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Journal of Proteome Research Mar 2023Accurate protein quantification is key to identifying protein markers, regulatory relationships between proteins, and pathophysiological mechanisms. Realizing this... (Review)
Review
Accurate protein quantification is key to identifying protein markers, regulatory relationships between proteins, and pathophysiological mechanisms. Realizing this potential requires sensitive and deep protein analysis of a large number of samples. Toward this goal, proteomics throughput can be increased by parallelizing the analysis of both precursors and samples using multiplexed data independent acquisition (DIA) implemented by the plexDIA framework: https://plexDIA.slavovlab.net. Here we demonstrate the improved precisions of retention time estimates within plexDIA and how this enables more accurate protein quantification. plexDIA has demonstrated multiplicative gains in throughput, and these gains may be substantially amplified by improving the multiplexing reagents, data acquisition, and interpretation. We discuss future directions for advancing plexDIA, which include engineering optimized mass-tags for high-plexDIA, introducing isotopologous carriers, and developing algorithms that utilize the regular structures of plexDIA data to improve sensitivity, proteome coverage, and quantitative accuracy. These advances in plexDIA will increase the throughput of functional proteomic assays, including quantifying protein conformations, turnover dynamics, modifications states and activities. The sensitivity of these assays will extend to single-cell analysis, thus enabling functional single-cell protein analysis.
Topics: Mass Spectrometry; Proteomics; Algorithms; Proteome
PubMed: 36735898
DOI: 10.1021/acs.jproteome.2c00721 -
Current Opinion in Neurobiology Apr 2023The highly heterogeneous nature of neuronal cell types and their connections presents a major challenge to the characterization of neural circuits at the protein level.... (Review)
Review
The highly heterogeneous nature of neuronal cell types and their connections presents a major challenge to the characterization of neural circuits at the protein level. New approaches now enable an increasingly sophisticated dissection of cell type- and cellular compartment-specific proteomes, as well as the profiling of the protein composition of specific synaptic connections. Here, we provide an overview of these approaches and discuss how they hold considerable promise toward unravelling the molecular mechanisms of neural circuit formation and function. Finally, we provide an outlook of technological developments that may bring the characterization of synaptic proteomes at the single-synapse level within reach.
Topics: Proteome; Proteomics; Synapses; Neurons; Neural Pathways
PubMed: 36805717
DOI: 10.1016/j.conb.2023.102690 -
Cell Reports Methods Oct 2023Here, we present a standardized, "off-the-shelf" proteomics pipeline working in a single 96-well plate to achieve deep coverage of cellular proteomes with high...
Here, we present a standardized, "off-the-shelf" proteomics pipeline working in a single 96-well plate to achieve deep coverage of cellular proteomes with high throughput and scalability. This integrated pipeline streamlines a fully automated sample preparation platform, a data-independent acquisition (DIA) coupled with high-field asymmetric waveform ion mobility spectrometer (FAIMS) interface, and an optimized library-free DIA database search strategy. Our systematic evaluation of FAIMS-DIA showing single compensation voltage (CV) at -35 V not only yields the deepest proteome coverage but also best correlates with DIA without FAIMS. Our in-depth comparison of direct-DIA database search engines shows that Spectronaut outperforms others, providing the highest quantifiable proteins. Next, we apply three common DIA strategies in characterizing human induced pluripotent stem cell (iPSC)-derived neurons and show single-shot mass spectrometry (MS) using single-CV (-35 V)-FAIMS-DIA results in >9,000 quantifiable proteins with <10% missing values, as well as superior reproducibility and accuracy compared with other existing DIA methods.
Topics: Humans; Proteomics; Tandem Mass Spectrometry; Reproducibility of Results; Induced Pluripotent Stem Cells; Proteome
PubMed: 37729920
DOI: 10.1016/j.crmeth.2023.100593 -
Journal of Proteome Research Jan 2021Protein -acylation (commonly known as palmitoylation) is a widespread reversible lipid modification, which plays critical roles in regulating protein localization,... (Review)
Review
Protein -acylation (commonly known as palmitoylation) is a widespread reversible lipid modification, which plays critical roles in regulating protein localization, activity, stability, and complex formation. The deregulation of protein -acylation contributes to many diseases such as cancer and neurodegenerative disorders. The past decade has witnessed substantial progress in proteomic analysis of protein -acylation, which significantly advanced our understanding of -acylation biology. In this review, we summarized the techniques for the enrichment of -acylated proteins or peptides, critically reviewed proteomic studies of protein -acylation at eight different levels, and proposed major challenges for the -acylproteomics field. In summary, proteome-scale analysis of protein -acylation comes of age and will play increasingly important roles in discovering new disease mechanisms, biomarkers, and therapeutic targets.
Topics: Acylation; Lipoylation; Protein S; Proteome; Proteomics
PubMed: 33253586
DOI: 10.1021/acs.jproteome.0c00409 -
Molecular & Cellular Proteomics : MCP Nov 2019The most straightforward applications of proteomics database searching involve intracellular proteins. Although intracellular gene products number in the thousands,... (Review)
Review
The most straightforward applications of proteomics database searching involve intracellular proteins. Although intracellular gene products number in the thousands, their well-defined post-translational modifications (PTMs) makes database searching practical. By contrast, cell surface and extracellular matrisome proteins pass through the secretory pathway where many become glycosylated, modulating their physicochemical properties, adhesive interactions, and diversifying their functions. Although matrisome proteins number only a few hundred, their high degree of complex glycosylation multiplies the number of theoretical proteoforms by orders of magnitude. Given that extracellular networks that mediate cell-cell and cell-pathogen interactions in physiology depend on glycosylation, it is important to characterize the proteomes, glycomes, and glycoproteomes of matrisome molecules that exist in a given biological context. In this review, we summarize proteomics approaches for characterizing matrisome molecules, with an emphasis on applications to brain diseases. We demonstrate the availability of methods that should greatly increase the availability of information on matrisome molecular structure associated with health and disease.
