Global Kinetic Analysis of Proteolysis Via Quantitative Targeted Proteomics

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 y9 choice

y7 Intensity (cps) 835.4492 1,046.5630

2,300 2,800 3,300 y5 Elution Time (sec) 635.3386 CE Intensity, counts Intensity, y10 b2 b5 y8 50 y9+2 1,117.6217 45 270.1754 y3 580.3547 949.5426 40 523.7418 35 385.2498 30 Collision m/z, Da energy

Intensity (cps) optimization Peptide identification 2,350 2,550 2,750 2,950 3,150

B i. ii. D i.

0.5 0.4 1.0 0.3 1.00 0.2 0.75

Intensity (AU) 0.1 0.50 0 0.50 0102030 40 50 60

Relative Intensity Relative Time (min) Measured IntensityMeasured 0.25 0.25 0 ii. 1.2 0 0.25 0.50 0.75 1.0 1 Total intensity Expected Intensity 0.8

ty (AU) ty 0.6 0.4 C i. ii. ensi Int 0.2

Caspase 0 Incubation 0102030405060 0 min Time (min) 5 min 10 min iii. 1.4 20 min 1.2 30 min 1 Intensity (cps) Intensity (cps) 0.8 0.6 0.4 Incubation Time (min) Elution Time (sec) Intensity (AU) 0.2 0 0 10 20 30 40 50 60 -(kcat/Km * Eo * t) Time (min) A/Ao = 1 - e

Fig. 1. Targeted proteomics of peptides enriched via subtiligase tagging enables global quantification of proteolysis. (A) Development of optimized transitions: Intense fragment ions from high-mass resolution peptide MS/MS spectra are analyzed via targeted proteomics for coelution at the expected retention time and optimized for ideal collision energies. Abu: L-aminobutyric acid. (B) Validation of peptide quantification: Protein from apoptotic and healthy cell lysates was mixed at 4∶0 (green), 2∶2 (red), or 1∶3 (blue) ratios, and N termini were quantified via our N-terminomics technology. i. The relative intensities of quantifiable peptides are plotted (dashed lines indicate ideal values) against the rank order of the combined peptide intensity for all three samples. ii. Mean intensities for each ratio show a linear dependence on amount of apoptotic cells (r2 > 0.99). (C) Determination of catalytic efficiency i. Integrations of signal intensity over time track the appearance of cleaved N termini. ii. Peptide intensities are fit to pseudo-first-order kinetic equations to determine the kinetic k ∕K efficiency ( cat M) for each substrate. (D) Statistical analysis reveals progress curves (i) below, (ii) within, and (iii) above the measureable range for catalytic efficiencies (2 CV) (black: idealized curves, colors: representative data).

