Global Kinetic Analysis of Proteolysis Via Quantitative Targeted Proteomics
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Global kinetic analysis of proteolysis via quantitative targeted proteomics Nicholas J. Agarda,b, Sami Mahrusa,c, Jonathan C. Trinidada, Aenoch Lynna,d, Alma L. Burlingamea, and James A. Wellsa,e,1 aDepartment of Pharmaceutical Chemistry, University of California, San Francisco, 1700 4th Street, San Francisco, CA 94114; bDepartment of Biocatalyst Characterization and Design, Codexis Inc., 200 Penobscot Drive, Redwood City, CA 94063-4718; cDepartment of Biomarker Research, Genentech, 1 DNA Way, South San Francisco, CA 94010; dDuke Translational Medicine Institute, Duke University Medical Center, Durham, NC 27710; and eDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, 1700 4th Street, San Francisco, CA 94114 Edited by* Robert T. Sauer, Massachusetts Institute of Technology, Cambridge, MA, and approved December 14, 2011 (received for review October 17, 2011) Mass spectrometry-based proteomics is a powerful tool for identi- ples with less complexity than those derived from the whole pro- fying hundreds to thousands of posttranslational modifications in teome, decreasing the likelihood of misassignment. Here, we complex mixtures. However, it remains enormously challenging to have applied SRM analysis to the N-terminal isolation technology k ∕K simultaneously assess the intrinsic catalytic efficiencies ( cat M)of to determine the time-course of caspase-mediated proteolysis. these modifications in the context of their natural interactors. Such From these data we calculated catalytic efficiencies for hundreds fundamental enzymological constants are key to determining sub- of caspase substrates in parallel. We believe these data will allow strate specificity and for establishing the timing and importance of a more quantitative systems-level understanding of the funda- cellular signaling. Here, we report the use of selected reaction mon- mental process of cell death, and move us closer to understanding itoring (SRM) for tracking proteolysis induced by human apoptotic the global enzymology of posttranslational modifications. 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, Results and Discussion we were able to determine hundreds of catalytic efficiencies in To globally assess caspase catalytic efficiencies in complex mix- parallel. Remarkably, we find the rates of substrate hydrolysis for tures, we quantified the appearance of caspase-cleaved N termini individual caspases vary greater than 500-fold indicating a sequen- in two complementary experiments. First, in cell lysates with tial process. Moreover, the rank-order of substrate cutting is similar endogenous caspases inactivated, we tracked the time-dependent in apoptotic cells, suggesting that cellular structures do not drama- activities of exogenously added executioner caspases-3 or -7, or tically alter substrate accessibility. Comparisons of extrinsic (TRAIL) extrinsic and intrinsic initiator caspases-8 or -9. Second, we com- and intrinsic (staurosporine) inducers of apoptosis revealed similar pared these results to the rates of appearance of caspase-cleaved substrate profiles, suggesting the final proteolytic demolitions pro- peptides in cells treated with different apoptosis-inducing drugs. ceed by similarly ordered plans. Certain biological processes were Experiments in cell lysates reveal the priority of substrate cleavage rapidly targeted by the caspases, including multiple components with a resolution that is unavailable in bulk cellular studies due of the endocyotic pathway and miRNA processing machinery. We to the stochasticity of mitochondrial permeablization and caspase believe this massively parallel and quantitative label-free approach activation (8). Additionally, the in vitro experiments allow us to to obtaining basic enzymological constants will facilitate the study specify the activities of individual caspases, a goal not readily of proteolysis and other posttranslational modifications in complex achieved in cellular studies where many caspases are simulta- mixtures. neously activated. Conversely, the cellular studies include exogen- ous factors, such as subcellular compartmentalization, that may apoptosis ∣ caspase ∣ enzymology ∣ mass spectrometry ∣ selected reaction affect cleavage rates. monitoring Quantitative Measurement of Caspase-Cleaved Substrates. Our ana- poptosis is a form of programmed cell death that serves to lysis of proteolysis substrates is based on a previously described Aeliminate unnecessary, infected, or tumorigenic cells from N-terminal isolation platform that compares cells or lysates eukaryotic organisms. While many intrinsic and extrinsic stimuli before and after initiating a proteolytic