Quantitative profiling of DNA damage and apoptotic pathways in UV damaged cells using PTMScan Direct.
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.
Cell Signaling Technology, 3 Trask Lane, Danvers, MA 01923, USA. mstokes@cellsignal.com.
Matthew P
MP
Stokes
Jeffrey C
JC
Silva
Xiaoying
X
Jia
Kimberly A
KA
Lee
Roberto D
RD
Polakiewicz
Michael J
MJ
Comb
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000528:radiation effects*; D001343:Autophagy / Q000528:radiation effects; D002453:Cell Cycle / Q000528:radiation effects; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D004249:DNA Damage; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational / Q000528:radiation effects*; D015398:Signal Transduction / Q000528:radiation effects*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014466:Ultraviolet Rays
D007202:Indicators and Reagents; D010455:Peptides