A field guide to the proteomics of post-translational modifications in DNA repair

Abstract

All cells incur DNA damage from exogenous and endogenous sources and possess pathways to detect and repair DNA damage. Post-translational modifications (PTMs), in the past 20 years, have risen to ineluctable importance in the study of the regulation of DNA repair mechanisms. For example, DNA damage response kinases are critical in both the initial sensing of DNA damage as well as in orchestrating downstream activities of DNA repair factors. Mass spectrometry-based proteomics revolutionized the study of the role of PTMs in the DNA damage response and has canonized PTMs as central modulators of nearly all aspects of DNA damage signaling and repair. This review provides a biologist-friendly guide for the mass spectrometry analysis of PTMs in the context of DNA repair and DNA damage responses. We reflect on the current state of proteomics for exploring new mechanisms of PTM-based regulation and outline a roadmap for designing PTM mapping experiments that focus on the DNA repair and DNA damage responses.

Keywords: LC-MS/MS, technology, bottom-up proteomics, technology, signal transduction, cell biology; phosphoproteomics, technology, post-translational modification analysis, technology, post-translational modifications, cell biology, mass spectrometry.

Figure 1:. PTMs Expand DNA Repair Protein Functionality.

Left to right: PTMs play important roles in DNA replication, recombination, and repair regulation. Proteins can be targeted for degradation by the proteasome, as in the case of CtIP (h, human) which is degraded in the G1 phase of the cell cycle [228]. Steric or electrostatic clashes may be induced by PTMs which impair protein:protein interactions, such as the Rad23:Png1 interaction in S. cerevisiae which is impaired by phosphorylation of Rad23 [47]. S. cerevisiae Rad9, a checkpoint mediator with important roles in HR regulation, engages with the scaffolding protein Dpb11 through Dpb11’s phosphopeptide-binding BRCT domains, a common theme in PTM mediated protein:protein interactions (see inset table for additional PTM-mediated interactions and binding domains). Modifications can also impair or enhance enzymatic activity, a theme evidenced by the checkpoint-dependent phosphorylation of Polε in S. cerevisiae, which limits exonuclease activity to prevent replication fork degradation [229]. Similarly, DNA binding potential can be altered by modification, as in the case of Rfa1 where its crotonylation limits this protein’s affinity for ssDNA [97]. Finally, some modifications may enhance protein stability or solubility, evidenced by the SUMOylation of Sae2 in S. cerevisiae [59].

Figure 2:. PTMs in the DNA Damage Response.

DNA damage signals PTM writers to confer PTMs on target proteins. Concomitantly, PTM erasers remove cognate PTMs. For example, kinases confer phosphorylation, which is removed by phosphatases. Figure 2 indicates a few notable PTM writers and erasers for each PTM type.

Figure 3:. Generalized Workflow for Mass Spectrometry Analysis of PTMs:

Following trypsinization and purification of PTM-modified peptides of interest from cell lysates (A). In (B), peptides are subjected to LC-MS/MS analysis where they are isolated according to their presence in a precursor (MS1) scan and subsequently fragmented to yield an MS2 “fingerprint.” An MS2 spectrum (C) contains multiple b (n-terminal fragment) and y (c-terminal fragment) ions. Depending on its location in the isolated peptide, the mass of the PTM of interest will often be present in b and y ion series, allowing for localization of the PTM of interest on the fragmented peptide. In (C), acquired MS2 spectra are computationally matched to theoretical spectra generated in silico using a search algorithm such as SEQUEST (Eng et al, 1994; Lundgren et al, 2009). In addition, PTMs are included in the search parameters as variable modifications. Often, confidence in PTM localization is scored using any of a number of available tools such as PTMProphet, available as a module within the Trans Proteomic Pipeline [168,230]. Shown in panel (D), quantitation of PTM dynamics between two or more experimental conditions can be achieved either by comparison of MS1 precursor ion intensities between two isotopically labelled biological isolates (SILAC; MS1-based quantitation, left panel) or comparison of MS2-labile reporter ion intensities (TMT; MS2-based quantitation, right panel).