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Cysteine-targeted proteomics analysis

ABSTRACT: The protein cysteine residue is one of the amino acids most susceptible to oxidative modifications, frequently caused by oxidative stress. Several applications have enabled cysteine-targeted proteomics analysis with simultaneous detection and quantitation. In this study, we employed a quantitative approach using a set of iodoacetyl-based cysteine reactive isobaric tags (iodoTMT) and evaluated the transient cellular oxidation ratio of free and reversibly modified cysteine thiols under DTT and hydrogen peroxide (H2O2) treatments.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney

SUBMITTER: Kazutaka Araki

LAB HEAD: Kazuhiko Fukui

PROVIDER: PXD004303 | Pride | 2016-07-18

REPOSITORIES: Pride

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			Action	DRS
	15f1391.mzML	Mzml
	15f1391.mzid	Mzid
	15f1392.mzML	Mzml
	15f1392.mzid	Mzid
	15f1393.mzML	Mzml

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Publications

Redox Sensitivities of Global Cellular Cysteine Residues under Reductive and Oxidative Stress.

Araki Kazutaka K Kusano Hidewo H Sasaki Naoyuki N Tanaka Riko R Hatta Tomohisa T Fukui Kazuhiko K Natsume Tohru T

Journal of proteome research 20160715 8

The protein cysteine residue is one of the amino acids most susceptible to oxidative modifications, frequently caused by oxidative stress. Several applications have enabled cysteine-targeted proteomics analysis with simultaneous detection and quantitation. In this study, we employed a quantitative approach using a set of iodoacetyl-based cysteine reactive isobaric tags (iodoTMT) and evaluated the transient cellular oxidation ratio of free and reversibly modified cysteine thiols under DTT and hyd ...[more]

PMID: 27350002

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Comparative proteomics using iodoTMT to identify changes in protein expression and cysteine oxidation in Leishmania mexicana

Project description:Two cell-lines of Leishmania mexicana were investigated using a TMT-based approach; wild-type and an attenuated H-line. Half of each sample was fully reduced and all cysteine residues were labelled with the cysteine-reactive iodoTMT reagent, while the other half was reacted with N-ethylmaleimide to block all free cysteines before the remaining reversibly modified cysteines were reduced and labelled with iodoTMT. This allowed direct comparisons to be made between the level of modified cysteines and the total cysteine count.

2018-11-23 | PXD010938 | Pride

Comparative genomic hybridization of diverse Enterococcus faecalis strains to determine the core species genome

Project description:Comparative genomic hybridization analysis of diverse Enterococcus faecalis strains to determine core species genome.<br>An additional data file, Gilmore_M-Genomotyping_Tables_12-21-2006.xls, containing information about data processing is available for download from <a href="ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-1090/">ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-1090/</a>

2007-06-04 | E-MEXP-1090 | biostudies-arrayexpress

Polyubiquitination analysis upon GSK3 inhibition in HeLa cell extracts

Project description:HeLa cell extracts with or without GSK3 enzyme inhibition were assayed using protein microarrays in order to detect GSK3-dependent changes in protein polyubiquitination. HeLa lysates in triplicates were supplemented with ubiquitin and incubated on protein microarrays (ProtoArray 5.0; Invitrogen) in the presence or absence of the GSK3 inhibitor SB-216763. Polyubiquitination of the arrayed proteins was detected using specific antibodies. ProtoArray 5.0 contains over 9,000 full-length human proteins purified and arrayed in duplicate under native conditions to maximize functionality.

2014-05-15 | E-GEOD-50629 | biostudies-arrayexpress

Comprehensive redox profiling of the thiol proteome of Clostridium difficile

Project description:The strictly anaerobic bacterium C. difficile has become one of the most problematic hospital acquired pathogens and a major burden for health care systems. Although antibiotics work effectively in most C. difficile infections (CDIs), their detrimental effect on the intestinal microbiome paves the way for recurrent episodes of CDI. To develop alternative, non-antibiotics-based treatment strategies, deeper knowledge on the physiology of C. difficile, stress adaptation mechanisms and regulation of virulence factors is mandatory. Focus of this work was to tackle the thiol proteome of C. difficile and its stress-induced alterations, since recent research reported the amino acid cysteine to play a central role in the metabolism of the pathogen. We developed a novel cysteine labeling approach to determine the redox state of protein thiols on a global scale. Applicability of the technique was demonstrated by inducing disulfide stress using the chemical diamide. The method can be transferred to any kind of redox challenge and was applied in this work to assess the effect of bile acids on the thiol proteome of C. difficile. We present redox-quantification of more than 1,500 thiol peptides and discuss the general difficulty of redox analyses of peptides possessing more than a single cysteine residue. The presented method will be especially useful when not only redox status shall be determined, but information on protein quantity is needed as well. Not least, our comprehensive dataset reveals protein cysteine sites particularly susceptible to oxidation and builds a groundwork for redox proteomics studies in C. difficile.

