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Plant Physiology May 2021Protein cysteine residues are susceptible to oxidative modifications that can affect protein functions. Proteomic techniques that comprehensively profile the cysteine... (Review)
Review
Protein cysteine residues are susceptible to oxidative modifications that can affect protein functions. Proteomic techniques that comprehensively profile the cysteine redoxome, the repertoire of oxidized cysteine residues, are pivotal towards a better understanding of the protein redox signaling. Recent technical advances in chemical tools and redox proteomic strategies have greatly improved selectivity, in vivo applicability, and quantification of the cysteine redoxome. Despite this substantial progress, still many challenges remain. Here, we provide an update on the recent advances in proteomic strategies for cysteine redoxome profiling, compare the advantages and disadvantages of current methods and discuss the outstanding challenges and future perspectives for plant redoxome research.
Topics: Cysteine; Metabolome; Oxidation-Reduction; Plant Proteins; Plants; Proteome; Proteomics
PubMed: 33793888
DOI: 10.1093/plphys/kiaa074 -
Expert Review of Proteomics Jun 2021Fibroblasts maintain tissue and organ homeostasis through output of extracellular matrix that affects nearby cell signaling within the stroma. Altered fibroblast... (Review)
Review
INTRODUCTION
Fibroblasts maintain tissue and organ homeostasis through output of extracellular matrix that affects nearby cell signaling within the stroma. Altered fibroblast signaling contributes to many disease states and extracellular matrix secreted by fibroblasts has been used to stratify patient by outcome, recurrence, and therapeutic resistance. Recent advances in imaging mass spectrometry allow access to single cell fibroblasts and their ECM niche within clinically relevant tissue samples.
AREAS COVERED
We review biological and technical challenges as well as new solutions to proteomic access of fibroblast expression within the complex tissue microenvironment. Review topics cover conventional proteomic methods for single fibroblast analysis and current approaches to accessing single fibroblast proteomes by imaging mass spectrometry approaches. Strategies to target and evaluate the single cell stroma proteome on the basis of cell signaling are presented.
EXPERT OPINION
The promise of defining proteomic signatures from fibroblasts and their extracellular matrix niches is the discovery of new disease markers and the ability to refine therapeutic treatments. Several imaging mass spectrometry approaches exist to define the fibroblast in the setting of pathological changes from clinically acquired samples. Continued technology advances are needed to access and understand the stromal proteome and apply testing to the clinic.
Topics: Extracellular Matrix; Fibroblasts; Humans; Mass Spectrometry; Proteome; Proteomics
PubMed: 34129411
DOI: 10.1080/14789450.2021.1941893 -
Expert Review of Proteomics 2016Aiming at clinical studies of human diseases, antibody-assisted assays have been applied to biomarker discovery and toward a streamlined translation from patient... (Review)
Review
Aiming at clinical studies of human diseases, antibody-assisted assays have been applied to biomarker discovery and toward a streamlined translation from patient profiling to assays supporting personalized treatments. In recent years, integrated strategies to couple and combine antibodies with mass spectrometry-based proteomic efforts have emerged, allowing for novel possibilities in basic and clinical research. Described in this review are some of the field's current and emerging immunocapture approaches from an affinity proteomics perspective. Discussed are some of their advantages, pitfalls and opportunities for the next phase in clinical and translational proteomics.
Topics: Animals; Antibodies; Humans; Immunoprecipitation; Limit of Detection; Mass Spectrometry; Protein Binding; Proteome; Proteomics; Translational Research, Biomedical
PubMed: 26558424
DOI: 10.1586/14789450.2016.1111141 -
Genomics, Proteomics & Bioinformatics Oct 2021In the past decade, relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in... (Review)
Review
In the past decade, relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in biological samples. Although its multiplexing capacity and flexibility make this a valuable technology for addressing various biological questions, its quantitative accuracy and precision still pose significant challenges to the reliability of its quantification results. Here, we give a detailed overview of the different kinds of isobaric mass tags and the advantages and disadvantages of the isobaric labeling method. We also discuss which precautions should be taken at each step of the isobaric labeling workflow, to obtain reliable quantification results in large-scale quantitative proteomics experiments. In the last section, we discuss the broad applications of the isobaric labeling technology in biological and clinical studies, with an emphasis on thermal proteome profiling and proteogenomics.
Topics: Proteome; Proteomics; Reproducibility of Results; Tandem Mass Spectrometry
PubMed: 35007772
DOI: 10.1016/j.gpb.2021.08.012 -
Molecular Systems Biology Jul 2021While informative, protein amounts and physical protein associations do not provide a full picture of protein function. This Commentary highlights the potential of...
While informative, protein amounts and physical protein associations do not provide a full picture of protein function. This Commentary highlights the potential of structural and stability proteomic technologies to derive new insights in biology and medicine.
