-
Experimental Neurology Sep 2022Extracellular vesicles (EVs) are small lipid bilayer particles ubiquitously released by almost every cell type. A specific and selective constituents of EVs loaded with... (Review)
Review
Extracellular vesicles (EVs) are small lipid bilayer particles ubiquitously released by almost every cell type. A specific and selective constituents of EVs loaded with variety of proteins, lipids, small noncoding RNAs, and long non-coding RNAs are reflective of cellular events, type, and physiologic/pathophysiologic status of the cell of origin. Moreover, these molecular contents carry information from the cell of origin to recipient cells, modulating intercellular communication. Recent studies demonstrated that EVs not only play a neuroprotective role by mediating the removal of toxic proteins, but also emerge as an important player in various neurodegenerative disease onset and progression through facilitating of misfolded proteins propagation. For this reason, neurodegenerative disease-associated differences in EV proteome relative to normal EVs can be used to fulfil diagnostic, prognostic, and therapeutic purposes. Nonetheless, characterizing EV proteome obtained from biological samples (brain tissue and body fluids, including urea, blood, saliva, and CSF) is a challenging task. Herein, we review the status of EV proteome profiling and the updated discovery of potential biomarkers for the diagnosis of neurodegenerative disease with an emphasis on the integration of high-throughput advanced mass spectrometry (MS) technologies for both qualitative and quantitative analysis of EVs in different clinical tissue/body fluid samples in past five years.
Topics: Extracellular Vesicles; Humans; Mass Spectrometry; Neurodegenerative Diseases; Proteome; Proteomics
PubMed: 35732219
DOI: 10.1016/j.expneurol.2022.114149 -
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 -
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 -
Proteomics. Clinical Applications Mar 2022The healthy human urine sediment proteome and metaproteome are investigated, to shed light on the variations of urine sediment proteins and metaproteins associated with...
PURPOSE
The healthy human urine sediment proteome and metaproteome are investigated, to shed light on the variations of urine sediment proteins and metaproteins associated with sex and age.
EXPERIMENTAL DESIGN
Urine sediment samples are collected from 19 healthy subjects. Protein identification and quantification are performed by liquid chromatography coupled high-resolution mass spectrometry.
RESULTS
A total of 2736 human proteins were identified, which were primarily associated with inflammatory response and energy metabolism. For the metaproteome, 65 genera were identified that were primarily involved in translation and carbohydrate metabolic processes. The median biological coefficient variation of the proteome/metaproteome of human urine sediment was 0.5/0.72, similar to the proteome of human urine supernatant. In addition, sex and age were observed to affect the proteome and metaproteome of healthy human urine sediment.
CONCLUSION AND CLINICAL RELEVANCE
The healthy human urine sediment were characterized, indicating that urine sediment might represent an alternative resource for disease research in addition to urine supernatant, but the influence of sex and age must be considered in the study design process.
Topics: Chromatography, Liquid; Humans; Mass Spectrometry; Proteome; Proteomics
PubMed: 34687263
DOI: 10.1002/prca.202100007 -
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 -
Methods in Molecular Biology (Clifton,... 2023Mass spectrometry-based proteomics provide a powerful tool for plant research, allowing global detection of steady-state levels of proteins under a given experimental...
Mass spectrometry-based proteomics provide a powerful tool for plant research, allowing global detection of steady-state levels of proteins under a given experimental setup. Here, we provide an optimized protocol for proteomic profiling using tandem mass tag (TMT) labeling followed by liquid chromatography-mass spectrometry (LC-MS/MS) to quantitate phosphopeptides and non-phosphopeptides from the same samples. The outlined protocol comprises a series of successive steps, namely, SDS (sodium dodecyl sulfate) protein extraction, protein precipitation, digestion, TMT labeling, phosphopeptide enrichment, high pH reversed-phase fractionation, LC-MS/MS analysis, protein identification, and data analysis. Our proteome-scale protocol requires 0.1 mg protein per sample and allows for the reliable and accurate quantification of more than 8000 proteins in Arabidopsis plant samples across multiple conditions, including low abundant peptides.
Topics: Chromatography, Liquid; Proteomics; Tandem Mass Spectrometry; Phosphopeptides; Proteome
PubMed: 36413327
DOI: 10.1007/978-1-0716-2784-6_22 -
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 -
Journal of Proteome Research May 2023
Topics: Proteome; Proteomics; Transcriptome
PubMed: 37143351
DOI: 10.1021/acs.jproteome.3c00219 -
Proteomics Oct 2022Mass spectrometry (MS) has emerged at the forefront of quantitative proteomic techniques. Liquid chromatography-mass spectrometry (LC-MS) can be used to determine... (Review)
Review
Mass spectrometry (MS) has emerged at the forefront of quantitative proteomic techniques. Liquid chromatography-mass spectrometry (LC-MS) can be used to determine abundances of proteins and peptides in complex biological samples. Several methods have been developed and adapted for accurate quantification based on chemical isotopic labeling. Among various chemical isotopic labeling techniques, isobaric tagging approaches rely on the analysis of peptides from MS2-based quantification rather than MS1-based quantification. In this review, we will provide an overview of several isobaric tags along with some recent developments including complementary ion tags, improvements in sensitive quantitation of analytes with lower abundance, strategies to increase multiplexing capabilities, and targeted analysis strategies. We will also discuss limitations of isobaric tags and approaches to alleviate these restrictions through bioinformatic tools and data acquisition methods. This review will highlight several applications of isobaric tags, including biomarker discovery and validation, thermal proteome profiling, cross-linking for structural investigations, single-cell analysis, top-down proteomics, along with applications to different molecules including neuropeptides, glycans, metabolites, and lipids, while providing considerations and evaluations to each application.
Topics: Proteomics; Proteome; Tandem Mass Spectrometry; Isotope Labeling; Peptides; Biomarkers; Lipids
PubMed: 35687565
DOI: 10.1002/pmic.202100256