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Proteomics Jul 2023Redox post-translational modifications on cysteine thiols (redox PTMs) have profound effects on protein structure and function, thus enabling regulation of various... (Review)
Review
Redox post-translational modifications on cysteine thiols (redox PTMs) have profound effects on protein structure and function, thus enabling regulation of various biological processes. Redox proteomics approaches aim to characterize the landscape of redox PTMs at the systems level. These approaches facilitate studies of condition-specific, dynamic processes implicating redox PTMs and have furthered our understanding of redox signaling and regulation. Mass spectrometry (MS) is a powerful tool for such analyses which has been demonstrated by significant advances in redox proteomics during the last decade. A group of well-established approaches involves the initial blocking of free thiols followed by selective reduction of oxidized PTMs and subsequent enrichment for downstream detection. Alternatively, novel chemoselective probe-based approaches have been developed for various redox PTMs. Direct detection of redox PTMs without any enrichment has also been demonstrated given the sensitivity of contemporary MS instruments. This review discusses the general principles behind different analytical strategies and covers recent advances in redox proteomics. Several applications of redox proteomics are also highlighted to illustrate how large-scale redox proteomics data can lead to novel biological insights.
Topics: Sulfhydryl Compounds; Proteomics; Protein Processing, Post-Translational; Proteins; Oxidation-Reduction; Proteome
PubMed: 37248656
DOI: 10.1002/pmic.202200194 -
The Analyst Jul 2023Although single cell RNA-seq has had a tremendous impact on biological research, a corresponding technology for unbiased mass spectrometric analysis of single cells has...
Although single cell RNA-seq has had a tremendous impact on biological research, a corresponding technology for unbiased mass spectrometric analysis of single cells has only recently become available. Significant technological breakthroughs including miniaturized sample handling have enabled proteome profiling of single cells. Furthermore, trapped ion mobility spectrometry (TIMS) in combination with parallel accumulation-serial fragmentation operated in data-dependent acquisition mode (DDA-PASEF) allowed improved proteome coverage from low-input samples. It has been demonstrated that modulating the ion flux in TIMS affects the overall performance of proteome profiling. However, the effect of TIMS settings on the analysis of low-input samples has been less investigated. Thus, we sought to optimize the conditions of TIMS with regard to ion accumulation/ramp times and ion mobility range for low-input samples. We observed that an ion accumulation time of 180 ms and monitoring a narrower ion mobility range from 0.7 to 1.3 V s cm resulted in a substantial gain in the depth of proteome coverage and in detecting proteins with low abundance. We used these optimized conditions for proteome profiling of sorted human primary T cells, which yielded an average of 365, 804, 1116, and 1651 proteins from single, five, ten, and forty T cells, respectively. Notably, we demonstrated that the depth of proteome coverage from a low number of cells was sufficient to delineate several essential metabolic pathways and the T cell receptor signaling pathway. Finally, we showed the feasibility of detecting post-translational modifications including phosphorylation and acetylation from single cells. We believe that such an approach could be applied to label-free analysis of single cells obtained from clinically relevant samples.
Topics: Humans; Proteome; Proteomics; Ion Mobility Spectrometry; Mass Spectrometry; Protein Processing, Post-Translational
PubMed: 37395315
DOI: 10.1039/d3an00080j -
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 -
Expert Review of Proteomics 2023Fertility rates in developing countries have declined over the past decades, and the trend of delayed fatherhood is rising as societies develop. The reasons behind the... (Review)
Review
INTRODUCTION
Fertility rates in developing countries have declined over the past decades, and the trend of delayed fatherhood is rising as societies develop. The reasons behind the decline in male fertility with advancing age remain mysterious, making it a compelling and crucial area for further research. However, the limited number of studies dedicated to unraveling this enigma poses a challenge. Thus, our objective is to illuminate some of the upregulated and downregulated mechanisms in the male testis during the aging process.
AREAS COVERED
Herein, we present a critical overview of the studies addressing the alterations of testicular proteome through the aging process, starting from sexually matured young males to end-of-life-expectancy aged males. The comparative studies of the proteomic testicular profile of men with and without spermatogenic impairment are also discussed and key proteins and pathways involved are highlighted.
EXPERT OPINION
The difficulty of making age-comparative studies, especially of advanced-age study subjects, makes this topic of study quite challenging. Another topic worth mentioning is the heterogeneous nature and vast cellular composition of testicular tissue, which makes proteome data interpretation tricky. The cell type sorting and comorbidities testing in the testicular tissue of the studied subjects would help mitigate these problems.
Topics: Male; Humans; Aged; Testis; Proteome; Proteomics; Spermatogenesis; Infertility, Male
PubMed: 37878493
DOI: 10.1080/14789450.2023.2274857 -
Analytical and Bioanalytical Chemistry Apr 2024Success of mass spectrometry characterization of the proteome of single cells allows us to gain a greater understanding than afforded by transcriptomics alone but... (Review)
Review
Success of mass spectrometry characterization of the proteome of single cells allows us to gain a greater understanding than afforded by transcriptomics alone but requires clear understanding of the tradeoffs between analytical throughput and precision. Recent advances in mass spectrometry acquisition techniques, including updated instrumentation and sample preparation, have improved the quality of peptide signals obtained from single cell data. However, much of the proteome remains uncharacterized, and higher throughput techniques often come at the expense of reduced sensitivity and coverage, which diminish the ability to measure proteoform heterogeneity, including splice variants and post-translational modifications, in single cell data analysis. Here, we assess the growing body of ultrasensitive single-cell approaches and their tradeoffs as researchers try to balance throughput and precision in their experiments.
