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Frontiers in Immunology 2023Proteomics is the characterization of the protein composition, the proteome, of a biological sample. It involves the large-scale identification and quantification of... (Review)
Review
Proteomics is the characterization of the protein composition, the proteome, of a biological sample. It involves the large-scale identification and quantification of proteins, peptides, and post-translational modifications. This review focuses on recent developments in mass spectrometry-based proteomics and provides an overview of available methods for sample preparation to study the innate immune system. Recent advancements in the proteomics workflows, including sample preparation, have significantly improved the sensitivity and proteome coverage of biological samples including the technically difficult blood plasma. Proteomics is often applied in immunology and has been used to characterize the levels of innate immune system components after perturbations such as birth or during chronic inflammatory diseases like rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). In cancers, the tumor microenvironment may generate chronic inflammation and release cytokines to the circulation. In these situations, the innate immune system undergoes profound and long-lasting changes, the large-scale characterization of which may increase our biological understanding and help identify components with translational potential for guiding diagnosis and treatment decisions. With the ongoing technical development, proteomics will likely continue to provide increasing insights into complex biological processes and their implications for health and disease. Integrating proteomics with other omics data and utilizing multi-omics approaches have been demonstrated to give additional valuable insights into biological systems.
Topics: Humans; Proteome; Proteomics; Peptides; Inflammation; Immune System
PubMed: 37868984
DOI: 10.3389/fimmu.2023.1254948 -
Nucleic Acids Research Jan 2024Advancements in mass spectrometry (MS)-based proteomics have greatly facilitated the large-scale quantification of proteins and microproteins, thereby revealing altered...
Advancements in mass spectrometry (MS)-based proteomics have greatly facilitated the large-scale quantification of proteins and microproteins, thereby revealing altered signalling pathways across many different cancer types. However, specialized and comprehensive resources are lacking for cancer proteomics. Here, we describe CancerProteome (http://bio-bigdata.hrbmu.edu.cn/CancerProteome), which functionally deciphers and visualizes the proteome landscape in cancer. We manually curated and re-analyzed publicly available MS-based quantification and post-translational modification (PTM) proteomes, including 7406 samples from 21 different cancer types, and also examined protein abundances and PTM levels in 31 120 proteins and 4111 microproteins. Six major analytical modules were developed with a view to describe protein contributions to carcinogenesis using proteome analysis, including conventional analyses of quantitative and the PTM proteome, functional enrichment, protein-protein associations by integrating known interactions with co-expression signatures, drug sensitivity and clinical relevance analyses. Moreover, protein abundances, which correlated with corresponding transcript or PTM levels, were evaluated. CancerProteome is convenient as it allows users to access specific proteins/microproteins of interest using quick searches or query options to generate multiple visualization results. In summary, CancerProteome is an important resource, which functionally deciphers the cancer proteome landscape and provides a novel insight for the identification of tumor protein markers in cancer.
Topics: Humans; Mass Spectrometry; Neoplasms; Protein Processing, Post-Translational; Proteome; Proteomics; Databases, Protein
PubMed: 37823596
DOI: 10.1093/nar/gkad824 -
Nature Communications Nov 2023Cellular activities are commonly associated with dynamic proteomic changes at the subcellular level. Although several techniques are available to quantify whole-cell...
Cellular activities are commonly associated with dynamic proteomic changes at the subcellular level. Although several techniques are available to quantify whole-cell protein turnover dynamics, such measurements often lack sufficient spatial resolution at the subcellular level. Herein, we report the development of prox-SILAC method that combines proximity-dependent protein labeling (APEX2/HRP) with metabolic incorporation of stable isotopes (pulse-SILAC) to map newly synthesized proteins with subcellular spatial resolution. We apply prox-SILAC to investigate proteome dynamics in the mitochondrial matrix and the endoplasmic reticulum (ER) lumen. Our analysis reveals a highly heterogeneous distribution in protein turnover dynamics within macromolecular machineries such as the mitochondrial ribosome and respiratory complexes I-V, thus shedding light on their mechanism of hierarchical assembly. Furthermore, we investigate the dynamic changes of ER proteome when cells are challenged with stress or undergoing stimulated differentiation, identifying subsets of proteins with unique patterns of turnover dynamics, which may play key regulatory roles in alleviating stress or promoting differentiation. We envision that prox-SILAC could be broadly applied to profile protein turnover at various subcellular compartments, under both physiological and pathological conditions.
Topics: Proteome; Proteomics; Mitochondria; Mitochondrial Ribosomes; Endoplasmic Reticulum
PubMed: 37940635
DOI: 10.1038/s41467-023-42861-8 -
American Journal of Physiology. Lung... Oct 2023Bronchopulmonary dysplasia (BPD) is a disease of prematurity related to the arrest of normal lung development. The objective of this study was to better understand how...