1914 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1117158109 Agard et al. Downloaded by guest on September 30, 2021 of at least three coeluting transitions at the retention time ob- lysates inactivated with iodoacetamide to quench both endogen- served at the peptide discovery stage qualified the assay for the ous active and zymogenic caspases. Excess iodoacetamide was peptide of interest (Dataset S1c). While we discovered 1,341 sub- reacted with dithiothreitol to ensure it would not inactivate the strates via our N-terminal labeling technology, only about half exogenously added caspases. Evaluation of caspase activities met the strict criteria for unambiguous identification for at least against a fluorescent substrate (DEVD-AFC) under these condi- three transitions (Q1/Q3 pairs). Thus, we focused our studies on tions confirmed that lysate did not competitively inhibit caspase these 676 caspase-derived peptides for which we had unambigu- activity (Fig. S2 A–C). This control shows that the presence of ∕ ous identification. To assess the reliability of this method for pep- additional substrates does not alter the relative kcat KM values, tide identification, we monitored an unfractionated sample for and that this kinetic treatment applies to lysates containing hun- the presence of unrelated transitions, transitions with rando- dreds of caspase substrates. mized Q1s (m∕z ratios), or transitions with altered retention We added recombinant caspases to the lysates, sampled 6–8 times (10 minutes) finding a false discovery rate of 0.9–1.2% time points over 1–2 h, and evaluated the areas of SRM peaks for transitions and 0.3–0.6% for peptides (≧3 transitions). We corresponding to targeted caspase-derived N termini using in- chose the five most intense transitions as qualified assays for each house scripts (Fig. 1C). The relative intensities of these data were peptide. fit to a pseudo-first order kinetic equation to allow simultaneous ∕ With the qualified transitions in hand, we assessed the ability calculation of kcat KM values for hundreds of substrates. We to quantify caspase-derived N termini in complex mixtures. found the caspase cleavage rates fell into three categories (Fig. 1D Lysates from apoptotic and healthy cells were mixed at varying and Fig. S2 D and E): (i) substrates that were cleaved below the ratios, the N termini were isolated, and the resulting intensity of measurable rate which appear linear (to which we could assign a each peptide was evaluated using our qualified assays (Fig. 1b). maximum rate), (ii) substrates cleaved within the measurable ∕ The intensities of the apoptotic peptides were linearly correlated range (to which we can calculate a kcat KM value), and (iii) sub- to the amount of apoptotic sample across the entire range of mea- strates that are saturated by the first time point (to which we can sured intensities (r2 > 0.99), indicating that our N-terminal isola- assign a minimum rate). Optimal progress curves covered tion and MS-analysis allow for direct quantitative comparisons. approximately one order of magnitude in catalytic efficiencies, and thus we expanded the dynamic range by varying the caspase Global Enzymatic Analysis of Caspase Activities. This quantitative concentration between 10 and 1,000 nM. analysis was used to generate progress curves for the proteolysis We profiled 676 potential apoptotic substrates for cleavage of individual caspase targets as a function of time. From these by the individual caspases -3, -7, and -8, and could determine ∕ curves we calculated the catalytic efficiency (kcat KM ) for each measurable catalytic efficiencies for 180, 58, and 66 substrates, substrate from well-established pseudo-first-order kinetic rela- respectively. Remarkably, the k ∕K values for substrates for cat M tionship (%Conversion ¼ 1 − e−ðkcat∕Km Eo tÞ) (16). While appli- each protease varied over nearly three orders of magnitude cation of this equation to the progress curve does not allow us to (Fig. 2A and Dataset S2). Proteolysis rates for individual sub- separate kcat from KM in the absence of substrate concentrations, strates analyzed by immunoblot were in good agreement with the – one can calculate the ratio of kcat to KM from the initial enzyme rates determined by the SRM method (Fig. 2B and Fig. S3 A D). concentration (Eo). We tracked cleavage kinetics in Jurkat-cell We also detected proteolysis for many additional substrates but

A i. ii. iii. 5 Caspase-3 5 Caspase-7 Caspase-8 CLCA 5 4 4 TARBP2 4 ARHGAP4 3 3 Log (kcat/Km (/M/s)) Log (kcat/Km (/M/s)) Log (kcat/Km (/M/s)) 3 2 2 0 50 100 150 200 0 20 40 60 020406080 Rank Order Rank Order Rank order

P4 P3 P2 P1 P1’ P2’ P3’ P4’ P4 P3 P2 P1 P1’ P2’ P3’ P4’ P4 P3 P2 P1 P1’ P2’ P3’ P4’

B 250 nM Casp-3 C 5 Caspase-3 vs. Caspase-7 Rates

t (min) 0 2 5 10203045 4.5

α -CLCA 4

3.5 α-TARBP2 * Log (C7 rate) 3 α-ARHGAP-4 α-GAPDH 2.5 3 3.5 4 4.5 5 5.5 Log (C3 Rate)

Fig. 2. In vitro activities of the apoptotic caspases. Triton lysates of Jurkat A3 cells were incubated with 10–1000 nM Caspase-3, -7, or -8, and sampled for 6–9 time points over 1–2h.A. Rank order of catalytic efficiencies and sequence specificity logos for: (i) caspase-3, (ii) -7, and (iii)-8.(B) Lysates treated with 250 nM caspase-3 were analyzed by immunoblot probing for fast (clathrin light chain A (CLCA)), medium (TARBP-2) or slow (ARHGAP4) substrates. (*—cross reactive band) Immunoblot against GAPDH confirms protein loading. (C) Rates for substrates cleaved by both caspase-3 and caspase-7 were compared. No marked correlation was found between the measured rates, though 48% of the observed data points were below the measurable range for both caspase-3 and BIOCHEMISTRY caspase-7 (2 × 103 and 5.3 × 102 ∕M∕s, respectively) (bottom leftmost point).

