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TMT6-switch - Identification and quantification of S-nitrosylation by cysteine reactive tandem mass tag switch assay

ABSTRACT: HPAEC lysate treated with GSNO followed by TMT6-switch

INSTRUMENT(S): instrument model, LTQ Velos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Christopher Murray

PROVIDER: PRD000411 | Pride | 2012-01-16

REPOSITORIES: Pride

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			Action	DRS
	PRIDE_Exp_Complete_Ac_16869.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_16869.pride.mztab.gz	Mztab
	PRIDE_Exp_Complete_Ac_16869.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_16870.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_16870.pride.mztab.gz	Mztab

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Publications

Identification and quantification of S-nitrosylation by cysteine reactive tandem mass tag switch assay.

Murray Christopher I CI Uhrigshardt Helge H O'Meally Robert N RN Cole Robert N RN Van Eyk Jennifer E JE

Molecular & cellular proteomics : MCP 20111129 2

Redox-switches are critical cysteine thiols that are modified in response to changes in the cell's environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine th ...[more]

PMID: 22126794

Similar Datasets

Measurement of S-nitrosylation occupancy in the myocardium with cysteine-reactive tandem mass tags: short communication.

Project description:RationaleS-nitrosylation (SNO) is a reversible, thiol-based protein modification that plays an important role in the myocardium by protecting critical cysteine residues from oxidation. However, little is known with regard to the percentage of a given protein that is modified by SNO (ie, SNO occupancy). Current methods allow for the relative quantification of SNO levels, but not for the determination of SNO occupancy.ObjectiveTo develop a method for the measurement of SNO occupancy, and apply this methodology to determine SNO occupancy in the myocardium.Methods and resultsWe developed a differential cysteinereactive tandem mass tag (cysTMT) labeling procedure for the measurement of SNO occupancy. To validate this cysTMT labeling method, we treated whole-heart homogenates with the S-nitrosylating agent S-nitrosoglutathione and determined maximal SNO occupancy. We also examined SNO occupancy under more physiological conditions and observed that SNO occupancy is low for most protein targets at baseline. Following ischemic preconditioning, SNO occupancy increased to an intermediate level compared to baseline and Snitrosoglutathione treatment, and this is consistent with the ability of SNO to protect against cysteine oxidation.ConclusionsThis novel cysTMT labeling approach provides a method for examining SNO occupancy in the myocardium. Using this approach, we demonstrated that IPC-induced SNO occupancy levels are sufficient to protect against oxidation.

| S-EPMC3483371 | biostudies-literature

Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection.

Project description:RationaleS-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin-switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent data set, which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations.ObjectiveTo investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome.Methods and resultsWe obtained SNO-modified cysteine data sets for wild-type and S-nitrosoglutathione reductase knockout mouse hearts (S-nitrosoglutathione reductase is a negative regulator of S-nitrosoglutathione production) and nitric oxide-induced human embryonic kidney cell using 2 labeling reagents: the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, whereas the remaining SNO sites were only labeled by 1 reagent. Characterization of the 2 distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. On the basis of this observation, we proposed a parallel dual-labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO sites in S-nitrosoglutathione reductase knockout hearts.ConclusionsUsing a protocol comprising 2 tags for dual-labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations.

| S-EPMC4979977 | biostudies-literature

Switch tandem repeats influence the choice of the alternative end-joining pathway in class switch recombination

Project description:Switch tandem repeats influence the choice of the alternative end-joining pathway in class switch recombination

2022-05-11 | GSE174296 | GEO

Improved isobaric tandem mass tag quantification by ion mobility mass spectrometry.

