Global kinetic analysis of proteolysis via quantitative targeted proteomics.
Mass spectrometry-based proteomics is a powerful tool for identifying hundreds to thousands of posttranslational modifications in complex mixtures. However, it remains enormously challenging to simultaneously assess the intrinsic catalytic efficiencies (k(cat)/K(M)) of these modifications in the context of their natural interactors. Such fundamental enzymological constants are key to determining substrate specificity and for establishing the timing and importance of cellular signaling. Here, we report the use of selected reaction monitoring (SRM) for tracking proteolysis induced by human apoptotic caspases-3, -7, -8, and -9 in lysates and living cells. By following the appearance of the cleaved peptides in lysate as a function of time, we were able to determine hundreds of catalytic efficiencies in parallel. Remarkably, we find the rates of substrate hydrolysis for individual caspases vary greater than 500-fold indicating a sequential process. Moreover, the rank-order of substrate cutting is similar in apoptotic cells, suggesting that cellular structures do not dramatically alter substrate accessibility. Comparisons of extrinsic (TRAIL) and intrinsic (staurosporine) inducers of apoptosis revealed similar substrate profiles, suggesting the final proteolytic demolitions proceed by similarly ordered plans. Certain biological processes were rapidly targeted by the caspases, including multiple components of the endocyotic pathway and miRNA processing machinery. We believe this massively parallel and quantitative label-free approach to obtaining basic enzymological constants will facilitate the study of proteolysis and other posttranslational modifications in complex mixtures.
Department of Pharmaceutical Chemistry, University of California, San Francisco, 1700 4th Street, San Francisco, CA 94114, USA.
Nicholas J
NJ
Agard
Sami
S
Mahrus
Jonathan C
JC
Trinidad
Aenoch
A
Lynn
Alma L
AL
Burlingame
James A
JA
Wells
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D035683:MicroRNAs / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects; D019311:Staurosporine / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology
D035683:MicroRNAs; D010455:Peptides; D053221:TNF-Related Apoptosis-Inducing Ligand; D020169:Caspases; D019311:Staurosporine