Agard et al. PNAS ∣ February 7, 2012 ∣ vol. 109 ∣ no. 6 ∣ 1915 Downloaded by guest on September 30, 2021 were unable to establish rates due to undetectable levels of N A termini at early time points or, rarely, due to nonfirst-order kinetics apparently caused by N-terminal instability and degrada- Relative Peptide Intensity 0.0 0.2 0.4 0.6 0.8 1.0 tion. A recent gel-based study determined that catalytic efficien- Stauro 0h Stauro 1h Stauro 2h Stauro 3h Stauro 4h Stauro 5h ∕ cies (kcat KM ) for the cleavage of eight purified mammalian substrates of caspase-3 ranged from 104 to 106 M−1 s−1, while the In vitro cleavage rate (/M/s) median rate for eleven noncognate Escherichia coli proteins was <2.0 E 3 >2.0 E 3 2 × 102 M−1 s−1 (17). These studies on purified proteins in vitro are consistent with a wide dynamic range of caspase-cleavage rates we see in a cellular context and suggest quantitative guide- B lines for assigning likely importance. Interestingly, treatment with caspase-9 plus APAF-1, a scaf- 100 folding protein necessary for caspase-9 activity (18), did not sig- 75 nificantly cleave any of the targeted substrates despite being fully 50 capable of cleaving a fluorescent substrate in vitro (Fig. S3E). We 25

