Cells have evolved complex mechanisms to halt cell cycle progression in response to genomic insult, collectively termed the DNA damage checkpoint [1
]. These checkpoint controls allow time for repair or bypass of the lesioned DNA, or, if the damage is too severe, to activate the apoptotic program [4
]. The Phosphatidylinositol 3-kinase-like kinases ATM and ATR are central upstream regulators of the DNA damage checkpoint, phosphorylating consensus SQ or TQ motifs on downstream effectors, such as the kinases Chk1 and Chk2 and the transcriptional regulator p53 [1
]. Chk1 and Chk2 kinases in turn phosphorylate substrates, including p53 and the Cdc25 phosphatases, which halt cell cycle progression through inhibition of cyclin dependent kinase activity [14
In addition to the core DNA damage checkpoint pathway, hundreds of other proteins have been identified that participate in a more generalized DNA damage response (DDR) [3
]. Proteomic analyses of DNA damage signaling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) have identified hundreds of candidate ATM/ATR substrates more highly phosphorylated in response to damage [37
]. These studies, along with years of more focused biochemical analysis, emphasize the far-reaching effects of the DDR in affecting protein post-translational modification across many signaling pathways controlling diverse cellular functions.
In addition to studies of the DNA damage response, antibody-based proteomic methods have also been employed to study signaling areas, such as cancer, neurodegenerative disease, growth and development and infectious disease [37
]. Other LC-MS-based proteomic methods of PTM analysis have employed sample fractionation and whole proteome analysis or global phosphopeptide enrichment methods, such as immobilized metal affinity chromatography (IMAC) with chelated iron or titanium dioxide beads [38
]. Such fractionation and enrichment strategies, combined with the sensitivity and speed of current LC-MS/MS systems, allow identification of hundreds to thousands of post-translationally modified peptides in a single LC-MS/MS run. These methods suffer, however, from the fact that the peptides identified and quantified are randomly sampled by the instrument and tend to focus on the more abundant peptides present in a sample, while critical signaling may be mediated by proteins expressed at exquisitely low levels. The results from these proteomic studies can therefore lack the specificity necessary to probe signaling networks in enough detail to sufficiently understand the mechanism of action or cellular response to a particular perturbation.
A novel method for identification and quantification of post-translationally modified peptides, termed PTMScan Direct, was recently published [67
]. This method enables focused study of peptides from proteins in the same signaling pathway or from the same protein type. Four PTMScan Direct reagents were previously designed that probed critical signaling nodes of many different pathways (the Multipathway Reagent), phosphorylation sites on kinases (the Ser/Thr Kinases Reagent and Tyr Kinases Reagent) and Phosphatidylinositol 3-kinase signaling networks (the Akt/PI3K Pathway Reagent). Each reagent allows quantification of hundreds of post-translationally modified peptides from proteins that reside in the desired signaling space in a single LC-MS/MS run. PTMScan Direct can be used on cell lines, tissues, xenografts or primary cells. The method has been validated for use in humans and mice, but could be extended to include any species with an available proteomic database (for peptide identification). This method therefore combines the specificity of traditional biochemical analysis with the speed, sensitivity and high number of endpoints inherent to LC-MS/MS analysis.
Two new PTMScan Direct Reagents have been formulated that target DNA damage and cell cycle pathways, as well as apoptotic and autophagolytic pathways (PTMScan Direct: DNA Damage/Cell Cycle and Apoptosis/Autophagy Reagents, respectively). Each PTMScan Direct reagent allows in-depth analysis of its targeted pathways by focusing the MS sampling on the immunoaffinity-enriched target peptides, eliminating the random sampling that can occur with traditional global enrichment strategies. Reagents were rigorously validated using both human samples, as well as mouse tissues, with only peptides that pass strict criteria (MS/MS score filtering; intensity filters; enrichment versus control filters; presence of a sequence targeted by the reagent, etc.) accepted as validated targets. The DNA Damage/Cell Cycle Reagent allows identification of 263 sites on 137 proteins, while the Apoptosis/Autophagy Reagent targets 175 sites of phosphorylation and caspase cleavage activation sites on 100 proteins.
These unique reagents have been used to study activation of the DNA damage checkpoint and induction of apoptosis in HeLa cells damaged with UV light. In response to UV damage of DNA, peptides from markers of checkpoint signaling (ATM, Chk1, Chk2), cell cycle arrest (p21), stress response (p38 MAPK, JNK) and apoptosis (caspase cleavage, cytochrome c) all changed in abundance in the PTMScan Direct studies. Changes to selected signaling nodes were confirmed by western blotting, highlighting the accuracy of the method. These reagents therefore provide detailed, reproducible analysis of hundreds of peptides from proteins involved in the response to DNA damage and damage-induced cell death in a single LC-MS/MS run and are powerful tools for LC-MS/MS-based proteomic analysis.