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Briefings in Bioinformatics Jan 2022Large-scale phosphoproteome profiling using mass spectrometry (MS) provides functional insight that is crucial for disease biology and drug discovery. However,...
Large-scale phosphoproteome profiling using mass spectrometry (MS) provides functional insight that is crucial for disease biology and drug discovery. However, extracting biological understanding from these data is an arduous task requiring multiple analysis platforms that are not adapted for automated high-dimensional data analysis. Here, we introduce an integrated pipeline that combines several R packages to extract high-level biological understanding from large-scale phosphoproteomic data by seamless integration with existing databases and knowledge resources. In a single run, PhosPiR provides data clean-up, fast data overview, multiple statistical testing, differential expression analysis, phosphosite annotation and translation across species, multilevel enrichment analyses, proteome-wide kinase activity and substrate mapping and network hub analysis. Data output includes graphical formats such as heatmap, box-, volcano- and circos-plots. This resource is designed to assist proteome-wide data mining of pathophysiological mechanism without a need for programming knowledge.
Topics: Data Mining; Mass Spectrometry; Phosphoproteins; Phosphorylation; Proteome; Proteomics; Software
PubMed: 34882763
DOI: 10.1093/bib/bbab510 -
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 -
The EMBO Journal Dec 2023Substantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high-resolution imaging as well as high-content...
Substantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high-resolution imaging as well as high-content molecular profiling technologies. The current work adds to these approaches by providing a comprehensive and quantitative protein expression map of 13 anatomically distinct brain regions covering more than 11,000 proteins. This was enabled by the optimization, characterization, and implementation of a high-sensitivity and high-throughput microflow liquid chromatography timsTOF tandem mass spectrometry system (LC-MS/MS) capable of analyzing more than 2,000 consecutive samples prepared from formalin-fixed paraffin embedded (FFPE) material. Analysis of this proteomic resource highlighted brain region-enriched protein expression patterns and functional protein classes, protein localization differences between brain regions and individual markers for specific areas. To facilitate access to and ease further mining of the data by the scientific community, all data can be explored online in a purpose-built R Shiny app (https://brain-region-atlas.proteomics.ls.tum.de).
Topics: Humans; Chromatography, Liquid; Proteomics; Paraffin Embedding; Tandem Mass Spectrometry; Proteins; Brain; Proteome
PubMed: 37916885
DOI: 10.15252/embj.2023114665 -
Nature Methods May 2023Major aims of single-cell proteomics include increasing the consistency, sensitivity and depth of protein quantification, especially for proteins and modifications of...
Major aims of single-cell proteomics include increasing the consistency, sensitivity and depth of protein quantification, especially for proteins and modifications of biological interest. Here, to simultaneously advance all these aims, we developed prioritized Single-Cell ProtEomics (pSCoPE). pSCoPE consistently analyzes thousands of prioritized peptides across all single cells (thus increasing data completeness) while maximizing instrument time spent analyzing identifiable peptides, thus increasing proteome depth. These strategies increased the sensitivity, data completeness and proteome coverage over twofold. The gains enabled quantifying protein variation in untreated and lipopolysaccharide-treated primary macrophages. Within each condition, proteins covaried within functional sets, including phagosome maturation and proton transport, similarly across both treatment conditions. This covariation is coupled to phenotypic variability in endocytic activity. pSCoPE also enabled quantifying proteolytic products, suggesting a gradient of cathepsin activities within a treatment condition. pSCoPE is freely available and widely applicable, especially for analyzing proteins of interest without sacrificing proteome coverage. Support for pSCoPE is available at http://scp.slavovlab.net/pSCoPE .
Topics: Proteome; Proteomics; Mass Spectrometry; Peptides; Macrophages
PubMed: 37012480
DOI: 10.1038/s41592-023-01830-1 -
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 -
Journal of Agricultural and Food... Aug 2022Barley is one of the key cereal grains for malting and brewing industries. However, climate variability and unprecedented weather events can impact barley yield and...
Barley is one of the key cereal grains for malting and brewing industries. However, climate variability and unprecedented weather events can impact barley yield and end-product quality. The genetic background and environmental conditions are key factors in defining the barley proteome content and malting characteristics. Here, we measure the barley proteome and malting characteristics of three barley lines grown in Western Australia, differing in genetic background and growing location, by applying liquid chromatography-mass spectrometry (LC-MS). Using data-dependent acquisition LC-MS, 1571 proteins were detected with high confidence. Quantitative data acquired using sequential window acquisition of all theoretical (SWATH) MS on barley samples resulted in quantitation of 920 proteins. Multivariate analyses revealed that the barley lines' genetics and their growing locations are strongly correlated between proteins and desired traits such as the malt yield. Linking meteorological data with proteomic measurements revealed how high-temperature stress in northern regions affects seed temperature tolerance during malting, resulting in a higher malt yield. Our results show the impact of environmental conditions on the barley proteome and malt characteristics; these findings have the potential to expedite breeding programs and malt quality prediction.
