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Journal of Proteome Research Dec 2023Top-down proteomics (TDP) aims to identify and profile intact protein forms (proteoforms) extracted from biological samples. True proteoform characterization requires... (Review)
Review
Top-down proteomics (TDP) aims to identify and profile intact protein forms (proteoforms) extracted from biological samples. True proteoform characterization requires that both the base protein sequence be defined and any mass shifts identified, ideally localizing their positions within the protein sequence. Being able to fully elucidate proteoform profiles lends insight into characterizing proteoform-unique roles, and is a crucial aspect of defining protein structure-function relationships and the specific roles of different (combinations of) protein modifications. However, defining and pinpointing protein post-translational modifications (PTMs) on intact proteins remains a challenge. Characterization of (heavily) modified proteins (>∼30 kDa) remains problematic, especially when they exist in a population of similarly modified, or kindred, proteoforms. This issue is compounded as the number of modifications increases, and thus the number of theoretical combinations. Here, we present our perspective on the challenges of analyzing kindred proteoform populations, focusing on annotation of protein modifications on an "average" protein. Furthermore, we discuss the technical requirements to obtain high quality fragmentation spectral data to robustly define site-specific PTMs, and the fact that this is tempered by the time requirements necessary to separate proteoforms in advance of mass spectrometry analysis.
Topics: Tandem Mass Spectrometry; Proteomics; Proteins; Protein Processing, Post-Translational; Amino Acid Sequence; Proteome
PubMed: 37937372
DOI: 10.1021/acs.jproteome.3c00416 -
Journal of Proteome Research May 2024
Topics: Proteomics; Single-Cell Analysis; Humans; Proteome; Protein Isoforms
PubMed: 38698756
DOI: 10.1021/acs.jproteome.4c00290 -
Nature Communications Sep 2023COVID-19 is characterised by systemic immunological perturbations in the human body, which can lead to multi-organ damage. Many of these processes are considered to be... (Meta-Analysis)
Meta-Analysis
COVID-19 is characterised by systemic immunological perturbations in the human body, which can lead to multi-organ damage. Many of these processes are considered to be mediated by the blood. Therefore, to better understand the systemic host response to SARS-CoV-2 infection, we performed systematic analyses of the circulating, soluble proteins in the blood through global proteomics by mass-spectrometry (MS) proteomics. Here, we show that a large part of the soluble blood proteome is altered in COVID-19, among them elevated levels of interferon-induced and proteasomal proteins. Some proteins that have alternating levels in human cells after a SARS-CoV-2 infection in vitro and in different organs of COVID-19 patients are deregulated in the blood, suggesting shared infection-related changes.The availability of different public proteomic resources on soluble blood proteome alterations leaves uncertainty about the change of a given protein during COVID-19. Hence, we performed a systematic review and meta-analysis of MS global proteomics studies of soluble blood proteomes, including up to 1706 individuals (1039 COVID-19 patients), to provide concluding estimates for the alteration of 1517 soluble blood proteins in COVID-19. Finally, based on the meta-analysis we developed CoViMAPP, an open-access resource for effect sizes of alterations and diagnostic potential of soluble blood proteins in COVID-19, which is publicly available for the research, clinical, and academic community.
Topics: Humans; COVID-19; Proteome; Proteomics; SARS-CoV-2; Cytoplasm
PubMed: 37739942
DOI: 10.1038/s41467-023-41159-z -
Protein and Peptide Letters 2024Human blood is a window of physiology and disease. Examination of biomarkers in blood is a common clinical procedure, which can be informative in diagnosis and prognosis... (Review)
Review
Human blood is a window of physiology and disease. Examination of biomarkers in blood is a common clinical procedure, which can be informative in diagnosis and prognosis of diseases, and in evaluating treatment effectiveness. There is still a huge demand on new blood biomarkers and assays for precision medicine nowadays, therefore plasma/serum proteomics has attracted increasing attention in recent years. How to effectively proceed with the biomarker discovery and clinical diagnostic assay development is a question raised to researchers who are interested in this area. In this review, we comprehensively introduce the background and advancement of technologies for blood proteomics, with a focus on mass spectrometry (MS). Analyzing existing blood biomarkers and newly-built diagnostic assays based on MS can shed light on developing new biomarkers and analytical methods. We summarize various protein analytes in plasma/serum which include total proteome, protein post-translational modifications, and extracellular vesicles, focusing on their corresponding sample preparation methods for MS analysis. We propose screening multiple protein analytes in the same set of blood samples in order to increase success rate for biomarker discovery. We also review the trends of MS techniques for blood tests including sample preparation automation, and further provide our perspectives on their future directions.