Bronchopulmonary dysplasia (BPD) is a disease of prematurity related to the arrest of normal lung development. The objective of this study was to better understand how proteome modulation and cell-type shifts are noted in BPD pathology. Pediatric human donors aged 1-3 yr were classified based on history of prematurity and histopathology consistent with "healed" BPD (hBPD, = 3) and "established" BPD (eBPD, = 3) compared with respective full-term born ( = 6) age-matched term controls. Proteins were quantified by tandem mass spectroscopy with selected Western blot validations. Multiplexed immunofluorescence (MxIF) microscopy was performed on lung sections to enumerate cell types. Protein abundances and MxIF cell frequencies were compared among groups using ANOVA. Cell type and ontology enrichment were performed using an in-house tool and/or EnrichR. Proteomics detected 5,746 unique proteins, 186 upregulated and 534 downregulated, in eBPD versus control with fewer proteins differentially abundant in hBPD as compared with age-matched term controls. Cell-type enrichment suggested a loss of alveolar type I, alveolar type II, endothelial/capillary, and lymphatics, and an increase in smooth muscle and fibroblasts consistent with MxIF. Histochemistry and Western analysis also supported predictions of upregulated ferroptosis in eBPD versus control. Finally, several extracellular matrix components mapping to angiogenesis signaling pathways were altered in eBPD. Despite clear parsing by protein abundance, comparative MxIF analysis confirms phenotypic variability in BPD. This work provides the first demonstration of tandem mass spectrometry and multiplexed molecular analysis of human lung tissue for critical elucidation of BPD trajectory-defining factors into early childhood. We provide new insights into the natural history of bronchopulmonary dysplasia in donor human lungs after the neonatal intensive care unit hospitalization. This study provides new insights into how the proteome and histopathology of BPD changes in early childhood, uncovering novel pathways for future study.
Topics: Child, Preschool; Infant, Newborn; Humans; Child; Bronchopulmonary Dysplasia; Immunohistochemistry; Proteome; Proteomics; Lung
PubMed: 37489262
DOI: 10.1152/ajplung.00130.2023 -
Journal of Lipid Research Sep 2023Lysosomal acid lipase (LAL) is the sole lysosomal enzyme responsible for the degradation of cholesteryl esters and triacylglycerols at acidic pH. Impaired LAL activity...
Lysosomal acid lipase (LAL) is the sole lysosomal enzyme responsible for the degradation of cholesteryl esters and triacylglycerols at acidic pH. Impaired LAL activity leads to LAL deficiency (LAL-D), a severe and fatal disease characterized by ectopic lysosomal lipid accumulation. Reduced LAL activity also contributes to the development and progression of non-alcoholic fatty liver disease (NAFLD). To advance our understanding of LAL-related liver pathologies, we performed comprehensive proteomic profiling of livers from mice with systemic genetic loss of LAL (Lal-/-) and from mice with hepatocyte-specific LAL-D (hepLal-/-). Lal-/- mice exhibited drastic proteome alterations, including dysregulation of multiple proteins related to metabolism, inflammation, liver fibrosis, and cancer. Global loss of LAL activity impaired both acidic and neutral lipase activities and resulted in hepatic lipid accumulation, indicating a complete metabolic shift in Lal-/- livers. Hepatic inflammation and immune cell infiltration were evident, with numerous upregulated inflammation-related gene ontology biological process terms. In contrast, both young and mature hepLal-/- mice displayed only minor changes in the liver proteome, suggesting that loss of LAL solely in hepatocytes does not phenocopy metabolic alterations observed in mice globally lacking LAL. These findings provide valuable insights into the mechanisms underlying liver dysfunction in LAL-D and may help in understanding why decreased LAL activity contributes to NAFLD. Our study highlights the importance of LAL in maintaining liver homeostasis and demonstrates the drastic consequences of its global deficiency on the liver proteome and liver function.
Topics: Mice; Animals; Sterol Esterase; Non-alcoholic Fatty Liver Disease; Proteome; Proteomics; Liver; Wolman Disease; Liver Cirrhosis; Triglycerides; Inflammation; Neoplasms
PubMed: 37595802
DOI: 10.1016/j.jlr.2023.100427 -
Genomics Jan 2024Primary Sjögren's syndrome (pSS) is a intricate autoimmune disease mainly characterized of immune-mediated destruction of exocrine tissues, such as salivary and...
OBJECTIVE
Primary Sjögren's syndrome (pSS) is a intricate autoimmune disease mainly characterized of immune-mediated destruction of exocrine tissues, such as salivary and lacrimal glands, occurring dry mouth and eyes. Although some breakthroughs in understanding pSS have been uncovered, many questions remain about its pathogenesis, especially the internal relations between exocrine glands and secretions.
