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Current Opinion in Chemical Biology Oct 2023Activity-based protein profiling (ABPP) is a powerful chemical approach for probing protein function and enzymatic activity in complex biological systems. This strategy... (Review)
Review
Activity-based protein profiling (ABPP) is a powerful chemical approach for probing protein function and enzymatic activity in complex biological systems. This strategy typically utilizes activity-based probes that are designed to bind a specific protein, amino acid residue, or protein family and form a covalent bond through a reactivity-based warhead. Subsequent analysis by mass spectrometry-based proteomic platforms that involve either click chemistry or affinity-based labeling to enrich for the tagged proteins enables identification of protein function and enzymatic activity. ABPP has facilitated elucidation of biological processes in bacteria, discovery of new antibiotics, and characterization of host-microbe interactions within physiological contexts. This review will focus on recent advances and applications of ABPP in bacteria and complex microbial communities.
Topics: Proteome; Gastrointestinal Microbiome; Proteomics; Microbiota; Bacteria
PubMed: 37429085
DOI: 10.1016/j.cbpa.2023.102351 -
Cell Chemical Biology Jul 2023Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic...
Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxICAT, Biotin Switch, and SP3-Rox, these methods typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. Here we establish the local cysteine capture (Cys-LoC) and local cysteine oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole-cell proteomic analysis. Application of the Cys-LOx method to LPS-stimulated immortalized murine bone marrow-derived macrophages (iBMDM), revealed previously unidentified, mitochondrially localized cysteine oxidative modifications upon pro-inflammatory activation, including those associated with oxidative mitochondrial metabolism.
Topics: Animals; Mice; Cysteine; Proteomics; Mitochondria; Proteome; Oxidation-Reduction
PubMed: 37419112
DOI: 10.1016/j.chembiol.2023.06.008 -
Journal of Proteome Research Aug 2023Thermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target and off-target... (Comparative Study)
Comparative Study
Thermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target and off-target proteins, complementing phenotypic-based drug screens. Detecting differences in thermal stability due to target engagement requires high quantitative accuracy and consistent detection. Isobaric tandem mass tags (TMTs) are used to multiplex samples and increase quantification precision in TPP analysis by data-dependent acquisition (DDA). However, advances in data-independent acquisition (DIA) can provide higher sensitivity and protein coverage with reduced costs and sample preparation steps. Herein, we explored the performance of different DIA-based label-free quantification approaches compared to TMT-DDA for thermal shift quantitation. Acute myeloid leukemia cells were treated with losmapimod, a known inhibitor of MAPK14 (p38α). Label-free DIA approaches, and particularly the library-free mode in DIA-NN, were comparable of TMT-DDA in their ability to detect target engagement of losmapimod with MAPK14 and one of its downstream targets, MAPKAPK3. Using DIA for thermal shift quantitation is a cost-effective alternative to labeled quantitation in the TPP pipeline.
Topics: Mass Spectrometry; Mitogen-Activated Protein Kinase 14; Proteome; Proteomics
PubMed: 37439223
DOI: 10.1021/acs.jproteome.3c00111 -
Stem Cell Research & Therapy Jul 2023Mesenchymal stromal cells (MSCs) have been shown to exert their therapeutic effects through the secretion of broad spectrum of paracrine factors, including extracellular...
BACKGROUND
Mesenchymal stromal cells (MSCs) have been shown to exert their therapeutic effects through the secretion of broad spectrum of paracrine factors, including extracellular vesicles (EVs). Accordingly, EVs are being pursued as a promising alternative to cell-based therapies. Menstrual blood-derived stromal cells (MenSCs) are a type of MSC that, due to their immunomodulatory and regenerative properties, have emerged as an innovative source. Additionally, new strategies of cell priming may potentially alter the concentration and cargo of released EVs, leading to modification of their biological properties. In this study, we aimed to characterize the EVs released by MenSCs and compare their therapeutic potential under three different preconditioning conditions (proinflammatory stimuli, physioxia, and acute hypoxia).
