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• Stokes, MP.
• Silva, JC.
• Jia, X
• Lee, KA.
• Polakiewicz, RD.
• Comb, MJ.
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• Stokes, MP.
• Silva, JC.
• Jia, X
• Lee, KA.
• Polakiewicz, RD.
• Comb, MJ.
• [+] on PubMed
• Stokes, MP.
• Silva, JC.
• Jia, X
• Lee, KA.
• Polakiewicz, RD.
• Comb, MJ.

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Int. J. Mol. Sci. 2013, 14(1), 286-307; doi:10.3390/ijms14010286

Article

Quantitative Profiling of DNA Damage and Apoptotic Pathways in UV Damaged Cells Using PTMScan Direct

Matthew P. Stokes* , Jeffrey C. Silva, Xiaoying Jia, Kimberly A. Lee, Roberto D. Polakiewicz and Michael J. Comb

Cell Signaling Technology, 3 Trask Lane, Danvers, MA 01923, USA

* Author to whom correspondence should be addressed.

Received: 31 October 2012; in revised form: 4 December 2012 / Accepted: 10 December 2012 / Published: 21 December 2012

(This article belongs to the Special Issue UV-Induced Cell Death 2012)

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Abstract: Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA.

Keywords: proteomics; liquid chromatography tandem mass spectrometry; DNA damage response; apoptosis; post-translational modification; PTMScan direct

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