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Quantitative analysis of in vivo methionine oxidation of the human proteome

ABSTRACT: A novel stable isotope labelling stategy was developed to quantify methionine oxidation in an unstressed human proteome. Cell extracts were oxidized with 18O labelled hydrogen peroxide following cell lysis in order to convert all unoxidized methionine residues to an oxidized version with a heavy label. The heavy labelled peptides were used to generate a custom search and quantification of the relative ratios between heavy and light labelled methionine sulfoxide containing peptides. Where the light labelled peptides were in vivo oxidized

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: John Bettinger

LAB HEAD: Sina Ghaemmaghami

PROVIDER: PXD014770 | Pride | 2019-07-26

REPOSITORIES: Pride

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Quantitative analysis of in vivo methionine oxidation of the human proteome

Project description:A novel stable isotope labelling strategy was developed to quantify methionine oxidation in an unstressed human proteome. Cell extracts were oxidized with 18O labelled hydrogen peroxide following cell lysis in order to convert all unoxidized methionine residues to an oxidized version with a heavy label. The heavy labelled peptides were used to generate a custom search and quantification of the relative ratios between heavy and light labelled methionine sulfoxide containing peptides. Where the light labelled peptides were in vivo oxidized

2019-12-09 | PXD014629 | Pride

Identification of PM-localized proteins

Project description:In order to assess the quality of alleged PM identifications from Arabidopsis, PM-enriched fractions were compared to PM-depleted fractions using 18O isotopic labeling and mass spectrometry. The two samples submitted are biological replicates. Keywords: Protein Localization via MS Two biological replicates were analyzed in order to assess sample complexity. PM-derived vesicles were digested in 18O water and compared to PM-depleted fractions digested in 16O (natural abundance) water. Relative ion intensities were then used to calculate a heavy/lite ratio with proteins showing enrichment in the upper phase having a ratio greater than 1 or 0 on a log2 scale.

2005-06-21 | E-GEOD-2830 | biostudies-arrayexpress

Accurate proteome-wide measurement of methionine oxidation in aging mouse brains

Project description:The oxidation of methionine side chains has emerged as an important posttranslational modification of proteins. A diverse array of low-throughput and targeted studies have suggested that the oxidation of methionine residues in select proteins can have diverse impacts on cell physiology, ranging from detrimental effects on protein structure and stability to functional roles in cell signaling. Despite its importance, the large-scale investigation of methionine oxidation in a complex matrix, such as the cellular proteome has been historically hampered by technical limitations. Herein we report a methodology, Methionine Oxidation by Blocking (MobB), that allows for accurate and precise quantification of low levels of methionine oxidation typically observed in vivo. To demonstrate the utility of this methodology, we applied MobB to the brain tissues of young (6 m.o.) and old (20 m.o.) mice and identified over 280 novel sites for in vivo methionine oxidation. We further demonstrated that oxidation stoichiometries for specific methionine residues are highly consistent between individual animals and methionine sulfoxides are enriched in clusters of functionally related gene products including membrane and extracellular proteins. However, we did not detect significant changes in methionine oxidation in brains of old mice, suggesting that oxidation levels are highly regulated during the course of aging.

2022-05-23 | PXD031238 | Pride

Dynamic metabolic reprogramming in dendritic cells: an early response to influenza infection that is essential for effector function

Project description:In response to influenza infection we found that dendritic cells (DCs), cells that are critical in mounting an effective immune response, undergo a profound metabolic shift. DCs alter the concentration and location of hundreds of proteins, including c-MYC, mediating a shift to a highly glycolytic phenotype that is also flexible in terms of fueling respiration.

