproductive function such as ciliogenesis/flagellum formation in germ cells (CCDC112 and TEX9), chromatin remodeling (C3orf62) and spermatozoon maturation (CCDC183). We also discussed the enigmatic case of MAGEB proteins, a poorly documented cancer/testis antigen subtype. Tools used and computational outputs produced during this study are freely accessible via ProteoRE (http://www.proteore.org), a Galaxy-based instance, for reuse purposes. We propose these five uPE1s should be investigated in priority by expert laboratories and hope that this inventory and shared resources will stimulate the interest of the community of reproductive biology.

FDR-controlled metabolite annotation for high-resolution imaging mass spectrometry.

High-mass-resolution imaging mass spectrometry promises to localize hundreds of metabolites in tissues, cell cultures, and agar plates with cellular resolution, but it is hampered by the lack of bioinformatics tools for automated metabolite identification. We report pySM, a framework for false discovery rate (FDR)-controlled metabolite annotation at the level of the molecular sum formula, for high-mass-resolution imaging mass spectrometry (https://github.com/alexandrovteam/pySM). We introduce a metabolite-signal match score and a target-decoy FDR estimate for spatial metabolomics.

Identification of 56 Proteins Involved in Embryo-Maternal Interactions in the Bovine Oviduct.

The bovine embryo develops in contact with the oviductal fluid (OF) during the first 4-5 days of pregnancy. The aim of this study was to decipher the protein interactions occurring between the developing embryo and surrounding OF. In-vitro produced 4-6 cell and morula embryos were incubated or not (controls) in post-ovulatory OF (OF-treated embryos) and proteins were then analyzed and quantified by high resolution mass spectrometry (MS) in both embryo groups and in OF. A comparative analysis of MS data allowed the identification and quantification of 56 embryo-interacting proteins originated from the OF, including oviductin (OVGP1) and several annexins (ANXA1, ANXA2, ANXA4) as the most abundant ones. Some embryo-interacting proteins were developmental stage-specific, showing a modulating role of the embryo in protein interactions. Three interacting proteins (OVGP1, ANXA1 and PYGL) were immunolocalized in the perivitelline space and in blastomeres, showing that OF proteins were able to cross the zona pellucida and be taken up by the embryo. Interacting proteins were involved in a wide range of functions, among which metabolism and cellular processes were predominant. This study identified for the first time a high number of oviductal embryo-interacting proteins, paving the way for further targeted studies of proteins potentially involved in the establishment of pregnancy in cattle.

Cell Type-Specific Expression of Testis Elevated Genes Based on Transcriptomics and Antibody-Based Proteomics.

One of the most complex organs in the human body is the testis, where spermatogenesis takes place. This physiological process involves thousands of genes and proteins that are activated and repressed, making testis the organ with the highest number of tissue-specific genes. However, the function of a large proportion of the corresponding proteins remains unknown and testis harbors many missing proteins (MPs), defined as products of protein-coding genes that lack experimental mass spectrometry evidence. Here, an integrated omics approach was used for exploring the cell type-specific protein expression of genes with an elevated expression in testis. By combining genome-wide transcriptomics analysis with immunohistochemistry, more than 500 proteins with distinct testicular protein expression patterns were identified, and these were selected for in-depth characterization of their in situ expression in eight different testicular cell types. The cell type-specific protein expression patterns allowed us to identify six distinct clusters of expression at different stages of spermatogenesis. The analysis highlighted numerous poorly characterized proteins in each of these clusters whose expression overlapped with that of known proteins involved in spermatogenesis, including 85 proteins with an unknown function and 60 proteins that previously have been classified as MPs. Furthermore, we were able to characterize the in situ distribution of several proteins that previously lacked spatial information and cell type-specific expression within the testis. The testis elevated expression levels both at the RNA and protein levels suggest that these proteins are related to testis-specific functions. In summary, the study demonstrates the power of combining genome-wide transcriptomics analysis with antibody-based protein profiling to explore the cell type-specific expression of both well-known proteins and MPs. The analyzed proteins constitute important targets for further testis-specific research in male reproductive disorders.

Human Proteome Project Mass Spectrometry Data Interpretation Guidelines 3.0.