2018-03-05 | PXD007278 | Pride

Quantitative profiling of protein sulfhydrome by the BTA-TMT

Project description:The redox-based modification of cysteine residues in proteins regulates their function in many biological processes. The gas molecule H2S has been shown to sulfhydrate redox sensitive cysteine residues resulting in an H2S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent. Here we developed TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. With this approach, we revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as novel sulfhydration substrate in pancreatic beta cells. Moreover, we identified metabolic pathways selectively being targets for H2S. Using this approach, we found that the enzymes involved in cellular energy pathway are primarily subjected to a Redox Thiol Switch (RTS), which is a switch of one modification to another on the same cysteine residue. We further showed that protein S-glutathioinylation, a specific reversible thiol modification in cysteine residues under oxidative stress conditions, which can be switched to S-sulfhydration. We propose that a RTSGS from S-glutathioinylation to S-sulfhydration is a key mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

2020-03-09 | PXD015307 | Pride

Notch1-driven epigenetic changes in a mouse model of T cell Acute Lymphoblastic Leukemia (T-ALL)

Project description:T-cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we used a mouse model of T-ALL through the overexpression of the intarcellular transcriptionally active part of Notch1 (N1-IC). This model faithfully recapitulates the major characteristics of the human disease. Comparison of the leukemic cells from peripheral tumors(thymoma) of this mouse model to normal thymic cells Double Positive (DP) for the markers CD4 and CD8 that express very low levels of Notch1 showed major expression changes (please see GSE34554) in pathways controlling the transition from physiology to disease. Further correlation of the data to ChIP-Seq data from the same cell populations led us to identify a hitherto unknown antagonism of the Notch1 oncogenic pathway and the polycomb complex (PRC2) in leukemia. Importantly exome sequencing in primary samples from human patients with T-ALL revealed that the PRC2 complex is frequently mutated and inactivated, further supporting the tumor suppressor role of the complex in this disease. Gene expression profiles from CD4+/8+ Double Positive (named DP) derived from normal thymus and Notch1-IC over-expressing tumors (named T-ALL) were analyzed for the genomewide enrichments of two major activating epigenetic marks (Histone 3 Lysine 9 acetylation (H3K9ac) and lysine 4 trimethylation (H3K4me3)), one reprressive mark (Histone 3 lysine 27 trimethylation, H3K27me3) and the major oncogene Notch1.

2012-01-10 | E-GEOD-34954 | biostudies-arrayexpress

Alterations of protein S-glutathionylation modification in macrophages under oxidative conditions

Project description:S-glutathionylation is an important regulatory posttranslational modification of protein cysteine (Cys) thiols, yet the role of specific cysteine residues as targets of modification is poorly understood. To identify the specific molecular targets and pathways of Grx1 that are susceptible to redox-dependent regulation, we applied a quantitative redox proteomics approach that permits site-specific profiling of S-glutathionylation at a proteome-wide scale in macrophages.

2021-11-01 | PXD027965 | Pride

ChiP-seq profiling of Drosophila melanogaster salivary glands to identify targets for NSL1 and MCRS2.

Project description:ChiP-seq profiling of Drosophila melanogaster salivary glands to identify targets for NSL1 and MCRS2.

2010-06-25 | E-MTAB-214 | biostudies-arrayexpress

Integrated analysis of transcript level regulation of metabolism during human adipocyte differentiation [ChIP-Seq]

Project description:Here, we have focused on studying the link between metabolic changes driven by the differentiation into mature adipocytes of a human preadipocyte cell line (SGBS) and their regulation, through a combined experimental and computational approach. By collecting data on gene expression, PPARg, CEBPa, LXR and H3K4me3 genome-wide ChIP-seq profles and transcriptome-wide microRNA target identification for miR-27a, miR29a and miR-222, and using constraint-based modeling to estimate metabolic reaction activity, we obtained a comprehensive set of information highlighting how epigenetic, transcriptional and post-transcriptional regulation impacts the metabolic network. Illumina Solexa sequencing: Six samples in total. Two ChIP-seq samples were prepared using an antibody against H3K4me3 active TSS chromatin marker from human SGBS preadipocyte and day 10 differentiated SGBS adipocyte cells. From day 10 differentiated SGBS cells additional three samples were prepared using an antibody against PPARg, CEBPa and LXRa to determine their genome-wide binding. One input control sample is included.

2013-11-19 | E-GEOD-41629 | biostudies-arrayexpress

ChIP-seq data of Epstein-Barr Virus Nuclear Antigen Leader ProteinM-BM- (EBNALP)

Project description:Epstein-Barr-Virus (EBV) Nuclear Antigens EBNALP and EBNA2 are co-expressed in EBV infected B-lymphocytes and are critical for Lymphoblastoid Cell Line (LCL) growth. EBNALP removes NCOR1 and RBPJ repressive complexes from promoter and enhancer sites and EBNA2 mostly activates transcription from distal enhancers. ChIP-seqs found EBNALP at 19,224 LCL sites, which were 33% promoter associated. EBNALP was associated with 10 transcription factor (TF) clusters that included YY1(63%), SP1(62%), PAX5(59%), BATF(50%), IRF4(49%), RBPJ(43%), ETS1(39%), PU.1(37%), RAD21(33%), NF-kB(31%), TBLR1(26%), ZNF143(24%), CTCF(23%), SMC3(21%), and EBF(17%). EBNALP sites had higher H3K4me3, H3K9ac, H3K27ac, H2Az, and RNA Pol II signals than EBNA2 sites and had similar transcription effects. EBNALP co-localized with 29% of 19,845 EBNA2 sites. EBNALP/EBNA2 sites were similar to EBNALP sites in promoter localization, associated cell TFs, Pol II, H3K4me3, H3K9ac, H3K27ac, and H2Az signals. EBNALP and EBNA2 promoter sites were more transcriptionally active than EBNALP or EBNA2 promoter sites. EBNALP was at the enhancer or promoter of myc and MYC affected genes, including cyclin D2, and bcl2. EBNALP at promoters with DNA looping and transcription factors, is positioned to deplete repressors from enhancers and promoters, enable chromatin remodeling, and transcription activation. Two EBNALP ChIP-seq replicates from IB4 LCL are analyzed in this study.

2013-07-31 | E-GEOD-49338 | biostudies-arrayexpress

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