Table of Contents
International Journal of Proteomics
Volume 2012 (2012), Article ID 514847, 9 pages
Research Article

Application of iTRAQ Reagents to Relatively Quantify the Reversible Redox State of Cysteine Residues

1Department of Biochemistry and Molecular Biology, University of Córdoba and IMIBIC, 14071 Córdoba, Spain
2Cardiovascular Proteomics Laboratory, National Center for Cardiovascular Research, 28026 Madrid, Spain
3Department of Biochemistry, University College Cork, Cork, Ireland

Received 12 April 2012; Accepted 30 April 2012

Academic Editor: Qiangwei Xia

Copyright © 2012 Brian McDonagh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Supplementary Figure 1: Identification of peptide CCSDVFNQVVK doubly labelled with NEM and IAM. ADH was first treated with NEM to alkylate free thiols and then reduced with DTT to freed disulfides and blocked with IAM. MS/MS traces from selected peptide fragments are shown in control (A); in 1 mM (B) and 5 mM (C) H2O2 treated samples. (1) Trace of fragment y6+. (2) Trace of fragment y9+. (3) Trace of fragment y10+(IAM-CSDVFNQVVK). (4) Trace of fragment b1+ (NEM-C). This data demonstrate that both successive cysteines can coexist in different redox states: one reduced and the other reversibly oxidised.

Supplementary Figure 2: Chromatograms corresponding to the base peak signals of most intense precursor ions of (A) control, (B) 1 mM H2O2 treatment, (C) 5 mM H2O2 treatment, and MS/MS trace from doubly sulfonic acid CCSDVFNQVVK fragments in control (D), 1 mM H2O2 treatment (E) and 5 mM H2O2 treatment (F). These results show how the peptide population of ADH changes upon treatment with H2O2 and how the doubly sulfonic acid modified peptide is only detected in the 5 mM H2O2 treated sample.

Supplementary Table 1: List of proteins and the Cys-containing peptides used for identification from the gram negative B. subtilis. Quantification is relative to control total and reversibly-oxidized thiols (total detectable thiols), taken as 1.0. The 116:114 is the total detectable thiols after exposure to 1 mM H2O2 compared to control. 118:114 and 121:114 are the ratios of reversibly-oxidized thiols in control and after H2O2 exposure, respectively, in comparison to total free thiols and reversibly-oxidized thiols in controls. Blast searches confirmed all peptides are found in B. subtilis.

  1. Supplementary Material