METHOD
Transcriptomic and proteomic analyses were conducted on salivary tissues and saliva in experimental Sjögren syndrome (ESS). The ESS model was established by immunization with salivary gland protein. The expression of mRNAs and proteins in salivary tissues and saliva were determined by high-throughput sequencing transcriptomic analysis and LC-MS/MS-based proteome, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to recognize dysregulated genes and proteins. The association between RNA and protein abundance was investigated to provides a comprehensive understanding of RNA-protein correlations in the pathogenesis of pSS.
RESULTS
As a result, we successfully established the ESS model. We recognized 3221 differentially expressed genes (DEGs) and 253 differentially expressed proteins (DEPs). The sample analysis showed that 61 proteins overlapped through the integrative analysis of transcriptomics and proteomics data. The enrichment pathway analysis of DEGs and DEPs in samples showed alterations in renin-angiotensin-system (RAS), lysosome, and apoptosis. Notably, we found that some genes, such as AGT, FN1, Klk1b26, Klk1, Klk1b5, Klk1b3 had a consistent trend in the regulation at the RNA and protein levels and might be potential diagnostic biomarkers of pSS.
CONCLUSION
Herein, we found critical processes and potential biomakers that may contribute to pSS pathogenesis by analyzing dysregulated genes and pathways. Additionally, the integrative multi-omics datasets provided additional insight into understanding complicated disease mechanisms.
Topics: Humans; Sjogren's Syndrome; Transcriptome; Proteome; Chromatography, Liquid; Proteomics; Tandem Mass Spectrometry; RNA
PubMed: 38128705
DOI: 10.1016/j.ygeno.2023.110767 -
Microbial Physiology 2024The denitrifying betaproteobacterium Aromatoleum aromaticum EbN1T is a facultative anaerobic degradation specialist and belongs to the environmental bacteria studied... (Review)
Review
The denitrifying betaproteobacterium Aromatoleum aromaticum EbN1T is a facultative anaerobic degradation specialist and belongs to the environmental bacteria studied best on the proteogenomic level. This review summarizes the current state of knowledge about the anaerobic and aerobic degradation (to CO2) of 47 organic growth substrates (23 aromatic, 21 aliphatic, and 3 amino acids) as well as the modes of respiratory energy conservation (denitrification vs. O2-respiration). The constructed catabolic network is comprised of 256 genes, which occupy ∼7.5% of the coding regions of the genome. In total, 219 encoded proteins have been identified by differential proteomics, yielding a proteome coverage of ∼74% of the network. Its degradation section is composed of 31 peripheral and 4 central pathways, with several peripheral modules (e.g., for 4-ethylphenol, 2-phenylethylamine, indoleacetate, and phenylpropanoids) discovered only after the complete genome [Arch Microbiol. 2005 Jan;183(1):27-36] and a first proteomic survey [Proteomics. 2007 Jun;7(13):2222-39] of A. aromaticum EbN1T were reported. The activation of recalcitrant aromatic compounds involves a suite of biochemically intriguing reactions ranging from C-H-bond activation (e.g., ethylbenzene dehydrogenase) via carboxylation (e.g., acetophenone carboxylase) to oxidative deamination (e.g., benzylamine), reductive dearomatization (benzoyl-CoA), and epoxide-forming oxygenases (e.g., phenylacetyl-CoA). The peripheral reaction sequences are substrate-specifically induced, mediated by specific transcriptional regulators with in vivo response thresholds in the nanomolar range. While lipophilic substrates (e.g., phenolics) enter the cells via passive diffusion, polar ones require active uptake that is driven by specific transporters. Next to the protein repertoire for canonical complexes I-III, denitrification, and O2-respiration (low- and high-affinity oxidases), the genome encodes an Ndh-II, a tetrathionate reductase, two ETF:quinone oxidoreductases, and two Rnf-type complexes, broadening the electron transfer flexibility of the strain. Taken together, the detailed catabolic network presented here forms a solid basis for future systems biology-level studies with A. aromaticum EbN1T.
Topics: Bacterial Proteins; Anaerobiosis; Metabolic Networks and Pathways; Aerobiosis; Proteome; Proteomics; Denitrification; Rhodocyclaceae
PubMed: 37816339
DOI: 10.1159/000534425 -
Frontiers in Endocrinology 2023There are no validated clinical or laboratory biomarkers to identify and differentiate endotypes of type 1 diabetes (T1D) or the risk of progression to chronic...
INTRODUCTION
There are no validated clinical or laboratory biomarkers to identify and differentiate endotypes of type 1 diabetes (T1D) or the risk of progression to chronic complications. Extracellular vesicles (EVs) have been studied as biomarkers in several different disease states but have not been well studied in T1D.
