Identification of yeast oxidized proteins - Chromatographic top-down approach for identification of carbonylated, fragmented and cross-linked proteins in yeast
ABSTRACT 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.
- SourceAvailable from: Joaquim Ros
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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). "
ABSTRACT: Detection and quantification of protein carbonyls present in biological samples has become a popular, albeit indirect, method to determine the existence of oxidative stress. Moreover, the rise of proteomics has allowed the identification of the specific proteins targeted by protein carbonylation. This review discusses these methodologies and proteomic strategies and then focuses on the relationship between protein carbonylation and aging and the parameters that may explain the increased sensitivity of certain proteins to protein carbonylation. © 2013 Wiley Periodicals, Inc. Mass Spec Rev.Mass Spectrometry Reviews 01/2014; 33(1). DOI:10.1002/mas.21375 · 8.05 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 , normal rat plasma , and normal human plasma  for the identification of carbonylated proteins    . "
ABSTRACT: The focus of this study was on the assessment of technology that might be of clinical utility in identification, quantification, characterization of carbonylation in human plasma proteins. Carbonylation is widely associated with oxidative stress diseases. Breast cancer patient samples were chosen as a stress positive case based on the fact that oxidative stress has been reported to be elevated in this disease. Measurements of 8-isoprostane in plasma confirmed that breast cancer patients in this study were indeed experiencing significant oxidative stress. Carbonyl groups in proteins from freshly drawn blood were derivatized with biotin hydrazide after which the samples were dialyzed and the biotinylated proteins subsequently selected, digested and labeled with iTRAQ™ heavy isotope coding reagent(s). Four hundred sixty proteins were identified and quantified, 95 of which changed 1.5 fold or more in concentration. Beyond confirming the utility of the analytical method, association of protein carbonylation was examined as well. Nearly one fourth of the selected proteins were of cytoplasmic, nuclear, or membrane origin. Analysis of the data by unbiased knowledge assembly methods indicated the most likely disease associated with the proteins was breast neoplasm. Pathway analysis showed the proteins which changed in carbonylation were strongly associated with Brca1, the breast cancer type-1 susceptibility protein. Pathway analysis indicated the major molecular functions of these proteins are defense, immunity and nucleic acid binding.Journal of proteomics 07/2011; 74(11):2395-416. DOI:10.1016/j.jprot.2011.07.014 · 3.93 Impact Factor
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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). "
ABSTRACT: Solar disinfection (SODIS) is a simple drinking water treatment method that improves microbiological water quality where other means are unavailable. It makes use of the deleterious effect of solar irradiation on pathogenic microbes and viruses. A positive impact on health has been documented in several epidemiological studies. However, the molecular mechanisms damaging cells during this simple treatment are not yet fully understood. Here we show that protein damage is crucial in the process of inactivation by sunlight. Protein damages in UVA-irradiated Escherichia coli cells have been evaluated by an immunoblot method for carbonylated proteins and an aggregation assay based on semi-quantitative proteomics. A wide spectrum of structural and enzymatic proteins within the cell is affected by carbonylation and aggregation. Vital cellular functions like the transcription and translation apparatus, transport systems, amino acid synthesis and degradation, respiration, ATP synthesis, glycolysis, the TCA cycle, chaperone functions and catalase are targeted by UVA irradiation. The protein damage pattern caused by SODIS strongly resembles the pattern caused by reactive oxygen stress. Hence, sunlight probably accelerates cellular senescence and leads to the inactivation and finally death of UVA-irradiated cells.Environmental Microbiology 11/2010; 12(11-11):2931-45. DOI:10.1111/j.1462-2920.2010.02268.x · 6.24 Impact Factor