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Current Opinion in Chemical Biology Feb 2022It is often unclear how genetic variation translates into cellular phenotypes, including how much of the coding variation can be recovered in the proteome. Proteogenomic... (Review)
Review
It is often unclear how genetic variation translates into cellular phenotypes, including how much of the coding variation can be recovered in the proteome. Proteogenomic analyses of heterogenous cell lines revealed that the genetic differences impact mostly the abundance and stoichiometry of protein complexes, with the effects propagating post-transcriptionally via protein interactions onto other subunits. Conversely, large scale binary interaction analyses of missense variants revealed that loss of interaction is widespread and caused by about 50% disease-associated mutations, while deep scanning mutagenesis of binary interactions identified thousands of interaction-deficient variants per interaction. The idea that phenotypes arise from genetic variation through protein-protein interaction is therefore substantiated by both forward and reverse interaction proteomics. With improved methodologies, these two approaches combined can close the knowledge gap between nucleotide sequence variation and its functional consequences on the cellular proteome.
Topics: Genetic Variation; Mutagenesis; Mutation; Protein Interaction Mapping; Proteome; Proteomics
PubMed: 34801969
DOI: 10.1016/j.cbpa.2021.102100 -
Journal of Proteome Research Mar 2018The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides...
The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human-readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard.
Topics: Amino Acid Sequence; Computational Biology; Databases, Protein; Humans; Information Dissemination; International Cooperation; Molecular Sequence Annotation; Protein Processing, Post-Translational; Proteome; Proteomics; Reproducibility of Results; Software; Tandem Mass Spectrometry
PubMed: 29397739
DOI: 10.1021/acs.jproteome.7b00851 -
Current Opinion in Chemical Biology Feb 2022Tight regulation of protein translation drives the proteome to undergo changes under influence of extracellular or intracellular signals. Despite mass spectrometry-based... (Review)
Review
Tight regulation of protein translation drives the proteome to undergo changes under influence of extracellular or intracellular signals. Despite mass spectrometry-based proteomics being an excellent method to study differences in protein abundance in complex proteomes, analyzing minute or rapid changes in protein synthesis and abundance remains challenging. Therefore, several dedicated techniques to directly detect and quantify newly synthesized proteins have been developed, notably puromycin-based, bio-orthogonal noncanonical amino acid tagging-based, and stable isotope labeling by amino acids in cell culture-based methods, combined with mass spectrometry. These techniques have enabled the investigation of perturbations, stress, or stimuli on protein synthesis. Improvements of these methods are still necessary to overcome various remaining limitations. Recent improvements include enhanced enrichment approaches and combinations with various stable isotope labeling techniques, which allow for more accurate analysis and comparison between conditions on shorter timeframes and in more challenging systems. Here, we aim to review the current state in this field.
Topics: Amino Acids; Isotope Labeling; Mass Spectrometry; Proteome; Proteomics
PubMed: 34364788
DOI: 10.1016/j.cbpa.2021.07.001 -
Antioxidants & Redox Signaling Mar 2017Oxidatively modified proteins are characterized by elevations in protein-resident carbonyls or 3-nitrotyrosine, measures of protein oxidation, or protein bound reactive...
Oxidatively modified proteins are characterized by elevations in protein-resident carbonyls or 3-nitrotyrosine, measures of protein oxidation, or protein bound reactive alkenals such as 4-hydroxy-2-nonenal, a measure of lipid peroxidation. Oxidatively modified proteins nearly always have altered structure and function. Redox proteomics is that branch of proteomics used to identify oxidized proteins and determine the extent and location of oxidative modifications in the proteomes of interest. This technique nearly always employs mass spectrometry as the major platform to achieve the goals of identifying the target proteins. Once identified, oxidatively modified proteins can be placed in specific molecular pathways to provide insights into protein oxidation and human disease. Both original research and review articles are included in this Forum on Redox Proteomics. The topics related to redox proteomics range from basic chemistry of sulfur radical-induced redox modifications in proteins, to the thiol secretome and inflammatory network, to reversible thiol oxidation in proteomes, to the role of glutamine synthetase in peripheral and central environments on inflammation and insulin resistance, to bioanalytical aspects of tyrosine nitrated proteins, to protein oxidation in human smokers and models thereof, and to Alzheimer disease, including articles on the brain ubiquitinylome and the "triangle of death" composed of oxidatively modified proteins involved in energy metabolism, mammalian target of rampamycin activation, and the proteostasis network. This Forum on Redox Proteomics is both timely and a critically important resource to highlight one of the key tools needed to better understand protein structure and function in oxidative environments in health and disease. Antioxid. Redox Signal. 26, 277-279.