METHODS
MenSCs were isolated from five healthy women. Following culturing to 80% confluence, MenSCs were exposed to different priming conditions: basal (21% O), proinflammatory stimuli (IFNγ and TNFα, 21% O), physioxia (1-2% O), and acute hypoxia (< 1% O) for 48-72 h. Conditioned media from MenSCs was collected after 48 h and EVs were isolated by a combination of ultra-filtration and differential centrifugation. An extensive characterization ranging from nano-flow cytometry (nFC) to quantitative high-throughput shotgun proteomics was performed. Bioinformatics analyses were used to derive hypotheses on their biological properties.
RESULTS
No differences in the morphology, size, or number of EVs released were detected between priming conditions. The proteome analysis associated with basal MenSC-EVs prominently revealed their immunomodulatory and regenerative capabilities. Furthermore, quantitative proteomic analysis of differentially produced MenSC-EVs provided sufficient evidence for the utility of the differential preconditioning in purpose-tailoring EVs for their therapeutic application: proinflammatory priming enhanced the anti-inflammatory, regenerative and immunomodulatory capacity in the innate response of EVs, physioxia priming also improves tissue regeneration, angiogenesis and their immunomodulatory capacity targeting on the adaptive response, while acute hypoxia priming, increased hemostasis and apoptotic processes regulation in MenSC-EVs, also by stimulating immunomodulation mainly through the adaptive response.
CONCLUSIONS
Priming of MenSCs under proinflammatory and hypoxic conditions affected the cargo proteome of EVs released, resulting in different therapeutic potential, and thus warrants experimental exploration with the aim to generate better-defined MSC-derived bioproducts.
Topics: Humans; Female; Proteomics; Proteome; Extracellular Vesicles; Mesenchymal Stem Cells; Hypoxia
PubMed: 37507751
DOI: 10.1186/s13287-023-03413-5 -
Platelets Dec 2023Research into the natural aging process of platelets has garnered much research interest in recent years, and there have long been associations drawn between the... (Review)
Review
Research into the natural aging process of platelets has garnered much research interest in recent years, and there have long been associations drawn between the proportion of newly formed platelets in the circulation and the risk of thrombosis. However, these observations have largely been demonstrated in patient groups in which there may be underlying systemic changes that effect platelet function. Recent advances in technology have allowed in-depth analysis of differently aged platelets isolated from the peripheral blood of healthy individuals and have demonstrated that aged platelets, often referred to as senescent platelets, undergo extensive changes in the transcriptome and proteome. Ultimately, these changes result in platelets whose functions have deteriorated such that they cannot partake in hemostatic responses to the same extent as newly formed platelets. Here, we review transcriptomic and proteomic research in platelet aging in the context of health and how this research sheds light upon alterations in platelet structure and function.
Topics: Humans; Aged; Transcriptome; Proteome; Proteomics; Blood Platelets; Aging
PubMed: 37070955
DOI: 10.1080/09537104.2023.2200838 -
Proteomics Jan 2024Increased throughput in proteomic experiments can improve accessibility of proteomic platforms, reduce costs, and facilitate new approaches in systems biology and...
Increased throughput in proteomic experiments can improve accessibility of proteomic platforms, reduce costs, and facilitate new approaches in systems biology and biomedical research. Here we propose combination of analytical flow rate chromatography with ion mobility separation of peptide ions, data-independent acquisition, and data analysis with the DIA-NN software suite, to achieve high-quality proteomic experiments from limited sample amounts, at a throughput of up to 400 samples per day. For instance, when benchmarking our workflow using a 500-μL/min flow rate and 3-min chromatographic gradients, we report the quantification of 5211 proteins from 2 μg of a mammalian cell-line standard at high quantitative accuracy and precision. We further used this platform to analyze blood plasma samples from a cohort of COVID-19 inpatients, using a 3-min chromatographic gradient and alternating column regeneration on a dual pump system. The method delivered a comprehensive view of the COVID-19 plasma proteome, allowing classification of the patients according to disease severity and revealing plasma biomarker candidates.