2021-09-08 | PXD013690 | Pride

Mechanisms of chlorate toxicity and resistance in Pseudomonas aeruginosa

Project description:Pseudomonas aeruginosa is an opportunistic pathogen known to cause both acute and chronic infections. P. aeruginosa often encounters hypoxic/anoxic environments within the host, which increases its tolerance to many conventional antibiotics. Towards identifying novel therapeutic treatments, we have been exploring the potential of chlorate, a pro-drug that kills hypoxic/anoxic, antibiotic-tolerant P. aeruginosa populations. Chlorate kills P. aeruginosa when it is enzymatically reduced by hypoxically-induced nitrate reductase, thereby generating the toxic oxidizing agent, chlorite. To better assess its therapeutic potential, we investigated mechanisms of chlorate toxicity and resistance in P. aeruginosa. We combined transposon mutagenesis and high-throughput sequencing to identify genes that alter P. aeruginosa fitness during chlorate treatment. We found that methionine sulfoxide reductase (msr) genes, whose products repair oxidized methionine residues, support survival during chlorate stress. We showed that chlorate treatment leads to proteome-wide methionine oxidation, which is exacerbated in a ∆msrA∆msrB strain. Highly abundant proteins were the likeliest targets of methionine oxidation, including proteins that are abundant under standard conditions (e.g. ribosomes) and proteins that increase in abundance in response to chlorate stress (e.g. protein chaperones). The addition of exogenous methionine partially rescued P. aeruginosa survival during chlorate treatment, suggesting that widespread methionine oxidation contributes to cell death. Finally, we found that decreased nitrate reductase activity is a common mechanism of chlorate resistance. Because nitrate respiration likely sustains P. aeruginosa anaerobic growth and survival within the host, developing chlorate resistance would be expected to hinder pathogen fitness in such environments.

2023-10-24 | PXD033396 | Pride

CD4+ Th immunogenicity of Ascaris spp.

Project description:Application of a reconstituted in vitro system to define the CD4+ Th immunogenicity of complex antigenic mixtures.

2020-03-04 | PXD015924 | Pride

Mps1 Phosphorylation of the Molecular Chaperone Hsp90 Regulates Mitotic Checkpoint

Project description:SUMMARY Mps1 protein kinase is required for accurate chromosome segregation during the mitotic checkpoint. The molecular chaperone Heat Shock Protein 90 (Hsp90) has been linked to Mps1 activity, however the molecular mechanismsunderlying this regulatory process remain elusive. We report that Mps1 directly phosphorylates a conserved threonine residue (T101 in yeast Hsp90 and T115 in human Hsp90) in the N domain of Hsp90. This phosphorylation regulates chaperone function by reducingHsp90 ATPase activity andfosteringits association with kinase client proteins including Mps1. Phosphorylation of T101is also essential for the mitotic checkpoint because it confers Mps1 stability and activity.Additionally,Mps1 phosphorylation of Hsp90 sensitizes cells to Hsp90 inhibitors and elevated Mps1 levels confer tumor selectivity on Hsp90 drugs.Finally,we identified Cdc14 as the phosphatasethat dephosphorylatesT101 and disruptsthe Mps1-Hsp90 interaction. This leads to degradation of Mps1,thusproviding a mechanism for its inactivation and mitotic exit.

2016-01-27 | PXD001969 | Pride

c-Abl Dependent Tyrosine Phosphorylation of theCo-chaperone Aha1 Regulates Interaction with Hsp90

Project description:The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity tailoring the chaperone function to specific “client” proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1) promoting its interaction with Hsp90. This also increases Hsp90 ATPase activity,enhancesHsp90 interaction with kinase clients,and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a new regulatory paradigm, we find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90 thereby, hypersensitizing cancer cells to Hsp90 inhibitors bothin vitro and ex vivo.

2015-08-06 | PXD001737 | Pride

Differential Enzymatic 16O/18O Labelling for the Detection of Cross-Linked Nucleic Acid-Protein Heteroconjugates

Project description:Enzymatic differential 16O/18O labelling of nucelic acid-protein heteroconjugates. Heteroconjugates are identified against a complex background using an automated analysis workflow.

2017-09-11 | PXD003673 | Pride

The Quantitative Changes in the Yeast Hsp70 and Hsp90 Interactomes upon DNA Damage

Project description:The molecular chaperones Hsp70 and Hsp90 participate in many important cellular processes, including how cells respond to DNA damage. In this study, we applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to understand the protein network through which Hsp70 and Hsp90 exert their effects on the DNA damage response (DDR). We characterized the interactomes of the yeast Hsp70 isoform Ssa1 and Hsp90 isoform Hsp82 before and after exposure to methyl methanesulfonate. We identified 256 chaperone interactors, 146 of which are novel. Although the majority of chaperone interaction remained constant under DNA damage, 5 proteins (Coq5, Ast1, Cys3, Ydr210c and Rnr4) increased in interaction with Ssa1 and/or Hsp82. This data is related to article “Quantitative proteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveals chaperone-dependent regulation of ribonucleotide reductase”.

2014-12-08 | PXD001284 | Pride

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