METHODS
As the initial step towards circulating biomarker identification in T1D, this pilot study aimed to provide an initial characterization of the proteomic and phosphoproteomic landscape of circulating EV-enriched preparations in participants with established T1D (N=10) and healthy normal volunteers (Controls) (N=7) (NCT03379792) carefully matched by age, race/ethnicity, sex, and BMI. EV-enriched preparations were obtained using EVtrap technology. Proteins were identified and quantified by LC-MS analysis. Differential abundance and coexpression network (WGCNA), and pathway enrichment analyses were implemented.
RESULTS
The detected proteins and phosphoproteins were enriched (75%) in exosomal proteins cataloged in the ExoCarta database. A total of 181 proteins and 8 phosphoproteins were differentially abundant in participants with T1D compared to controls, including some well-known EVproteins (i.e., CD63, RAB14, BSG, LAMP2, and EZR). Enrichment analyses of differentially abundant proteins and phosphoproteins of EV-enriched preparations identified associations with neutrophil, platelet, and immune response functions, as well as prion protein aggregation. Downregulated proteins were involved in MHC class II signaling and the regulation of monocyte differentiation. Potential key roles in T1D for C1q, plasminogen, IL6ST, CD40, HLA-DQB1, HLA-DRB1, CD74, NUCB1, and SAP, are highlighted. Remarkably, WGCNA uncovered two protein modules significantly associated with pancreas size, which may be implicated in the pathogenesis of T1D. Similarly, these modules showed significant enrichment for membrane compartments, processes associated with inflammation and the immune response, and regulation of viral processes, among others.
DISCUSSION
This study demonstrates the potential of proteomic and phosphoproteomic signatures of EV-enriched preparations to provide insight into the pathobiology of T1D. The WGCNA analysis could be a powerful tool to discriminate signatures associated with different pathobiological components of the disease.
Topics: Humans; Diabetes Mellitus, Type 1; Proteome; Proteomics; Pilot Projects; Biomarkers; Phosphoproteins; Extracellular Vesicles
PubMed: 37576973
DOI: 10.3389/fendo.2023.1219293 -
Science Advances Feb 2024Characterizing the tumor microenvironment at the molecular level is essential for understanding the mechanisms of tumorigenesis and evolution. However, the specificity...
Characterizing the tumor microenvironment at the molecular level is essential for understanding the mechanisms of tumorigenesis and evolution. However, the specificity of the blood proteome in localized region of the tumor and its linkages with other systems is difficult to investigate. Here, we propose a spatially multidimensional comparative proteomics strategy using glioma as an example. The blood proteome signature of tumor microenvironment was specifically identified by in situ collection of arterial and venous blood from the glioma region of the brain for comparison with peripheral blood. Also, by integrating with different dimensions of tissue and peripheral blood proteomics, the information on the genesis, migration, and exchange of glioma-associated proteins was revealed, which provided a powerful method for tumor mechanism research and biomarker discovery. The study recruited multidimensional clinical cohorts, allowing the proteomic results to corroborate each other, reliably revealing biological processes specific to gliomas, and identifying highly accurate biomarkers.
Topics: Humans; Proteomics; Brain Neoplasms; Proteome; Glioma; Biomarkers; Tumor Microenvironment
PubMed: 38363834
DOI: 10.1126/sciadv.adk1721 -
Nature Communications Dec 2023Modified vaccinia Ankara (MVA) virus does not replicate in human cells and is the vaccine deployed to curb the current outbreak of mpox. Here, we conduct a multiplexed...
Modified vaccinia Ankara (MVA) virus does not replicate in human cells and is the vaccine deployed to curb the current outbreak of mpox. Here, we conduct a multiplexed proteomic analysis to quantify >9000 cellular and ~80% of viral proteins throughout MVA infection of human fibroblasts and macrophages. >690 human proteins are down-regulated >2-fold by MVA, revealing a substantial remodelling of the host proteome. >25% of these MVA targets are not shared with replication-competent vaccinia. Viral intermediate/late gene expression is necessary for MVA antagonism of innate immunity, and suppression of interferon effectors such as ISG20 potentiates virus gene expression. Proteomic changes specific to infection of macrophages indicate modulation of the inflammatory response, including inflammasome activation. Our approach thus provides a global view of the impact of MVA on the human proteome and identifies mechanisms that may underpin its abortive infection. These discoveries will prove vital to design future generations of vaccines.
Topics: Humans; Vaccinia; Proteome; Proteomics; Vaccinia virus; Cell Death; Antiviral Agents
PubMed: 38065956
DOI: 10.1038/s41467-023-43299-8