Topics: Animals; Humans; Oxidation-Reduction; Proteome; Proteomics
PubMed: 27835924
DOI: 10.1089/ars.2016.6919 -
The FEBS Journal Nov 2013The elucidation of the subcellular distribution of proteins under different conditions is a major challenge in cell biology. This challenge is further complicated by the... (Review)
Review
The elucidation of the subcellular distribution of proteins under different conditions is a major challenge in cell biology. This challenge is further complicated by the multicompartmental and dynamic nature of protein localization. To address this issue, quantitative proteomics workflows have been developed to reliably identify the protein complement of whole organelles, as well as for protein assignment to subcellular location and relative protein quantification based on different cell culture conditions. Here, we review quantitative MS-based approaches that combine cellular fractionation with proteomic analysis. The application of these methods to the characterization of organellar composition and to the determination of the dynamic nature of protein complexes is improving our understanding of protein functions and dynamics.
Topics: Cell Fractionation; Cell Line; Computational Biology; Humans; Mass Spectrometry; Organelles; Proteome; Proteomics; Subcellular Fractions
PubMed: 24034475
DOI: 10.1111/febs.12502 -
Microbial Biotechnology Nov 2013With the advent of high-resolution mass spectrometry together with sophisticated data analysis and interpretation algorithms, determination of protein synthesis and... (Review)
Review
With the advent of high-resolution mass spectrometry together with sophisticated data analysis and interpretation algorithms, determination of protein synthesis and degradation rates (i.e. protein turnover) on a proteome-wide scale by employing stable isotope-labelled amino acids has become feasible. These dynamic data provide a deeper understanding of protein homeostasis and stress response mechanisms in microorganisms than well-established 'steady state' proteomics approaches. In this article, we summarize the technological challenges and solutions both on the biochemistry/mass spectrometry and bioinformatics level for turnover proteomics with a focus on chromatographic techniques. Although the number of available case studies for Corynebacterium glutamicum and related actinobacteria is still very limited, our review illustrates the potential of protein turnover studies for an improved understanding of questions in the area of biotechnology and biomedicine. Here, new insights from investigations of growth phase transition and different stress dynamics including iron, acid and heat stress for pathogenic but also for industrial actinobacteria are presented. Finally, we will comment on the advantages of integrated software solutions for biologists and briefly discuss the remaining technical challenges and upcoming possibilities for protein turnover analysis.
Topics: Bacterial Proteins; Corynebacterium glutamicum; Proteome; Proteomics
PubMed: 23425033
DOI: 10.1111/1751-7915.12035 -
Molecular & Cellular Proteomics : MCP Jan 2016Cysteine occupies a unique place in protein chemistry. The nucleophilic thiol group allows cysteine to undergo a broad range of redox modifications beyond classical... (Review)
Review
Cysteine occupies a unique place in protein chemistry. The nucleophilic thiol group allows cysteine to undergo a broad range of redox modifications beyond classical thiol-disulfide redox equilibria, including S-sulfenylation (-SOH), S-sulfinylation (-SO(2)H), S-sulfonylation (-SO(3)H), S-nitrosylation (-SNO), S-sulfhydration (-SSH), S-glutathionylation (-SSG), and others. Emerging evidence suggests that these post-translational modifications (PTM) are important in cellular redox regulation and protection against oxidative damage. Identification of protein targets of thiol redox modifications is crucial to understanding their roles in biology and disease. However, analysis of these highly labile and dynamic modifications poses challenges. Recent advances in the design of probes for thiol redox forms, together with innovative mass spectrometry based chemoproteomics methods make it possible to perform global, site-specific, and quantitative analyses of thiol redox modifications in complex proteomes. Here, we review chemical proteomic strategies used to expand the landscape of thiol redox modifications.