Topics: Proteome; Proteomics; Peptides; Mass Spectrometry; Protein Processing, Post-Translational
PubMed: 38358530
DOI: 10.1007/s00216-024-05171-6 -
Mass Spectrometry Reviews 2023The Human Plasma Proteome has always been the most investigated compartment in proteomics-based biomarker discovery, and is considered the largest and deepest version of... (Review)
Review
The Human Plasma Proteome has always been the most investigated compartment in proteomics-based biomarker discovery, and is considered the largest and deepest version of the human proteome, reflecting the state of the body in health and disease. Even if efforts have been always dedicated to the refinement of proteomic approaches to investigate more deeply the plasma proteome, it should not be forgotten that also highly abundant plasma proteins, like human serum albumin (HSA), often neglected in these studies, might provide fundamental physiological functions in plasma, and should be better considered. This review summarizes the important roles of HSA in the context of cardiovascular diseases (CVD), and in particular in heart failure. Notwithstanding much attention has been historically directed toward the association of HSA levels and CVD risk, the advances in the field of mass spectrometry research allow also a better characterization of the effects of oxidative modifications that could alter not only the structure but also the function of HSA.
Topics: Humans; Albumins; Cardiovascular Diseases; Heart Failure; Proteome; Proteomics
PubMed: 34747521
DOI: 10.1002/mas.21743 -
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 -
Expert Review of Proteomics 2023Positional proteomics provides proteome-wide information on protein termini and their modifications, uniquely enabling unambiguous identification of site-specific,...
INTRODUCTION
Positional proteomics provides proteome-wide information on protein termini and their modifications, uniquely enabling unambiguous identification of site-specific, limited proteolysis. Such proteolytic cleavage irreversibly modifies protein sequences resulting in new proteoforms with distinct protease-generated neo-N and C-termini and altered localization and activity. Misregulated proteolysis is implicated in a wide variety of human diseases. Protein termini, therefore, constitute a huge, largely unexplored source of specific analytes that provides a deep view into the functional proteome and a treasure trove for biomarkers.
AREAS COVERED
We briefly review principal approaches to define protein termini and discuss recent advances in method development. We further highlight the potential of positional proteomics to identify and trace specific proteoforms, with a focus on proteolytic processes altered in disease. Lastly, we discuss current challenges and potential for applying positional proteomics in biomarker and pre-clinical research.
EXPERT OPINION
Recent developments in positional proteomics have provided significant advances in sensitivity and throughput. In-depth analysis of proteolytic processes in clinical cohorts thus appears feasible in the near future. We argue that this will provide insights into the functional state of the proteome and offer new opportunities to utilize proteolytic processes altered or targeted in disease as specific diagnostic, prognostic and companion biomarkers.
Topics: Humans; Protein Processing, Post-Translational; Proteome; Proteomics; Proteolysis; Peptide Hydrolases; Biomarkers
PubMed: 37869791
DOI: 10.1080/14789450.2023.2272046 -
Phytomedicine : International Journal... Aug 2023Natural products are an important source for discovering novel drugs due to their various pharmacological activities. Salvia miltiorrhiza Burge (Danshen) has been shown...
BACKGROUND
Natural products are an important source for discovering novel drugs due to their various pharmacological activities. Salvia miltiorrhiza Burge (Danshen) has been shown to have promising therapeutic potential in the management of heart diseases, making it a candidate for cardiovascular drug discovery. Currently, there is limited quantitative analysis of the phosphorylation levels of Danshen-derived natural products on a proteome-wide, which may bias the study of their mechanisms of action.
PURPOSE
This study aimed to evaluate the global signaling perturbation induced by Danshen-derived bioactive compounds and their potential relationship with myocardial ischemia/reperfusion (IR) injury therapy.
STUDY DESIGN
We employed quantitative proteome and phosphoproteome analysis to identify dysregulated signaling in IR injury hearts from mice. We compared changes induced by Danshen-derived compounds based on IR-associated phospho-events, using an integrative approach that maps relative abundance of proteins and phosphorylation sites.
METHODS
Isobaric chemical tandem mass tags (TMT) labeled multiplexing strategy was used to generate unbiased quantitative proteomics and phosphoproteomics data. Highly accurate and precise TMT quantitation was performed using the Orbitrap Fusion Tribrid Mass Spectrometer with synchronous precursor selection MS3 detection mode. Mass spectrometric raw files were analyzed with MaxQuant (2.0.1.0) and statistical and bioinformatics analysis was conducted with Perseus (1.6.15).
RESULTS
We quantified 3661 proteins and over 11,000 phosphosites in impaired heart tissue of the IR mice model, expanding our knowledge of signaling pathways and other biological processes disrupted in IR injury. Next, 1548 and 5545 differently expressed proteins and phosphosites were identified by quantifying the proteome and phosphoproteome of H9c2 cells treated by five Danshen bioactive compounds respectively. Results revealed the vast differences in abilities of five Danshen-derived bioactive compounds to regulate phosphorylation modifications in cardiomyocytes, with dihydrotanshinone I (DHT) showing potential for protecting against IR injury by modulating the AMPK/mTOR signaling pathway.
CONCLUSIONS
This study provides a new strategy for analyzing drug/natural product-regulated phosphorylation modification levels on a proteome-wide scale, leading to a better understanding of cell signaling pathways and downstream phenotypic responses.
Topics: Mice; Animals; Salvia miltiorrhiza; Proteome; Proteomics; Phosphorylation; Myocytes, Cardiac
PubMed: 37307738
DOI: 10.1016/j.phymed.2023.154897 -
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