Topics: Hordeum; Phenotype; Plant Breeding; Proteome; Proteomics
PubMed: 35981222
DOI: 10.1021/acs.jafc.2c03816 -
Molecular & Cellular Proteomics : MCP Sep 2023Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population of incompletely differentiated immune cells. They are known to suppress T cell activity and...
Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population of incompletely differentiated immune cells. They are known to suppress T cell activity and are implicated in multiple chronic diseases, which make them an attractive cell population for drug discovery. Here, we characterized the baseline proteomes and phospho-proteomes of mouse MDSC differentiated from a progenitor cell line to a depth of 7000 proteins and phosphorylation sites. We also validated the cellular system for drug discovery by recapitulating and identifying known and novel molecular responses to the well-studied MDSC drugs entinostat and mocetinostat. We established a high-throughput drug screening platform using a MDSC/T cell coculture system and assessed the effects of ∼21,000 small molecule compounds on T cell proliferation and IFN-γ secretion to identify novel MDSC modulator. The most promising candidates were validated in a human MDSC system, and subsequent proteomic experiments showed significant upregulation of several proteins associated with the reduction of reactive oxygen species (ROS). Proteome-wide solvent-induced protein stability assays identified Acyp1 and Cd74 as potential targets, and the ROS-reducing drug phenotype was validated by measuring ROS levels in cells in response to compound, suggesting a potential mode of action. We anticipate that the data and chemical tools developed in this study will be valuable for further research on MDSC and related drug discovery.
Topics: Mice; Humans; Animals; Myeloid-Derived Suppressor Cells; High-Throughput Screening Assays; Proteome; Proteomics; Reactive Oxygen Species
PubMed: 37586548
DOI: 10.1016/j.mcpro.2023.100632 -
Journal of the American Society For... Oct 2023Bacteria are orders of magnitude smaller than mammalian cells, and while single cell proteomics (SCP) currently detects and quantifies several thousands of proteins per...
Bacteria are orders of magnitude smaller than mammalian cells, and while single cell proteomics (SCP) currently detects and quantifies several thousands of proteins per mammalian cell, it is not clear whether conventional SCP methods will be suitable for bacteria. Here we report on the first successful attempt to detect proteins from individual bacteria, with validation of our findings by comparison with two bacteria samples and bulk proteomics data. Data are available via ProteomeXchange with the identifier PXD043473.
Topics: Bacteria; Escherichia coli; Proteome; Proteomics
PubMed: 37713396
DOI: 10.1021/jasms.3c00242 -
Emerging Topics in Life Sciences May 2021Plants rapidly respond to environmental fluctuations through coordinated, multi-scalar regulation, enabling complex reactions despite their inherently sessile nature. In... (Review)
Review
Plants rapidly respond to environmental fluctuations through coordinated, multi-scalar regulation, enabling complex reactions despite their inherently sessile nature. In particular, protein post-translational signaling and protein-protein interactions combine to manipulate cellular responses and regulate plant homeostasis with precise temporal and spatial control. Understanding these proteomic networks are essential to addressing ongoing global crises, including those of food security, rising global temperatures, and the need for renewable materials and fuels. Technological advances in mass spectrometry-based proteomics are enabling investigations of unprecedented depth, and are increasingly being optimized for and applied to plant systems. This review highlights recent advances in plant proteomics, with an emphasis on spatially and temporally resolved analysis of post-translational modifications and protein interactions. It also details the necessity for generation of a comprehensive plant cell atlas while highlighting recent accomplishments within the field.
Topics: Mass Spectrometry; Plants; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 33620075
DOI: 10.1042/ETLS20200270 -
International Journal of Molecular... Feb 2020Proteomics is a large-scale study of proteins, aiming at the description and characterization of all expressed proteins in biological systems. The expressed proteins are... (Review)
Review
Proteomics is a large-scale study of proteins, aiming at the description and characterization of all expressed proteins in biological systems. The expressed proteins are typically highly complex and large in abundance range. To fulfill high accuracy and sensitivity of proteome analysis, the hybrid platforms of multidimensional (MD) separations and mass spectrometry have provided the most powerful solution. Multidimensional separations provide enhanced peak capacity and reduce sample complexity, which enables mass spectrometry to analyze more proteins with high sensitivity. Although two-dimensional (2D) separations have been widely used since the early period of proteomics, three-dimensional (3D) separation was barely used by low reproducibility of separation, increased analysis time in mass spectrometry. With developments of novel microscale techniques such as nano-UPLC and improvements of mass spectrometry, the 3D separation becomes a reliable and practical selection. This review summarizes existing offline and online 3D-LC platforms developed for proteomics and their applications. In detail, setups and implementation of those systems as well as their advances are outlined. The performance of those platforms is also discussed and compared with the state-of-the-art 2D-LC. In addition, we provide some perspectives on the future developments and applications of 3D-LC in proteomics.
Topics: Animals; Chromatography, Liquid; Humans; Liver; Mass Spectrometry; Online Systems; Proteome; Proteomics; Reproducibility of Results
PubMed: 32102244
DOI: 10.3390/ijms21041524