process (3, 9, 10). Briefly, can initiate apoptosis, these ultimately converge on the activation free N termini in lysates are enzymatically labeled with a biotiny- of a related family of aspartate-specific cysteine proteases, the lated peptide ester, captured on neutravidin beads, and trypsinized caspases, that execute widespread proteolysis and induce nonin- to produce N-terminal peptides. The peptides are released by flammatory death (1). We and others have surveyed N termini site-specific proteolysis with Tobacco Etch Virus (TEV) protease, that occur in apoptotic cells and collectively reported more than and the N-terminal sequence identified via LC-MS/MS. Here, we 1,000 caspase-derived cleavages (2–5). This explosion of proteomic further optimized the tagging peptide to contain an aminobutyric data has defined a vast array of caspase substrates proteolyzed dur- acid residue at the P1 position instead of the serine-tyrosine tag ing apoptosis. While these data identify caspase targets, and in previously employed (Fig. S1) (4). This improvement resulted in some cases the sites of proteolysis, they fail to reveal the relative fewer tag-specific fragments and provided a nonnatural mass sig- rates of cleavage, a parameter necessary to establish the order of nature. Cleavages after aspartic acid residues are rare in healthy proteolytic events and their importance in extracts and intact cells. cell lysates (approximately 1% of N termini), so virtually all aspar- The recent application of selected reaction monitoring (SRM) methods, traditionally used for metabolite identification, to Author contributions: N.J.A., S.M., J.C.T., A.L.B., and J.A.W. designed research; N.J.A., S.M., proteomic studies has enabled the simultaneous label-free quan- and J.C.T. performed research; N.J.A., S.M., J.C.T., A.J.L., and A.L.B. contributed new tification of hundreds of peptides (6, 7). Our development of a reagents/analytic tools; N.J.A., S.M., J.C.T., and J.A.W. analyzed data; N.J.A. and J.A.W. N-terminal enrichment platform (3) is ideally suited to the appli- wrote the paper. cation of SRM to apoptotic proteolysis. Using this platform, The authors declare no conflict of interest. we have characterized approximately 1,000 caspase-derived pep- *This Direct Submission article had a prearranged editor. tides from human apoptotic cells forming a basis from which to 1To whom correspondence should be addressed. E-mail: [email protected]. establish high-confidence mass spectrometric assays for SRM. This article contains supporting information online at www.pnas.org/lookup/suppl/ BIOCHEMISTRY Additionally, our positive enrichment technology generates sam- doi:10.1073/pnas.1117158109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1117158109 PNAS ∣ February 7, 2012 ∣ vol. 109 ∣ no. 6 ∣ 1913–1918 Downloaded by guest on September 30, 2021 tic-cleavages identified in apoptotic cells (typically 20–50% of total inal isolation protocol (3, 7). Additionally, the positive enrich- identified N termini) are due to caspase activities. ment for N termini significantly decreases sample complexity, To expand this technology to kinetic analysis required quanti- facilitating the development of unambiguous peptide quantifica- fication of these isolated N termini as a function of time. Tradi- tion assays. tional isotope encoded approaches to MS-based quantification To track hundreds of caspase proteolysis events across many (e.g. SILAC and iTRAQ) can monitor relative peptide abun- conditions, we developed assays to quantify N-terminal peptides dance (11, 12). However, these approaches are expensive, chal- isolated from apoptotic Jurkat cells. Investigation of intrinsic lenging to expand to more than a few samples, and often fail to (staurosporine) or extrinsic (TRAIL) inducers of apoptosis quantify the same peptide across multiple samples due to variable identified 1,341 peptides with caspase-like cleavage sites from sampling at the MS-level (13, 14). Thus we investigated selected a total of 3,892 high-confidence peptides (false discovery rate of reaction monitoring (SRM) (6), as a targeted label-free quanti- <1%) (Fig. 1A and Dataset S1 a and b) (15). We analyzed the fication approach for tracking caspase-mediated proteolysis. same fractions across the same chromatography on a QTRAP SRM quantification necessitates both prior identification of the mass spectrometer, monitoring up to 10 coeluting parent ion/ species of interest and reliable MS fragmentation patterns, two fragment ion (Q1/Q3) pairs (transitions) and optimizing the tran- requirements addressed by previous applications of our N-term- sition’s collision energies (Supporting Information). The presence Abu-LAPNVTYSLPR Fragments A y10 y9 y6 y8 y7 SRM y6 y5 736.3791 +2 b5 y9 qualification & transition