Dataset Information

Redox proteomics in NO-mediated protection on hypoxic cardiomyocyte

ABSTRACT: We developed a mass spectrometry (MS)-based quantitative approach using a set of novel iodoacetyl-based cysteine-reactive isobaric tags (iodoTMT) endowed with unique irreversible Cys-reactivities to differentially quantify the multiple redox-modified forms of a Cys site in the original cellular context. The established method was than applied to map global Cys-redoxomic regulation in NO-mediated ischemic cardioprotection. Upon cell lysis, iodoacetamide was first added to the cell lysates to block free thiols. Reversible Cys modifications were then selectively reduced and labeled by different isobaric sets of iodoTMT. After tryptic digestion, the iodoTMT-labeled peptides were immuno-enriched, and analyzed by LTQ-Orbitrap Velos (Thermo Fisher Scientific) coupled with nanoACQUITY (Waters). All MS and MS/MS raw data were processed with Proteome Discoverer version 1.3 (Thermo Fisher Scientific), and the peptides were identified from the MS/MS spectra searched against the UniProtKB/Swiss-Prot database using the Mascot 2.3.02 (Matrix Science) with the following constraints: only tryptic peptides with up to two missed cleavage sites; taxonomy: Rattus; and mass accuracy of 10 ppm for the parent ion and 0.05 Da for the fragment ions. iodoTMT labeled (C), carbamidomethyl (C), deamidation (NQ), oxidation (M), and acetyl (protein N-term) were specified as dynamic modifications. False discovery rates were calculated by target-decoy strategy with or without using Mascot Percolator. All peptide hits were filtered with a 1% FDR cutoff.

INSTRUMENT(S): LTQ Orbitrap Velos Pro

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Cell Culture

SUBMITTER: Kuan-Ting Pan

LAB HEAD: Kay-Hooi Khoo

PROVIDER: PXD000274 | Pride | 2016-06-27

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	H9c2_HG_iodoTMT_3hr_0224-2.mzML	Mzml
	H9c2_HG_iodoTMT_3hr_0224-3.mzML	Mzml
	PRIDE_Exp_Complete_Ac_29617.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_29617.pride.mztab.gz	Mztab
	PRIDE_Exp_Complete_Ac_29617.xml.gz	Xml

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Publications

Mass spectrometry-based quantitative proteomics for dissecting multiplexed redox cysteine modifications in nitric oxide-protected cardiomyocyte under hypoxia.

Pan Kuan-Ting KT Chen Yi-Yun YY Pu Tsung-Hsien TH Chao Yu-Shu YS Yang Chun-Yi CY Bomgarden Ryan D RD Rogers John C JC Meng Tzu-Ching TC Khoo Kay-Hooi KH

Antioxidants & redox signaling 20131023 9

<h4>Aims</h4>Distinctive states of redox-dependent cysteine (Cys) modifications are known to regulate signaling homeostasis under various pathophysiological conditions, including myocardial injury or protection in response to ischemic stress. Recent evidence further implicates a dynamic interplay among these modified forms following changes in cellular redox environment. However, a precise delineation of multiplexed Cys modifications in a cellular context remains technically challenging. To this ...[more]

PMID: 24152285

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