Topics: Animals; Humans; Proteomics; Peptides; Proteome; Chromatography, Liquid; COVID-19; Mammals
PubMed: 37287406
DOI: 10.1002/pmic.202300100 -
Bioorganic Chemistry Feb 2024Protein trafficking is a fundamental process with profound implications for both intracellular and intercellular functions. Proximity labeling (PL) technology has... (Review)
Review
Protein trafficking is a fundamental process with profound implications for both intracellular and intercellular functions. Proximity labeling (PL) technology has emerged as a powerful tool for capturing precise snapshots of subcellular proteomes by directing promiscuous enzymes to specific cellular locations. These enzymes generate reactive species that tag endogenous proteins, enabling their identification through mass spectrometry-based proteomics. In this comprehensive review, we delve into recent advancements in PL-based methodologies, placing particular emphasis on the label-and-fractionation approach and TransitID, for mapping proteome trafficking. These methodologies not only facilitate the exploration of dynamic intracellular protein trafficking between organelles but also illuminate the intricate web of intercellular and inter-organ protein communications.
Topics: Proteomics; Organelles; Mass Spectrometry; Proteome; Protein Transport
PubMed: 38134520
DOI: 10.1016/j.bioorg.2023.107041 -
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 -
Aging Cell Jun 2024An early diagnosis of Alzheimer's disease is crucial as treatment efficacy is limited to the early stages. However, the current diagnostic methods are limited to mid or...
An early diagnosis of Alzheimer's disease is crucial as treatment efficacy is limited to the early stages. However, the current diagnostic methods are limited to mid or later stages of disease development owing to the limitations of clinical examinations and amyloid plaque imaging. Therefore, this study aimed to identify molecular signatures including blood plasma extracellular vesicle biomarker proteins associated with Alzheimer's disease to aid early-stage diagnosis. The hippocampus, cortex, and blood plasma extracellular vesicles of 3- and 6-month-old 5xFAD mice were analyzed using quantitative proteomics. Subsequent bioinformatics and biochemical analyses were performed to compare the molecular signatures between wild type and 5xFAD mice across different brain regions and age groups to elucidate disease pathology. There was a unique signature of significantly altered proteins in the hippocampal and cortical proteomes of 3- and 6-month-old mice. The plasma extracellular vesicle proteomes exhibited distinct informatic features compared with the other proteomes. Furthermore, the regulation of several canonical pathways (including phosphatidylinositol 3-kinase/protein kinase B signaling) differed between the hippocampus and cortex. Twelve potential biomarkers for the detection of early-stage Alzheimer's disease were identified and validated using plasma extracellular vesicles from stage-divided patients. Finally, integrin α-IIb, creatine kinase M-type, filamin C, glutamine γ-glutamyltransferase 2, and lysosomal α-mannosidase were selected as distinguishing biomarkers for healthy individuals and early-stage Alzheimer's disease patients using machine learning modeling with approximately 79% accuracy. Our study identified novel early-stage molecular signatures associated with the progression of Alzheimer's disease, thereby providing novel insights into its pathogenesis.
Topics: Animals; Alzheimer Disease; Mice; Proteomics; Mice, Transgenic; Biomarkers; Humans; Disease Models, Animal; Proteome; Male
PubMed: 38436501
DOI: 10.1111/acel.14137 -
The Journal of Investigative Dermatology Aug 2023Hand eczema (HE) is a prevalent skin disease. However, the classification of HE into different subtypes remains challenging. A limited number of previous studies have...
Hand eczema (HE) is a prevalent skin disease. However, the classification of HE into different subtypes remains challenging. A limited number of previous studies have employed invasive biopsy-based strategies; yet, studies of the HE proteome using noninvasive tape-stripping methodology have not been reported. In this study, we wanted to assess whether global proteomic analysis of skin tape strip samples can be used for subclassification of patients with HE. Tape strips were collected from patients with HE and healthy skin. Liquid chromatography-mass spectrometry proteomics was performed, and the global protein expression was analyzed. We identified 2,919 proteins in stratum corneum-derived skin cells from tape strip samples. Compared with healthy skin, the lesional samples from patients with HE exhibited increased expression of immune-related markers and a decreased expression of structural barrier proteins. The difference between HE subtypes was restricted to the lesional skin areas and included an increased expression of skin barrier-related proteins independently of the concurrent AD. In conclusion, we found that the noninvasive tape strip method used in combination with liquid chromatography-mass spectrometry proteomics can be used for analysis of skin protein expression in patients with HE. Thus, the method shows potential for assessing the proteomic differences between subtypes of HE and biomarker discovery.
Topics: Humans; Proteome; Proteomics; Skin; Epidermis; Eczema; Biomarkers
PubMed: 36773646
DOI: 10.1016/j.jid.2022.12.024