Topics: Humans; Proteomics; Biomarkers; Mass Spectrometry; Blood Proteins; Proteome; Protein Processing, Post-Translational; Extracellular Vesicles; Plasma
PubMed: 38869039
DOI: 10.2174/0109298665286952240212053723 -
International Journal of Molecular... Feb 2024The liver is the central metabolic organ and produces 85-90% of the proteins found in plasma. Accordingly, the plasma proteome is an attractive source of liver disease... (Review)
Review
The liver is the central metabolic organ and produces 85-90% of the proteins found in plasma. Accordingly, the plasma proteome is an attractive source of liver disease biomarkers that reflects the different cell types present in this organ, as well as the processes such as responses to acute and chronic injury or the formation of an extracellular matrix. In the first part, we summarize the biomarkers routinely used in clinical evaluations and their biological relevance in the different stages of non-malignant liver disease. Later, we describe the current proteomic approaches, including mass spectrometry and affinity-based techniques, that allow a more comprehensive assessment of the liver function but also require complex data processing. The many approaches of analysis and interpretation and their potential caveats are delineated. While these advances hold the promise to transform our understanding of liver diseases and support the development and validation of new liver-related drugs, an interdisciplinary collaboration is needed.
Topics: Humans; Proteome; Proteomics; Biomarkers; Liver Diseases
PubMed: 38396688
DOI: 10.3390/ijms25042008 -
Journal of Proteome Research Jun 2024We introduce single cell Proteoform imaging Mass Spectrometry (), which realizes the benefit of direct solvent extraction and MS detection of intact proteins from single...
We introduce single cell Proteoform imaging Mass Spectrometry (), which realizes the benefit of direct solvent extraction and MS detection of intact proteins from single cells dropcast onto glass slides. Sampling and detection of whole proteoforms by individual ion mass spectrometry enable a scalable approach to single cell proteomics. This new platform addresses the throughput bottleneck in single cell proteomics and boosts the cell processing rate by several fold while accessing protein composition with higher coverage.
Topics: Single-Cell Analysis; Proteomics; Humans; Mass Spectrometry; Proteome
PubMed: 38497708
DOI: 10.1021/acs.jproteome.4c00075 -
Cells Nov 2023Voluntary striated muscles are characterized by a highly complex and dynamic proteome that efficiently adapts to changed physiological demands or alters considerably... (Review)
Review
Voluntary striated muscles are characterized by a highly complex and dynamic proteome that efficiently adapts to changed physiological demands or alters considerably during pathophysiological dysfunction. The skeletal muscle proteome has been extensively studied in relation to myogenesis, fiber type specification, muscle transitions, the effects of physical exercise, disuse atrophy, neuromuscular disorders, muscle co-morbidities and sarcopenia of old age. Since muscle tissue accounts for approximately 40% of body mass in humans, alterations in the skeletal muscle proteome have considerable influence on whole-body physiology. This review outlines the main bioanalytical avenues taken in the proteomic characterization of skeletal muscle tissues, including top-down proteomics focusing on the characterization of intact proteoforms and their post-translational modifications, bottom-up proteomics, which is a peptide-centric method concerned with the large-scale detection of proteins in complex mixtures, and subproteomics that examines the protein composition of distinct subcellular fractions. Mass spectrometric studies over the last two decades have decisively improved our general cell biological understanding of protein diversity and the heterogeneous composition of individual myofibers in skeletal muscles. This detailed proteomic knowledge can now be integrated with findings from other omics-type methodologies to establish a systems biological view of skeletal muscle function.
