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Analysis of reduction capability of GrxS12 on SufB and its cysteine residues

ABSTRACT: To elucidate the reduction of GrxS12 on SufB, an in vitro reducing system was utilized. The mass spectrometry analysis of the GrxS12-reduced SufB samples identified and quantified a large number of SufB peptides containing a total of eight Cys residues. The Cys-containing peptides of SufB exhibited a notable reduction in relative intensity when reduced by GrxS12C34S, in comparison to those reduced by the normal GrxS12. When these peptides were reduced by GrxS12C92S, the identities of GSSG-oxidized peptides were obviously reduced, whereas those of H2O2-oxidized peptides remained unaltered. The oxidation level of SufB under GSSG and H2O2 treatments was quantified by mass spectrometry. It was observed that four Cys sites (Cys108, Cys176, Cys415, and Cys477) of SufB exhibited sulfenation, sulfination, and sulfonation at varying degrees. With regard to Cys108 and Cys415, sulfenation represents the predominant form of oxidation, whereas the majority of Cys176 and Cys477 underwent sulfonation. Importantly, the level of oxidation at Cys415 was significantly higher than that observed at other three Cys sites.

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ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell

SUBMITTER: Yanshuang Liu

LAB HEAD: Shaojun Dai

PROVIDER: PXD058901 | Pride | 2026-01-16

REPOSITORIES: Pride

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Project description:In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx- and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized over 1.3-fold by ARF, while BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than GSH system, support distinct function of Trx and GSH in cellular redox control, and show for the first time in mammalian cells selective targeting peptidyl Cys and biological pathways due to deficient function of the Trx system. ICAT-labeled Cys peptides were analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Peptide eluents were monitored in an MS survey scan followed by ten data-dependent MS/MS scans on an LTQ-Orbitrap ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). The LTQ was used to acquire MS/MS spectra (2 m/z isolation width, 35% collision energy, 5,000 AGC target, 200 ms maximum ion time). The Orbitrap was used to collect MS scans (300-1600 m/z, 1,000,000 AGC target, 500 ms maximum ion time, resolution 30,000). All data were converted from .raw files to the .dta format using ExtractMS version 2.0 (Thermo Finnigan, San Jose, CA). The acquired MS/MS spectra were searched against a concatenated target-decoy human RefSeq (release 37 - September 2009 - 38108 target proteins) database of the National Center for Biotechnology Information using the SEQUEST Sorcerer algorithm (version 3.11, SAGE-N) searching parameters included partially tryptic restriction, parent ion mass tolerance (+/- 20 ppm), dynamic modifications of oxidized Met (+15.9949 Da), differential ICAT-modified Cys (+9.0302 Da) and static ICAT modification of Cys (+227.1270 Da). The peptides were classified by charge state and tryptic state (fully and partial) and first filtered by mass accuracy (10 ppm for high-resolution MS), and then dynamically by increasing XCorr and deltaCn values to reduce protein false discovery rate to less than 1%, according to the target-decoy strategy.

			Action	DRS
	20241109-LYS-SufB-IAM.msf	Msf
	LYSG1-1.raw	Raw
	LYSG1-2.raw	Raw
	LYSG1-3.raw	Raw
	LYSG1-4.raw	Raw

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Analysis of reduction capability of GrxS12 on SufB and its cysteine residues

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