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): instrument model

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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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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Project description:E3 ubiquitin ligases mediate the last step of the ubiquitination pathwayin the ubiquitin-proteasome system (UPS). By targeting transcriptional regulators for their turnover, E3s play a crucial role in every aspect of plant biology. In plants, SKP1/CULLIN1/F-BOX PROTEIN (SCF)-type E3 ubiquitin ligases are essential for the perception and signaling of several key hormones including auxins and jasmonates (JAs). F-box proteins, TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and CORONATINE INSENSITIVE 1 (COI1) bind directly transcriptional repressors AUX/IAAs and JAZs in an auxin- and JAs-depending manner, respectively, which permits the perception of the hormones and transcriptional activation of signaling pathways. Redox modification of proteins mainly by S-nitrosation of cysteines via nitric oxide (NO) has emerged as a valued regulatory mechanism in physiological processes requiring its rapid and versatile integration. Previously, we demonstrated that TIR1 and ASK1 (Arabidopsis thaliana SKP1) are targets of S-nitrosation, and these (NO)-dependent post-translational modifications enhance protein-protein interactions, and positively regulate SCFTIR1 complex assembly and expression of auxin response genes. In this work, we provide evidence on the modulation of SCFCOI1 complex by different S-nitrosation events. We demonstrated that S-nitrosation of ASK1 Cys118 enhanced ASK1-COI1 protein-protein interaction. Overexpression of non-nitrosable ask1 mutant protein impaired the activation of JA-responsive genes mediated by SCFCOI1 illustrating the functional relevance of this redox-mediated regulation in planta. Additionally, in silico analysis positioned COI1 as a promising S-nitrosation target. The regulation of SCF components involved in hormonal perception by S- nitrosation may represent a key strategy to determine the precise time and site-dependent activation of each hormonal signaling pathway, and highlights NO as a pivotal molecular player in these scenarios.

Dataset Information

Redox proteomics in NO-mediated protection on hypoxic cardiomyocyte

Publications

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

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