Project description:RationaleIsobaric tandem mass tags are an attractive alternative to mass difference tags and label-free approaches for quantitative proteomics due to the high degree of multiplexing that can be performed with their implementation. A drawback of tandem mass tags are that the co-isolation and co-fragmentation of labeled peptide precursors can result in chimeric tandem mass (MS/MS) spectra that can underestimate the fold-change expression of each peptide. Ion mobility (IM) separations coupled to quadrupole time-of-flight (Q-TOF) instruments have the potential to mitigate MS/MS spectra chimeracy since IM-MS has the ability to separate ions based on charge, m/z, and collision cross section (CCS).MethodsTwo complex protein mixtures, labeled with DiLeu isobaric tandem mass tags in opposite ratios, were mixed together and analyzed by data-dependent LC/IM-MS/MS. The accuracy of reporters from interfering pairs was compared with and without IM separation.ResultsIM separation was able to mitigate isobaric interference from differentially charged interfering ion pairs, as well as pairs of the same charge. Of the eight example precursors shown, only one had reporters that remained compressed below the significance threshold after IM separation.ConclusionsThe results of this investigation demonstrate proof-of-principle that IM separation of tagged precursors prior to MS/MS fragmentation can help mitigate quantitative inaccuracies caused by isobaric interference. Future improvements of the method would include software for automated correction and use of higher resolution IM instrumentations.

| S-EPMC4000571 | biostudies-literature

mTMT: An Alternative, Nonisobaric, Tandem Mass Tag Allowing for Precursor-Based Quantification.

Project description:Stable isotope labeling of peptides is the basis for numerous mass-spectrometry-based quantification strategies. Isobaric tagging and metabolic labeling, namely, tandem mass tagging (TMT) and SILAC, are among the most widely used techniques for relative protein quantification. Here we report an alternative, precursor-based quantification method using nonisobaric TMT variants: TMTzero (TMT0) and superheavy TMT (shTMT). We term this strategy mass difference tandem mass tagging (mTMT). These TMT variants differ by 11 mass units; however, peptides labeled with these reagents coelute, analogous to SILAC-labeled peptide pairs. As a proof-of-concept, we profiled the proteomes of two cell lines that are frequently used in neuroscience studies, SH-SY5Y and SVGp12, using mTMT and standard SILAC-labeling approaches. We show similar quantified proteins and peptides for each method, with highly correlated fold-changes between workflows. We conclude that mTMT is a suitable alternative for precursor-based protein quantification.

| S-EPMC7294932 | biostudies-literature

29-Plex tandem mass tag mass spectrometry enabling accurate quantification by interference correction.

Project description:Tandem mass tag (TMT) mass spectrometry is a mainstream isobaric chemical labeling strategy for profiling proteomes. Here we present a 29-plex TMT method to combine the 11-plex and 18-plex labeling strategies. The 29-plex method was examined with a pooled sample composed of 1×, 3×, and 10× Escherichia coli peptides with 100× human background peptides, which generated two E. coli datasets (TMT11 and TMT18), displaying the distorted ratios of 1.0:1.7:4.2 and 1.0:1.8:4.9, respectively. This ratio compression from the expected 1:3:10 ratios was caused by co-isolated TMT-labeled ions (i.e., noise). Interestingly, the mixture of two TMT sets produced MS/MS spectra with unique features for the noise detection: (i) in TMT11-labeled spectra, TMT18-specific reporter ions (e.g., 135N) were shown as the noise; (ii) in TMT18-labeled spectra, the TMT11/TMT18-shared reporter ions (e.g., 131C) typically exhibited higher intensities than TMT18-specific reporter ions, due to contaminated TMT11-labeled ions in these shared channels. We further estimated the noise levels contributed by both TMT11- and TMT18-labeled peptides, and corrected reporter ion intensities in every spectrum. Finally, the anticipated 1:3:10 ratios were largely restored. This strategy was also validated using another 29-plex sample with 1:5 ratios. Thus the 29-plex method expands the TMT throughput and enhances the quantitative accuracy.

| S-EPMC9588555 | biostudies-literature

Switch tandem repeats influence the choice of the alternative end-joining pathway in class switch recombination