The Human Proteome Organization's (HUPO) Human Proteome Project (HPP) developed Mass Spectrometry (MS) Data Interpretation Guidelines that have been applied since 2016. These guidelines have helped ensure that the emerging draft of the complete human proteome is highly accurate and with low numbers of false-positive protein identifications. Here, we describe an update to these guidelines based on consensus-reaching discussions with the wider HPP community over the past year. The revised 3.0 guidelines address several major and minor identified gaps. We have added guidelines for emerging data independent acquisition (DIA) MS workflows and for use of the new Universal Spectrum Identifier (USI) system being developed by the HUPO Proteomics Standards Initiative (PSI). In addition, we discuss updates to the standard HPP pipeline for collecting MS evidence for all proteins in the HPP, including refinements to minimum evidence. We present a new plan for incorporating MassIVE-KB into the HPP pipeline for the next (HPP 2020) cycle in order to obtain more comprehensive coverage of public MS data sets. The main checklist has been reorganized under headings and subitems, and related guidelines have been grouped. In sum, Version 2.1 of the HPP MS Data Interpretation Guidelines has served well, and this timely update to version 3.0 will aid the HPP as it approaches its goal of collecting and curating MS evidence of translation and expression for all predicted ∼20 000 human proteins encoded by the human genome.

Peptimapper: proteogenomics workflow for the expert annotation of eukaryotic genomes.

BACKGROUND: Accurate structural annotation of genomes is still a challenge, despite the progress made over the past decade. The prediction of gene structure remains difficult, especially for eukaryotic species, and is often erroneous and incomplete. We used a proteogenomics strategy, taking advantage of the combination of proteomics datasets and bioinformatics tools, to identify novel protein coding-genes and splice isoforms, assign correct start sites, and validate predicted exons and genes. RESULTS: Our proteogenomics workflow, Peptimapper, was applied to the genome annotation of Ectocarpus sp., a key reference genome for both the brown algal lineage and stramenopiles. We generated proteomics data from various life cycle stages of Ectocarpus sp. strains and sub-cellular fractions using a shotgun approach. First, we directly generated peptide sequence tags (PSTs) from the proteomics data. Second, we mapped PSTs onto the translated genomic sequence. Closely located hits (i.e., PSTs locations on the genome) were then clustered to detect potential coding regions based on parameters optimized for the organism. Third, we evaluated each cluster and compared it to gene predictions from existing conventional genome annotation approaches. Finally, we integrated cluster locations into GFF files to use a genome viewer. We identified two potential novel genes, a ribosomal protein L22 and an aryl sulfotransferase and corrected the gene structure of a dihydrolipoamide acetyltransferase. We experimentally validated the results by RT-PCR and using transcriptomics data. CONCLUSIONS: Peptimapper is a complementary tool for the expert annotation of genomes. It is suitable for any organism and is distributed through a Docker image available on two public bioinformatics docker repositories: Docker Hub and BioShaDock. This workflow is also accessible through the Galaxy framework and for use by non-computer scientists at https://galaxy.protim.eu . Data are available via ProteomeXchange under identifier PXD010618.

Genome editing reveals reproductive and developmental dependencies on specific types of vitellogenin in zebrafish (Danio rerio).

Oviparous vertebrates produce multiple forms of vitellogenin (Vtg), the major source of yolk nutrients, but little is known about their individual contributions to reproduction and development. This study utilized clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing to assess essentiality and functionality of zebrafish (Danio rerio) type-I and type-III Vtgs. A multiple CRISPR approach was employed to knockout (KO) all genes encoding type-I vtgs (vtg1, 4, 5, 6, and 7) simultaneously (vtg1-KO), and the type-III vtg (vtg3) individually (vtg3-KO). Results of polymerase chain reaction (PCR) genotyping and sequencing, quantitative PCR, liquid chromatography-tandem mass spectrometry, and Western blot analysis showed that only vtg6 and vtg7 escaped Cas9 editing. In fish whose remaining type-I vtgs were incapacitated (vtg1-KO), and in vtg3-KO fish, significant increases in Vtg7 transcript and protein levels occurred in liver and eggs, revealing a heretofore-unknown mechanism of genetic compensation regulating Vtg homeostasis. Egg numbers per spawn were elevated more than 2-fold in vtg1-KO females, and egg fertility was approximately halved in vtg3-KO females. Substantial mortality was evident in vtg3-KO eggs/embryos after only 8 hr of incubation and in vtg1-KO embryos after 5 days. Hatching rate and timing were markedly impaired in embryos from vtg mutant mothers and pericardial and yolk sac/abdominal edema and spinal lordosis were evident in the larvae, with feeding and motor activities also being absent in vtg1-KO larvae. By late larval stages, vtg mutations were either completely lethal (vtg1-KO) or nearly so (vtg3-KO). These novel findings offer the first experimental evidence that different types of vertebrate Vtg are essential and have disparate requisite functions at different times during both reproduction and development.

Deciphering the Dark Proteome: Use of the Testis and Characterization of Two Dark Proteins.