Topics: Animals; Cysteine; Humans; Mass Spectrometry; Oxidation-Reduction; Protein Processing, Post-Translational; Proteome; Proteomics; Reproducibility of Results; Sulfhydryl Compounds
PubMed: 26518762
DOI: 10.1074/mcp.O115.056051 -
Proteomics Jan 2017Given superior analytical features, MS proteomics is well suited for the basic investigation and clinical diagnosis of human disease. Modern MS enables detailed... (Review)
Review
Given superior analytical features, MS proteomics is well suited for the basic investigation and clinical diagnosis of human disease. Modern MS enables detailed functional characterization of the pathogenic biochemical processes, as achieved by accurate and comprehensive quantification of proteins and their regulatory chemical modifications. Here, we describe how high-accuracy MS in combination with high-resolution chromatographic separations can be leveraged to meet these analytical requirements in a mechanism-focused manner. We review the quantification methods capable of producing accurate measurements of protein abundance and posttranslational modification stoichiometries. We then discuss how experimental design and chromatographic resolution can be leveraged to achieve comprehensive functional characterization of biochemical processes in complex biological proteomes. Finally, we describe current approaches for quantitative analysis of a common functional protein modification: reversible phosphorylation. In all, current instrumentation and methods of high-resolution chromatography and MS proteomics are poised for immediate translation into improved diagnostic strategies for pediatric and adult diseases.
Topics: Humans; Mass Spectrometry; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 27775219
DOI: 10.1002/pmic.201600079 -
Cell Reports Methods Feb 2024Method development for mass spectrometry (MS)-based thermal shift proteomic assays have advanced to probe small molecules with known and unknown protein-ligand... (Review)
Review
Method development for mass spectrometry (MS)-based thermal shift proteomic assays have advanced to probe small molecules with known and unknown protein-ligand interaction mechanisms and specificity, which is predominantly used in characterization of drug-protein interactions. In the discovery of target and off-target protein-ligand interactions, a thorough investigation of method development and their impact on the sensitivity and accuracy of protein-small molecule and protein-protein interactions is warranted. In this review, we discuss areas of improvement at each stage of thermal proteome profiling data analysis that includes processing of MS-based data, method development, and their effect on the overall quality of thermal proteome profiles. We also overview the optimization of experimental strategies and prioritization of an increased number of independent biological replicates over the number of evaluated temperatures.
Topics: Proteome; Proteomics; Ligands; Mass Spectrometry; Data Analysis
PubMed: 38412830
DOI: 10.1016/j.crmeth.2024.100717 -
STAR Protocols Jun 2020identification of chromatin interactors can reveal unexpected pathways relevant to physiology and human disease. Inspired by the DNA mediated chromatin pull-down...
identification of chromatin interactors can reveal unexpected pathways relevant to physiology and human disease. Inspired by the DNA mediated chromatin pull-down (Dm-ChP) technology (also known as iPOND [isolation of proteins on nascent DNA]) for the proteomic characterization of nascent DNA, we have recently reported a new experimental protocol that allows for the identification of proteins on total DNA (iPOTD) for bulk chromatome profiling and identification of chromatin-bound proteins. Here, we detail a step-by-step protocol to survey the cellular chromatin-bound proteome in a simple, robust, and unbiased manner. For complete details on the use and execution of this protocol, please refer to Aranda et al. (2019).
Topics: Cells, Cultured; Chromatin; Genetic Techniques; Genome; Humans; Proteome; Proteomics
PubMed: 33111072
DOI: 10.1016/j.xpro.2020.100014