Topics: Animals; Biomarkers; Extracellular Matrix; Extracellular Matrix Proteins; Glycomics; Glycoproteins; Glycosylation; Humans; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 31471497
DOI: 10.1074/mcp.R119.001543 -
Mass Spectrometry Reviews Nov 2019Progress in proteomics research has led to a demand for powerful analytical tools with high separation efficiency and sensitivity for confident identification and... (Review)
Review
Progress in proteomics research has led to a demand for powerful analytical tools with high separation efficiency and sensitivity for confident identification and quantification of proteins, posttranslational modifications, and protein complexes expressed in cells and tissues. This demand has significantly increased interest in capillary electrophoresis-mass spectrometry (CE-MS) in the past few years. This review provides highlights of recent advances in CE-MS for proteomics research, including a short introduction to top-down mass spectrometry and native mass spectrometry (native MS), as well as a detailed overview of CE methods. Both the potential and limitations of these methods for the analysis of proteins and peptides in synthetic and biological samples and the challenges of CE methods are discussed, along with perspectives about the future direction of CE-MS. @ 2019 Wiley Periodicals, Inc. Mass Spec Rev 00:1-16, 2019.
Topics: Animals; Electrophoresis, Capillary; Humans; Mass Spectrometry; Proteome; Proteomics; Spectrometry, Mass, Electrospray Ionization
PubMed: 31407381
DOI: 10.1002/mas.21599 -
Analytical Chemistry Jul 2022Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample...
Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10,000 cells using the in-solution digestion method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.
Topics: Digestion; Peptides; Proteome; Proteomics; Water
PubMed: 35817413
DOI: 10.1021/acs.analchem.1c05487 -
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 -
Biochemical and Biophysical Research... Oct 2022The balance between the actions of protein kinases and phosphatases is crucial for neuronal functions, including synaptic plasticity. Although the phosphorylation and...
The balance between the actions of protein kinases and phosphatases is crucial for neuronal functions, including synaptic plasticity. Although the phosphorylation and dephosphorylation of neuronal proteins are regulated by synaptic plasticity, no systematic analyses of this have yet been conducted. We performed a phosphoproteomic analysis of hippocampal synaptic plasticity using a nano-Acquity/Synapt LC-MS/MS system. Neuronal proteins were extracted from hippocampal tissues and cultured neurons exposed to long-term potentiation (LTP) or long-term depression (LTD). Filter-aided sample preparation (FASP) was performed to remove residual anionic detergents for complete tryptic digestion. Phosphopeptides were then enriched using TiO chromatography, followed by immunoaffinity chromatography with an anti-phosphotyrosine antibody. Among the 1500 phosphopeptides identified by LC-MS/MS, 374 phosphopeptides were detected simultaneously in both hippocampal tissues and cultured neurons. Semi-quantification counting the number of spectra of each phosphopeptide showed that 42 of 374 phosphopeptides changed significantly depending on synaptic plasticity. In conclusion, a new proteomic method using sequential enrichment of phosphopeptides and semi-quantification enabled the phosphoproteomic analysis of hippocampal synaptic plasticity.
Topics: Chromatography, Liquid; Hippocampus; Long-Term Synaptic Depression; Neuronal Plasticity; Phosphopeptides; Proteome; Proteomics; Tandem Mass Spectrometry
PubMed: 35981422
DOI: 10.1016/j.bbrc.2022.07.051 -
Biomolecules Nov 2023Mitochondria are ancient endosymbiotic double membrane organelles that support a wide range of eukaryotic cell functions through energy, metabolism, and cellular... (Review)
Review
Mitochondria are ancient endosymbiotic double membrane organelles that support a wide range of eukaryotic cell functions through energy, metabolism, and cellular control. There are over 1000 known proteins that either reside within the mitochondria or are transiently associated with it. These mitochondrial proteins represent a functional subcellular protein network (mtProteome) that is encoded by mitochondrial and nuclear genomes and significantly varies between cell types and conditions. In neurons, the high metabolic demand and differential energy requirements at the synapses are met by specific modifications to the mtProteome, resulting in alterations in the expression and functional properties of the proteins involved in energy production and quality control, including fission and fusion. The composition of mtProteomes also impacts the localization of mitochondria in axons and dendrites with a growing number of neurodegenerative diseases associated with changes in mitochondrial proteins. This review summarizes the findings on the composition and properties of mtProteomes important for mitochondrial energy production, calcium and lipid signaling, and quality control in neural cells. We highlight strategies in mass spectrometry (MS) proteomic analysis of mtProteomes from cultured cells and tissue. The research into mtProteome composition and function provides opportunities in biomarker discovery and drug development for the treatment of metabolic and neurodegenerative disease.
Topics: Humans; Proteome; Neurodegenerative Diseases; Proteomics; Mitochondria; Neurons; Mitochondrial Proteins
PubMed: 38002320
DOI: 10.3390/biom13111638