Project description:Switch tandem repeats influence the choice of the alternative end-joining pathway in class switch recombination

| PRJNA729240 | ENA

Mouse,GC-1,TMT（Tandem Mass Tag）tag quantification

Project description:Tandem Mass Tag (TMT) technology is an in vitro peptide labeling technique developed by Thermo Fisher Scientific, a peptide labeling technology developed in vitro. The technology uses six (TMTsixplex™ Isobaric Label Reagent Set) or 10 (TMT10plex™ Isobaric Label Reagent Set) isotope labels by labeling peptide specific amino acid sites. In TMT experiments, the relative and absolute amounts of proteins in up to 10 different samples can be compared flexibly by tandem mass spectrometry analysis. In TMT experiments, whole proteins from tissue cells are obtained by protein extraction, and the whole proteins are enzymatically cleaved into peptides by trypsin, and the TMT reagents are used to label all the obtained peptides. The TMT reagent consists of three main components: a reporter group, an equilibrium group and a peptide reaction group.

2022-01-19 | PXD030967 | Pride

Proteome-wide detection of cysteine nitrosylation targets and motifs using bio-orthogonal cleavable linker based enrichment and switch technique (Cys-BOOST)

Project description:Cysteine (Cys) reversible post-translational modifications (PTMs) are emerging as important players in cellular signaling and redox homeostasis. Here we present Cys-BOOST a novel strategy for LC-MS/MS based quantitative analysis of reversibly modified Cys using switch technique, enrichment via bio-orthogonal cleavable linker and quantification using tandem mas tag (TMT) reagents. We performed direct comparison of Cys-BOOST (n=3) with iodo-TMT (n=3) by analyzing the total CysCys from HeLa cell extracts. As a result higher sensitivity (25,019 vs 9,966 Cys peptides), specificity (98 vs 74 %) and technical reproducibility were obtained by Cys-BOOST. In addition, the application of Cys-BOOST for the analysis of Cys nitrosylation (SNO) in S-nitrosoglutathione (GSNO) treated and non-treated HeLa cell extracts lead to the identification of unprecedented number of SNO proteins (3,537), SNO peptides (9,314) and unique SNO sites (8,304). Based on the quantitative data we describe SNO consensus motifs for endogenous SNO and SNO sites with differential reactivity to GSNO. Collectively, our findings suggest Cys-BOOST as a concurrent method of choice for Cys PTM analysis.

2019-05-21 | PXD011131 | Pride

Structural analysis of cysteine S-nitrosylation: a modified acid-based motif and the emerging role of trans-nitrosylation.

Project description:S-Nitrosylation, the selective and reversible addition of nitric oxide (NO) moiety to cysteine (Cys) sulfur in proteins, regulates numerous cellular processes. In recent years, proteomic approaches that are capable of identifying nitrosylated Cys residues have been developed. However, the features underlying the specificity of Cys modification with NO remain poorly defined. Previous studies suggested that S-nitrosylated Cys may be flanked by an acid-base motif or hydrophobic areas and show high reactivity, low pK(a), and high sulfur atom exposure. In the current study, we prepared an extensive, manually curated data set of proteins with S-nitrosothiols, accounting for a variety of biochemical functions, organisms of origin, and physiological responses to NO. Analysis of this generic NO-Cys data set revealed that proximal acid-base motif, Cys pK(a), sulfur atom exposure, and Cys conservation or hydrophobicity in the vicinity of the modified Cys do not define the specificity of S-nitrosylation. Instead, this analysis revealed a revised acid-base motif, which is located more distantly to the Cys and has its charged groups exposed. We hypothesize that, rather than being strictly used for direct activation of Cys, the modified acid-base motif is engaged in protein-protein interactions thereby contributing to trans-nitrosylation as an important and widespread mechanism for reversible modification of Cys with NO moiety. For proteins lacking the revised motif, we discuss alternative mechanisms including a potential role of nitrosoglutathione as a trans-acting agent.

| S-EPMC3061812 | biostudies-literature

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