Topics: Biophysics; Proteome; Proteomics
PubMed: 34293219
DOI: 10.15252/msb.202110442 -
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 -
Current Protocols in Protein Science Feb 2019Stable isotope labeling by amino acids in cell culture (SILAC) has become very popular as a quantitative proteomic method since it was firstly introduced by Matthias... (Review)
Review
Stable isotope labeling by amino acids in cell culture (SILAC) has become very popular as a quantitative proteomic method since it was firstly introduced by Matthias Mann's group in 2002. It is a metabolic labeling strategy in which isotope-labeled amino acids are metabolically incorporated in vivo into proteins during translation. After natural (light) or heavy amino acid incorporation, differentially labeled samples are mixed immediately after cell lysis and before any further processing, which minimizes quantitative errors caused by handling different samples in parallel. In this unit, we describe protocols for basic duplex SILAC, triplex SILAC for use in nondividing cells such as neurons, and for measuring amounts of newly synthesized proteins. © 2018 by John Wiley & Sons, Inc.
Topics: Animals; Humans; Isotope Labeling; Nerve Tissue Proteins; Neurons; Proteome; Proteomics
PubMed: 30238645
DOI: 10.1002/cpps.74 -
Journal of Chromatography. A Nov 2017Microscale separation (e.g., liquid chromatography or capillary electrophoresis) coupled with mass spectrometry (MS) has become the primary tool for advanced proteomics,... (Review)
Review
Microscale separation (e.g., liquid chromatography or capillary electrophoresis) coupled with mass spectrometry (MS) has become the primary tool for advanced proteomics, an indispensable technology for gaining understanding of complex biological processes. In recent decades significant advances have been achieved in MS-based proteomics. However, the current proteomics platforms still face an analytical challenge in overall sensitivity towards nanoproteomics applications for starting materials of less than 1μg total proteins (e.g., cellular heterogeneity in tissue pathologies). Herein, we review recent advances in microscale separation techniques and integrated sample processing strategies that improve the overall sensitivity and proteome coverage of the proteomics workflow, and their contributions towards nanoproteomics applications.
Topics: Chromatography, Liquid; Electrophoresis, Capillary; Mass Spectrometry; Proteome; Proteomics
PubMed: 28765000
DOI: 10.1016/j.chroma.2017.07.055 -
Analytical Chemistry Jan 2023Large-scale proteome analysis requires rapid and high-throughput analytical methods. We recently reported a new paradigm in proteome analysis where direct infusion and...
Large-scale proteome analysis requires rapid and high-throughput analytical methods. We recently reported a new paradigm in proteome analysis where direct infusion and ion mobility are used instead of liquid chromatography (LC) to achieve rapid and high-throughput proteome analysis. Here, we introduce an improved direct infusion shotgun proteome analysis protocol including label-free quantification (DISPA-LFQ) using CsoDIAq software. With CsoDIAq analysis of DISPA data, we can now identify up to ∼2000 proteins from the HeLa and 293T proteomes, and with DISPA-LFQ, we can quantify ∼1000 proteins from no more than 1 μg of sample within minutes. The identified proteins are involved in numerous valuable pathways including central carbon metabolism, nucleic acid replication and transport, protein synthesis, and endocytosis. Together with a high-throughput sample preparation method in a 96-well plate, we further demonstrate the utility of this technology for performing high-throughput drug analysis in human 293T cells. The total time for data collection from a whole 96-well plate is approximately 8 h. We conclude that the DISPA-LFQ strategy presents a valuable tool for fast identification and quantification of proteins in complex mixtures, which will power a high-throughput proteomic era of drug screening, biomarker discovery, and clinical analysis.
Topics: Humans; Proteome; Proteomics; Chromatography, Liquid; Software
PubMed: 36527718
DOI: 10.1021/acs.analchem.2c02249 -
Nucleic Acids Research Jan 2022Proteome-pI 2.0 is an update of an online database containing predicted isoelectric points and pKa dissociation constants of proteins and peptides. The isoelectric...
Proteome-pI 2.0 is an update of an online database containing predicted isoelectric points and pKa dissociation constants of proteins and peptides. The isoelectric point-the pH at which a particular molecule carries no net electrical charge-is an important parameter for many analytical biochemistry and proteomics techniques. Additionally, it can be obtained directly from the pKa values of individual charged residues of the protein. The Proteome-pI 2.0 database includes data for over 61 million protein sequences from 20 115 proteomes (three to four times more than the previous release). The isoelectric point for proteins is predicted by 21 methods, whereas pKa values are inferred by one method. To facilitate bottom-up proteomics analysis, individual proteomes were digested in silico with the five most commonly used proteases (trypsin, chymotrypsin, trypsin + LysC, LysN, ArgC), and the peptides' isoelectric point and molecular weights were calculated. The database enables the retrieval of virtual 2D-PAGE plots and customized fractions of a proteome based on the isoelectric point and molecular weight. In addition, isoelectric points for proteins in NCBI non-redundant (nr), UniProt, SwissProt, and Protein Data Bank are available in both CSV and FASTA formats. The database can be accessed at http://isoelectricpointdb2.org.
Topics: Amino Acid Sequence; Computational Biology; Databases, Protein; Electrophoresis, Gel, Two-Dimensional; Isoelectric Point; Molecular Weight; Peptides; Proteome; Proteomics
PubMed: 34718696
DOI: 10.1093/nar/gkab944