note that some substrates are not detected by the N-terminal % of maximum signal 0 isolation method because of the small size of the tryptic fragment 02468 Time (h) or inaccessibility of the N terminus for labeling. Such is the case Caspase activity Cell viability of pro-caspase-3, a known caspase-9 substrate. Nonetheless, it is striking that no caspase-9 substrates were found and our data C suggests it has many fewer substrates than the others, including Staurosporine initiator caspase-8. Time (h) 012345 In aggregate we were able to assess catalytic efficiencies for α-Casp-3 20–40% of the targeted substrates within the measurable range α (5 × 102 to 2 × 105 M−1 s−1) for individual caspases. Investigation -Casp-7 of the primary sequences of the fastest approximately 20% of Time to half maximal intensity substrates for each caspase showed specificities similar to those α-PARP found against synthetic peptide substrates (19). Notably while the α-ARHGAP4 primary sequence specificities of caspases-3 and -7 were highly similar, the efficiencies of cleavage by these two enzymes were α-GAPDH not highly correlated (Fig. 2C). Interestingly, 60–80% of the tar- 3 −1 −1 gets showed efficiencies less than 2 × 10 M s , a cleavage rate 0102030 within 10-fold the range observed for caspase-3 cleaving noncog- # of peptides nate substrates derived from E. coli (17). This may suggest that Fig. 3. Cleavage of caspase substrates during apoptosis. (A) Jurkat A3 cells these poorly cleaved substrates play little role in apoptosis beyond were treated with 2 μM staurosporine for 0–5 h, lysed, and N termini were cellular dismantling. Nonetheless, poor rates of hydrolysis alone quantified. A heat map analysis of N termini was plotted ranking cleavages cannot rule out that limited proteolysis of certain targets may still by t1∕2 from slowest to fastest. Right: The cellular cleavage events are com- have important physiological consequences. pared to in vitro cleavages. The substrates are distributed into five equally sized bins, and the number of in vitro cleavages less than and greater than −1 −1 Endocytosis and miRNA Processing are Rapidly Inactivated by Cas- 2E3 M s are plotted. (B) Caspase activity and cell viability were monitored pase-Cleavage. In an effort to gain a global perspective into the over the time-course of staurosporine treatment. (C) Western blot analysis function of caspase-cleavages we analyzed the gene ontology of ra- of example caspase substrates during staurosporine treatment. The GAPDH targeted antibody confirms equal protein loading. pidly cleaved substrates using GO Miner (20). We found particular enrichment for proteins involved with RNA processing, negative signals reached a maximum between 3–5 h of treatment with regulators of metabolic processes, and endosomal trafficking. staurosporine. The appearance of caspase-cleaved substrates Within our dataset, two components of the miRNA processing correlates directly with luminescence-based measures of caspase pathway, DGCR8 and TARBP2, are rapidly targeted by multiple activity and cell viability (Fig. 3B) and with the cleavage rates of caspases. Recently, others have reported DICER to be cleaved by the apoptotic caspases and hallmark substrates (Fig. 3C). We as- caspases (21) and miRNA levels are known to fall during apoptosis sessed the relative priority in cellular cleavages by determining (22). Interestingly, several of the endosomal proteins were required for EGFR signaling (Fig. S4). Consistent with this observation, we the time to reach half maximal signal (Fig. S6D). These data cor- found that TRAIL-treatment blocked clathrin-mediated endo- related well with the catalytic efficiencies determined from the cytosis and altered endocytosis-dependent signaling. By contrast, addition of individual caspases to extracts which are plotted ad- signaling through AKT, a membrane-associated process was not jacent to the heat map analysis (Fig. 3A). For example, substrates 2 0 × 103 −1 −1 rapidly disrupted. Recently, Ashkenazi and coworkers reported cleaved at greater than . M s by at least one of the that cleavage of the clathrin adaptor subunit AP2α is important for apoptotic caspases (corresponding to t1∕2 of approximately 1 h sustaining TRAIL signaling by blocking receptor recycling (23). at expected physiological conditions of 100 nM caspase) were also Our data show more targets are cut early in this pathway suggesting among the set of rapidly cleaved cellular substrates. This obser- this to be a critical early step in apoptosis. vation is remarkable as a number of factors could have grossly perturbed this correlation, including stochastic induction of the Caspase Activities in Live Cells. To assess the cellular relevance of caspases, subcellular localization, and the possible activities of catalytic efficiencies determined in cell extracts, we monitored other caspases and unknown proteases during apoptosis (24). the rate of appearance of caspase-cleaved N termini in Jurkat These data suggest that even though the apoptotic caspases are cells treated with inducers of intrinsic (staurosporine) or extrinsic initially activated in the cytosol, they can access cellular compart- (TRAIL) apoptosis (Fig. 3A, Figs. S5 and S6 A–C, and ments very quickly in the apoptotic process. Notably, while clea- Dataset S3). Heat map analysis of the cleaved substrates revealed vage rates in lysates varied by more than 3 orders of magnitude, that, after an induction period, there was a consistent monotonic in cellular studies most substrates are processed with similar increase in the amounts of N termini generated. Generally the rates. Induction of apoptosis is stochastic (8), so analyses of cells

