Abstract
BackgroundProtein enrichment by sub-cellular fractionation was combined with differential-in-gel-electrophoresis (DIGE) to address the detection of the low abundance chromatin proteins in the budding yeast proteome. Comparisons of whole-cell extracts and chromatin fractions were used to provide a measure of the degree of chromatin association for individual proteins, which could be compared across sample treatments. The method was applied to analyze the effect of the DNA damaging agent methyl methanesulfonate (MMS) on levels of chromatin-associated proteins.ResultsUp-regulation of several previously characterized DNA damage checkpoint-regulated proteins, such as Rnr4, Rpa1 and Rpa2, was observed. In addition, several novel DNA damage responsive proteins were identified and assessed for genotoxic sensitivity using either DAmP (decreased abundance by mRNA perturbation) or knockout strains, including Acf2, Arp3, Bmh1, Hsp31, Lsp1, Pst2, Rnr4, Rpa1, Rpa2, Ste4, Ycp4 and Yrb1. A strain in which the expression of the Ran-GTPase binding protein Yrb1 was reduced was found to be hypersensitive to genotoxic stress.ConclusionThe described method was effective at unveiling chromatin-associated proteins that are less likely to be detected in the absence of fractionation. Several novel proteins with altered chromatin abundance were identified including Yrb1, pointing to a role for this nuclear import associated protein in DNA damage response.
Highlights
Protein enrichment by sub-cellular fractionation was combined with differential-in-gel-electrophoresis (DIGE) to address the detection of the low abundance chromatin proteins in the budding yeast proteome
Candidate proteins were selected for identification on the basis of chromatin enrichment factor (EF), defined for a given spot as the average ratio of spot volume in the chromatin fraction vs. the whole cell extract in DIGE images [Additional File 1, Figure S1]
Enrichment factors were calculated for paired chromatin and whole cell extracts (WCE) samples in the four DIGE gels using the Biological variation analysis (BVA) analysis module within the DeCyderTM software package
Summary
Protein enrichment by sub-cellular fractionation was combined with differential-in-gel-electrophoresis (DIGE) to address the detection of the low abundance chromatin proteins in the budding yeast proteome. C. elegans sperm [13] As demonstrated in such studies based on chromatography and/or mass spectrometrybased analysis of digested peptides, initial fractionation coupled with downstream proteomics methods is extremely valuable for addressing the relatively low abundance of many chromatin-associated proteins, especially in the context of large-scale protein identification. It can still be challenging to address differential expression using fractionated chromatin, as technical variability during its preparation can interfere with multiplex sampling and stringent statistical evaluation is needed to minimize false discovery rates In addressing this aspect, gel-based proteomics is a promising approach to accommodate multiplex experimentation effectively while minimizing systemic experimental variation. The DIGE method is extremely useful for identifying various protein forms resulting from posttranslational modifications such as phosphorylation [14] and evaluating their relative abundance
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