For the C-HPP consortium, dark proteins include not only uPE1, but also missing proteins (MPs, PE2-4), smORFs, proteins from lncRNAs, and products from uncharacterized transcripts. Here, we investigated the expression of dark proteins in the human testis by combining public mRNA and protein expression data for several tissues and performing LC-MS/MS analysis of testis protein extracts. Most uncharacterized proteins are highly expressed in the testis. Thirty could be identified in our data set, of which two were selected for further analyses: (1) A0AOU1RQG5, a putative cancer/testis antigen specifically expressed in the testis, where it accumulates in the cytoplasm of elongated spermatids; and (2) PNMA6E, which is enriched in the testis, where it is found in the germ cell nuclei during most stages of spermatogenesis. Both proteins are coded on Chromosome X. Finally, we studied the expression of other dark proteins, uPE1 and MPs, in a series of human tissues. Most were highly expressed in the testis at both the mRNA and protein levels. The testis appears to be a relevant organ to study the dark proteome, which may have a function related to spermatogenesis and germ cell differentiation. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium under the data set identifier PXD009598.

Validating Missing Proteins in Human Sperm Cells by Targeted Mass-Spectrometry- and Antibody-based Methods.

The present study is a contribution to the "neXt50 challenge", a coordinated effort across C-HPP teams to identify the 50 most tractable missing proteins (MPs) on each chromosome. We report the targeted search of 38 theoretically detectable MPs from chromosomes 2 and 14 in Triton X-100 soluble and insoluble sperm fractions from a total of 15 healthy donors. A targeted mass-spectrometry-based strategy consisting of the development of LC-PRM assays (with heavy labeled synthetic peptides) targeting 92 proteotypic peptides of the 38 selected MPs was used. Out of the 38 selected MPs, 12 were identified with two or more peptides and 3 with one peptide after extensive SDS-PAGE fractionation of the two samples and with overall low-intensity signals. The PRM data are available via ProteomeXchange in PASSEL (PASS01013). Further validation by immunohistochemistry on human testes sections and cytochemistry on sperm smears was performed for eight MPs with antibodies available from the Human Protein Atlas. Deep analysis of human sperm still allows the validation of MPs and therefore contributes to the C-HPP worldwide effort. We anticipate that our results will be of interest to the reproductive biology community because an in-depth analysis of these MPs may identify potential new candidates in the context of human idiopathic infertilities.

Quantitative proteome profiling of dystrophic dog skeletal muscle reveals a stabilized muscular architecture and protection against oxidative stress after systemic delivery of MuStem cells.

Proteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell based therapy represents a promising approach for clinical applications to cure Duchenne muscular dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome 4 months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope-coded protein labeling was used to compile quantitative changes in the protein expression profiles of muscle in transplanted Golden Retriever muscular dystrophy (GRMD) dogs as compared to Golden Retriever muscular dystrophy dogs. A total of 492 proteins were quantified, including 25 that were overrepresented and 46 that were underrepresented after MuStem cell transplantation. Interestingly, this study demonstrates that somatic stem cell therapy impacts on the structural integrity of the muscle fascicle by acting on fibers and its connections with the extracellular matrix. We also show that cell infusion promotes protective mechanisms against oxidative stress and favors the initial phase of muscle repair. This study allows us to identify putative candidates for tissue markers that might be of great value in objectively exploring the clinical benefits resulting from our cell-based therapy for DMD. All MS data have been deposited in the ProteomeXchange with identifier PXD001768 (http://proteomecentral.proteomexchange.org/dataset/PXD001768).

Looking for Missing Proteins in the Proteome of Human Spermatozoa: An Update.

The Chromosome-Centric Human Proteome Project (C-HPP) aims to identify "missing" proteins in the neXtProt knowledgebase. We present an in-depth proteomics analysis of the human sperm proteome to identify testis-enriched missing proteins. Using protein extraction procedures and LC-MS/MS analysis, we detected 235 proteins (PE2-PE4) for which no previous evidence of protein expression was annotated. Through LC-MS/MS and LC-PRM analysis, data mining, and immunohistochemistry, we confirmed the expression of 206 missing proteins (PE2-PE4) in line with current HPP guidelines (version 2.0). Parallel reaction monitoring acquisition and sythetic heavy labeled peptides targeted 36 ≪one-hit wonder≫ candidates selected based on prior peptide spectrum match assessment. 24 were validated with additional predicted and specifically targeted peptides. Evidence was found for 16 more missing proteins using immunohistochemistry on human testis sections. The expression pattern for some of these proteins was specific to the testis, and they could possibly be valuable markers with fertility assessment applications. Strong evidence was also found of four "uncertain" proteins (PE5); their status should be re-examined. We show how using a range of sample preparation techniques combined with MS-based analysis, expert knowledge, and complementary antibody-based techniques can produce data of interest to the community. All MS/MS data are available via ProteomeXchange under identifier PXD003947. In addition to contributing to the C-HPP, we hope these data will stimulate continued exploration of the sperm proteome.