Global kinetic analysis of proteolysis via quantitative targeted proteomics

Abstract

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.

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 efficiency (k_cat/K_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).

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 1–2 h. 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 caspase-7 (2 × 10³ and 5.3 × 10² /M/s, respectively) (bottom leftmost point).

Fig. 3.

Cleavage of caspase substrates during apoptosis. (A) Jurkat A3 cells were treated with 2 μM staurosporine for 0–5 h, lysed, and N termini were quantified. A heat map analysis of N termini was plotted ranking cleavages by t_1/2 from slowest to fastest. Right: The cellular cleavage events are compared 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 2E3 M^-1 s^-1 are plotted. (B) Caspase activity and cell viability were monitored over the time-course of staurosporine treatment. (C) Western blot analysis of example caspase substrates during staurosporine treatment. The GAPDH targeted antibody confirms equal protein loading.

Fig. 4.

Correlation between cleavage rates in staurosporine- and TRAIL-mediated cell death. (A) Time to reach half-maximal signal (t_1/2) for peptides identified in stuarosporine and TRAIL-mediated deaths were imaged and approximated by a biphasic fit. Substrates cleaved in staurosporine-mediated apoptosis with a t_1/2 less than 2 h are boxed, and those cleaved rapidly in TRAIL-treated cells, but slowly in staurosporine-mediated cells are circled. (B) Substrates cleaved faster than 2.0 × 10³ M^-1 s^-1 during in vitro experiments (red) and those cleaved at less than 2.0 × 10³ M^-1 s^-1 (blue) are plotted.