1916 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1117158109 Agard et al. Downloaded by guest on September 30, 2021 treated with apoptotic inducers include cells that have been ex- roles in apoptosis, transient caspase activation occurs during cellular posed to the caspases for significantly varying periods of time. As differentiation, including the transformations of monocytes to a result, the cellular data do not distinguish between the very fast macrophages and pluripotent stem cells to more specialized cell and the fast substrates, but only the slower substrates that appear types (25, 26). Perhaps these rapidly cleaved substrates are also later. We believe an advantage of the in vitro cleavage rates is important during transient caspase activation in differentiation. they better reflect the timing of cleavages that occur at the single The use of label-free SRM technologies to characterize enzyme- cell level because the induction of proteolysis is synchronized. substrate kinetics and the dynamics of cellular modifications has Comparing the proteolysis rates between TRAIL- and stuar- tremendous potential to prioritize posttranslational modifications osporine-mediated apoptosis reveals a modest biphasic correla- and provide fundamental enzymological constants for systems-level tion (Fig. 4A). This is consistent with cell viability and immuno- cellular modeling. blot data suggesting that staurosporine-mediated death is slower to initiate, but less stochastic than TRAIL-mediated death Methods Summary (Fig. 3 B–C. and Fig. S5). Mapping of the observed cleavage rates Cell Culture and Lysate Preparation. Jurkat clone A3 and U87-MG onto this cross correlation shows that most of the substrates (ATCC) were cultured as per ATCC guidelines. For in vitro clea- rapidly cleaved in vitro occur prior to the second phase of staur- vage experiments Jurkat cells were lysed (2 × 108 cells∕mL) in osporine-mediated death, while slowly cleaved substrates were triton X-100 (0.1%), with HEPES (100 mM pH 7.4), and protease equally distributed across both phases (Fig. 4b). Interestingly, five inbibitors [EDTA (1 mM), AEBSF (1 mM), PMSF (1 mM), and of six substrates with half-lives less than 2 h in stuarosporine- iodoacetamide (10 mM)] for 15 min at 4 °C, quenched by the mediated death (dashed rectangle Fig. 4A) were involved with addition of DTT (20 mM) for 15 min 4 °C. For caspase-9 assays, mRNA processing, while three of four substrates cleaved rapidly KCl (10 mM), MgCl2 (1.5 mM), and sucrose (1.5%) were in- in TRAIL-mediated death but slowly via the intrinsic pathway cluded in the lysis buffer. The lysates were clarified by centrifuga- (circle Fig. 4A) were involved in endocytosis. Thus, while the de- tion (4;100 × g, 5 min, 2x). For analysis of cellular cleavages, cells molition programs of staurosporine and TRAIL-mediated death were treated with TRAIL (2 nM, Peprotech) or Staurosporine are highly similar, each has a set of inducer-specific preferred (2 μM, LC labs) for 0–6 h, lysed (8 × 108 cells∕mL) in SDS (4%), substrates. Bicine (400 mM pH 8.0), with protease inhbitors [EDTA (1 mM), AEBSF (1 mM), PMSF (1 mM), E-64 (0.1 mM), and zVAD-fmk Conclusions and Perspective. These studies present a global approach (0.1 mM)], and DNA was fragmented via probe sonication. The to characterize the kinetics of protease-substrate pairs in their native lysates were diluted with triton X-100 followed by water (final states allowing us to prioritize substrates for this important class of concentrations 1% SDS, 1% triton X-100, 100 mM Bicine), pel- posttranslational modifications. Interestingly, in addition to their leted to remove any insoluble material (7;500 × g, 15 min), re- duced by treatment with TCEP hydrochloride (2 mM) at 95 °C A 3 for 10 min, and alkylated with iodoacetamide (4 mM) in the dark for 1 h.

In Vitro Cleavage Assays and N-terminal Isolation. Lysates were trea- 2 ted with recombinant caspases-3, -7, -8, or -9 plus APAF-1, for the indicated periods of time at RT, and quenched with zVAD-fmk (0.1 mM). TEVest4B (Fig. S3E) (10 mM in DMSO) was added to TRAIL (h) ∕ μ

1/2 the lysates (10% v v), followed by subtiligase (1 M) and incu-

t 1 bated at RT for 1 h. Samples were precipitated in CH3CN (80% v∕v), resolubilized in guanidine hydrochloride (8 M, 10 mg∕mL final protein concentration) and N termini were isolated as 0 described (9). 012345

t1/2 Staurosporine (h) LC/MS/MS. For discovery experiments, peptides were analyzed via two-dimensional reversed-phase LC/MS/MS. Samples were frac- B 3 tionated by offline high pH reversed-phase chromatography with a 70 min gradient on a 1 × 150 mm XBridge C18 3.5 μM with a flow rate of 75 μL∕ min. Fractions were analyzed by low pH reversed-phase chromatography with a 90 min gradient on a 2 0.1 × 100 mm column and a flow rate of 1 μL∕ min on a nanoAC- QUITY UPLC system (Waters). The capillary column was coupled to a QSTAR Elite mass spectrometer (Applied Biosys-

TRAIL (h) tems) for peptide identification or a QTRAP 5500 mass spectro- 1/2 t 1 meter (AB Sciex) for transition qualification. For analysis of cleavage kinetics, samples were analyzed via coupling of the same system to a QTRAP 5500 mass spectrometer.

