Identification of yeast oxidized proteins - Chromatographic top-down approach for identification of carbonylated, fragmented and cross-linked proteins in yeast

Department of Chemistry, Purdue University, ウェストラファイエット, Indiana, United States
Journal of Chromatography A (Impact Factor: 4.17). 03/2007; 1141(1):22-31. DOI: 10.1016/j.chroma.2006.11.009
Source: PubMed


The effects of oxidative stress on the yeast proteome were studied using hydrogen peroxide as the stress agent. Oxidized proteins were isolated by (1) biotinylation of oxidized proteins with biotin hydrazide, (2) affinity selection using monomeric avidin affinity chromatography, and (3) further fractionated by reversed-phase liquid chromatography (RPLC) on a C(8) column. Oxidized protein fractions from RPLC were then trypsin digested and the peptide cleavage fragments identified by tandem mass spectrometry (MS/MS). Slightly over 400 proteins were identified. Sites of carbonyl formation were found in roughly one fourth of these proteins. Oxidation on other amino acids in carbonylated peptides was seen in 32 cases while carbonylation was absent in 96 of the oxidized proteins observed. Although there are large numbers of potential oxidation sites, oxidation seemed to be restricted to a small area in most of the proteins identified. Sometimes multiple amino acids in the same tryptic peptide were oxidized. A second trend was that more than 8% of the proteins identified appeared in more than one of the RPLC fractions. Based on the position of the peptides identified in the primary structure of protein candidates derived from databases it was concluded that this occurred by fragmentation of a parent protein. It is not clear from the data whether the fragmentation process was of enzymatic or oxidative origin. Finally, peptides from two or more proteins occurred together in more than one reversed phase fraction with 2% of the proteins identified. This data was interpreted to mean that this was the result of protein cross-linking.

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    • "For instance, carbonylated peptides can be captured by a solid-phase hydrazide reagent (Roe et al., 2007) or by immobilized oxalyldihydrazide on a microchip (Hollins, Soper, & Feng, 2012). In any case, the enriched proteins can be further digested and the peptides obtained identified by LC-MS/ MS (Soreghan et al., 2003; Grimsrud et al., 2007; Meany et al., 2007; Mirzaei & Regnier, 2007; Tsaytler et al., 2008). Using a biotin-hydrazide based approach, Soreghan and co-workers identified 100 carbonylated proteins, including low abundance receptors, in brain homogenates of mice of different ages (Soreghan et al., 2003). "
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    Mass Spectrometry Reviews 01/2014; 33(1). DOI:10.1002/mas.21375 · 7.71 Impact Factor
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    • "The objective of the work reported here was to explore the possibility that methods could be developed that measure the levels of oxidative stress induced posttranslational modifications (OSi~PTMs) in blood proteins and identify the molecular function of these proteins or the biological processes with which they are associated. This was achieved at the analytical level by adapting highly selective proteomics methods that have been used with yeast [15], normal rat plasma [16], and normal human plasma [10] for the identification of carbonylated proteins [17] [18] [19] [20]. "
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    • "An increase in protein size was interpreted as a sign of protein aggregation, a decrease as protein fragmentation (Davies, 1987; Davies and Delsignore, 1987). Other authors used advanced protein identification methods instead of gel electrophoresis to detect oxidatively crosslinked and fragmented proteins (Mirzaei and Regnier, 2007). "
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