Topics: Humans; Proteome; Proteomics; Muscle Fibers, Skeletal; Muscle, Skeletal; Mass Spectrometry
PubMed: 37947638
DOI: 10.3390/cells12212560 -
Molecular & Cellular Proteomics : MCP Aug 2023Protein aggregation of amyloid-β peptides and tau are pathological hallmarks of Alzheimer's disease (AD), which are often resistant to detergent extraction and thus... (Meta-Analysis)
Meta-Analysis
Protein aggregation of amyloid-β peptides and tau are pathological hallmarks of Alzheimer's disease (AD), which are often resistant to detergent extraction and thus enriched in the insoluble proteome. However, additional proteins that coaccumulate in the detergent-insoluble AD brain proteome remain understudied. Here, we comprehensively characterized key proteins and pathways in the detergent-insoluble proteome from human AD brain samples using differential extraction, tandem mass tag (TMT) labeling, and two-dimensional LC-tandem mass spectrometry. To improve quantification accuracy of the TMT method, we developed a complement TMT-based strategy to correct for ratio compression. Through the meta-analysis of two independent detergent-insoluble AD proteome datasets (8914 and 8917 proteins), we identified 190 differentially expressed proteins in AD compared with control brains, highlighting the pathways of amyloid cascade, RNA splicing, endocytosis/exocytosis, protein degradation, and synaptic activity. To differentiate the truly detergent-insoluble proteins from copurified background during protein extraction, we analyzed the fold of enrichment for each protein by comparing the detergent-insoluble proteome with the whole proteome from the same AD samples. Among the 190 differentially expressed proteins, 84 (51%) proteins of the upregulated proteins (n = 165) were enriched in the insoluble proteome, whereas all downregulated proteins (n = 25) were not enriched, indicating that they were copurified components. The vast majority of these enriched 84 proteins harbor low-complexity regions in their sequences, including amyloid-β, Tau, TARDBP/TAR DNA-binding protein 43, SNRNP70/U1-70K, MDK, PTN, NTN1, NTN3, and SMOC1. Moreover, many of the enriched proteins in AD were validated in the detergent-insoluble proteome by five steps of differential extraction, proteomic analysis, or immunoblotting. Our study reveals a resource list of proteins and pathways that are exclusively present in the detergent-insoluble proteome, providing novel molecular insights to the formation of protein pathology in AD.
Topics: Humans; Alzheimer Disease; Proteome; Detergents; Proteomics; Tandem Mass Spectrometry; Brain; Ribonucleoprotein, U1 Small Nuclear
PubMed: 37356496
DOI: 10.1016/j.mcpro.2023.100608 -
Journal of Proteome Research Apr 2024Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches....
Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches. Thus, multienzyme strategies are used to maximize sequence coverage in post-translational modification profiling. We present fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin. All three enzymes are remarkably fast, producing near-complete digests in 1-5 min, and cost 200-1000× less than proteomics-grade trypsin. Using FragPipe resolved a major challenge by drastically reducing the duration of the required "unspecific" searches. In-depth analyses of proteinase K, subtilisin, and thermolysin Jurkat digests identified 7374, 8178, and 8753 unique proteins with average sequence coverages of 21, 29, and 37%, including 10,000s of amino acids not reported in PeptideAtlas' >2400 experiments. While we could not identify distinct cleavage patterns, machine learning could distinguish true protease products from random cleavages, potentially enabling the prediction of cleavage products. Finally, proteinase K, subtilisin, and thermolysin enabled label-free quantitation of 3111, 3659, and 4196 unique Jurkat proteins, which in our hands is comparable to trypsin. Our data demonstrate that broad-specificity proteases enable quantitative proteomics of uncharted areas of the proteome. Their fast kinetics may allow "on-the-fly" digestion of samples in the future.
Topics: Peptide Hydrolases; Proteomics; Trypsin; Proteome; Endopeptidase K; Thermolysin; Subtilisins
PubMed: 38457694
DOI: 10.1021/acs.jproteome.3c00852 -
Genome Biology Sep 2023Quantitative proteomics is an indispensable tool in life science research. However, there is a lack of reference materials for evaluating the reproducibility of...
BACKGROUND
Quantitative proteomics is an indispensable tool in life science research. However, there is a lack of reference materials for evaluating the reproducibility of label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based measurements among different instruments and laboratories.
RESULTS
Here, we develop the Quartet standard as a proteome reference material with built-in truths, and distribute the same aliquots to 15 laboratories with nine conventional LC-MS/MS platforms across six cities in China. Relative abundance of over 12,000 proteins on 816 mass spectrometry files are obtained and compared for reproducibility among the instruments and laboratories to ultimately generate proteomics benchmark datasets. There is a wide dynamic range of proteomes spanning about 7 orders of magnitude, and the injection order has marked effects on quantitative instead of qualitative characteristics.
CONCLUSION
Overall, the Quartet offers valuable standard materials and data resources for improving the quality control of proteomic analyses as well as the reproducibility and reliability of research findings.
Topics: Chromatography, Liquid; Proteomics; Reproducibility of Results; Tandem Mass Spectrometry; Proteome
PubMed: 37674236
DOI: 10.1186/s13059-023-03048-y