0 012345 Data Analysis. Peptide identification was performed as de-

t1/2 Staurosporine (h) scribed.(3) Prospective transitions were generated using in-house scripts and fractionated peptides were qualified by monitoring Fig. 4. Correlation between cleavage rates in staurosporine- and TRAIL- for >3 coeluting transitions within 2.5 min of the expected reten- t mediated cell death. (A) Time to reach half-maximal signal ( 1∕2) for peptides tion time (Supporting Information). Integrations for qualified pep- identified in stuarosporine and TRAIL-mediated deaths were imaged and tides were performed using Supplementary Script 4, and approximated by a biphasic fit. Substrates cleaved in staurosporine-mediated apoptosis with a t1∕2 less than 2 h are boxed, and those cleaved rapidly in normalized via a set of qualified peptides monitoring endogenous TRAIL-treated cells, but slowly in staurosporine-mediated cells are circled. (noncaspase-cleaved) N termini. For cellular cleavage experi-

3 −1 −1 4 BIOCHEMISTRY (B) Substrates cleaved faster than 2.0 × 10 M s during in vitro experiments ments, peptides with significant cleavage (Integrationfinal > 3 −1 −1 (red) and those cleaved at less than 2.0 × 10 M s (blue) are plotted. Integration0) were analyzed. For in vitro cleavage experiments

Agard et al. PNAS ∣ February 7, 2012 ∣ vol. 109 ∣ no. 6 ∣ 1917 Downloaded by guest on September 30, 2021 peptides with significant cleavage were fit to pseudo-first-order tated in GO Miner were manually evaluated for references to the kinetic equations via Graphpad Prizm. Spectra from peptides indicated functional categories. with statistically significant fits (r2 > 0.9) were manually validated to investigate the presence of interfering peaks, or peak mis- Analysis of Endocytosis. Analysis of endocytosis in Jurkat A3 cells assignment. was performed as described (23). To assess phosphorylation of U87-MG cells they were grown in serum-free media for 12 h, Immunoblotting of Caspase Cleavages and Cell Viability Assays. Small treated with or without TRAIL (10 nM) for 2.5 h, and EGF portions of the mass spectrometry samples were reserved prior to (3 ng∕mL, Cell Signaling) was added. Cells were lysed in 1% SDS biotinylation for immunoblot analysis. Lysates were normalized with sonication, and probed with appropriate antibodies. Antibo- to approximately 4 mg∕mL prior to analysis by SDS-PAGE and western blot. The antibodies used in western blots included: dies used include anti-p-Tyr-100 (9411), anti-AKT pT308 (4056), anti-Casp-3 (9662), anti-Casp-7 (9494), anti-PARP (9542), anti- and anti-ERK1/2 pT202/pY204 (9106) from Cell Signaling and GAPDH (2118) from Cell Signaling, anti-DGCR-8 (H00054487- anti-pEGFR pY1173 (101668) from Santa Cruz Biotechnology. A01), anti-ARHGAP4(H00000393-M01), and anti-TARBP2 (H00006895-M01) from Novus Biotech, and anti-CLCA (X16), a ACKNOWLEDGMENTS. We would like to thank David Wildes, Prof. Frances generous gift from Professor Frances Brodsky. Cell viability and Brodsky, and the University of California, San Francisco Mass Spectrometry caspase activity assays were performed with Cell Titer glo and for invaluable discussions. We would like to thank Julie Zorn and Prof. Dennis Caspase glo (Promega) per the manufacturers recommendations. Wolan for providing the recombinant caspases. This work has been supported by National Institutes of Health F32 AI077177 (N.J.A.), R01 GM081051 (J.A.W.). Mass spectrometry was performed at the Bio-Organic Biomedical Bioinformatics. Cleaved substrates were analyzed by GO Miner Mass Spectrometry Resource at UCSF (A.L.B., Director) supported by the using the collection of all substrates with measureable or limited Biomedical Research Technology Program of the National Institutes of Health rates as the total dataset, and substrates known to be cleaved at National Center for Research Resources, NIH NCRR P41RR001614 and 2 −1 −1 >5.3 × 10 M s as the enriched dataset. Substrates not anno- 1S10RR026662.

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