We have combined the differential proteomics technique of DIGE with a chromatin fractionation and enrichment strategy, and applied it to investigate the response to genotoxic agents in budding yeast cells. Our approach facilitated the selective screening of important chromatin-associated proteins that can otherwise be difficult to observe by typical proteomics approaches, and was successful in identifying functionally relevant target proteins. Moreover, the method was effective for the differential analysis of yeast cells following chemical treatment, as demonstrated by the MMS exposure experiment. While the fractionation method used was effective at enrichment of chromatin binding factors, a number of the observed proteins were likely mitochondrial, suggesting that more specific fractionation methods could be applied. One possible approach would be to first isolate nuclei prior to chromatin enrichment. Overall, the described method was successful in permitting the differential analysis of chromatin binding proteins using a gel-based proteomics technique, largely overcoming the technical limitations for analyzing lower-abundance chromatin proteins.
While the methodology was effective at identifying known and potentially novel proteins involved in DNA damage response, the technique does not provide comprehensive coverage. Future refinements to the methodology may be able to increase the number of factors identified in similar studies. The gel methods could be expanded to increase the pH range over which proteins can be separated effectively, more sensitive mass spectrometers may be used to increase the success rate of protein identification, a greater degree of replication and experimental precision may be utilized to detect proteins undergoing small changes in abundance and/or localization.
Characterization of differentially expressed proteins based on DF analysis was extended using an analysis of the chromatin enrichment factors (EF), providing a quantitative estimate of protein localization not typically available within proteomics studies. The method was also informative in addressing changes in protein localization, as demonstrated in the change of enrichment factor depending on treatment. Chromatin fractionation was able to consistently reveal a large population of chromatin-associated proteins using a relatively straightforward sampling procedure, in which intact complexes are maintained, as indicated by the co-detection of functionally related chromatin proteins (i.e. Tables and ). A technical strength of DIGE itself, compared to MS-based methods, is that it is able to distinguish differences in response to compounds such as MMS for different protein isoforms or post-translational variants, as revealed in the Rnr4 isoforms in this study. In contrast, quantitative MS-based methods largely rely on digested peptides [44
], making it more challenging to distinguish variable forms, as the peptides on which the change is located need to be correctly identified, quantified, and compared with peptides representing other forms of the protein.
With respect to the budding yeast DNA damage response, this study was in broad agreement with previous high-throughput studies on this response, using a variety of approaches such as microarray analysis [45
], phenotyping of deletion strains [46
] and quantitative phosphoproteomics [5
]. The microarray study showed the over-expression of the RNR complex (which is composed of four subunits Rnr1, Rnr2, Rnr3 and Rnr4) as the most significantly changed along with other key proteins such as Din7, Dun1, Rad54 and Rad51. The phosphoproteome study screened the possible phosphorylation-mediated targets of Mec1/Tel1 and Rad53 kinases [5
], and identified proteins involved in DNA replication, cytokinesis, transcription, mitosis, RNA export, stress response, transcription, and nuclear transport. Compared to the above studies, our approach focused on a subset of the budding yeast proteome that is highly associated with chromatin. In addition to the confirmation of known checkpoint-regulated factors (e.g. Rpa1, Rpa2, Rnr4), several new proteins related to DNA damage response pathways have been identified. One such factor is the Ran-GTPase binding protein Yrb1, a component of the nuclear import-export system [43
], in which the ternary complex of Gsp1, Yrb1 and Rna1 controls the GTP/GDP balance across the nuclear membrane. We propose here that Yrb1 protein may represent a link between the nuclear transport system and DNA damage responses, as implied by a recent model for G1-S cell cycle arrest during checkpoint execution [47
]. It will now be of interest to determine which proteins dependent on Yrb1-mediated nucleocytoplasmic trafficking act downstream of this factor in affording protection to genotoxic agents.
In conclusion, we present a simple fractionation and DIGE-based approach for chromatin proteomics, which can be broadly applied to investigate biological responses to chemical stress and other factors. This method was successfully applied to investigate changes that occur following exposure to the genotoxic agent MMS, confirming that it is effective in identifying novel proteins involved in cellular processes, such as the response to DNA damage.