Global analysis of cellular proteolysis by selective enzymatic labeling of protein N-termini

Abstract

Proteolysis is a critical modification leading to alteration of protein function with important outcomes in many biological processes. However, for the majority of proteases, we have an incomplete understanding of both cellular substrates and downstream effects. Here, we describe detailed protocols and applications for using the rationally engineered peptide ligase, subtiligase, to specifically label and capture protein N-termini generated by proteases either induced or added to complex biological samples. This method allows identification of the protein targets as well as their precise cleavage locations. This approach has revealed >8000 proteolytic sites in healthy and apoptotic cells including >1700 caspase cleavages. One can further determine substrate preferences through rate analysis with quantitative mass spectrometry, physiological substrate specificities, and even infer the identity of proteases operating in the cell. In this chapter, we also describe how this experimental method can be generalized to investigate proteolysis in any biological sample.

Keywords: Apoptosis; Caspase; Degradomics; Mass spectrometry; Proteolysis; Proteomics; Selected reaction monitoring; Subtiligase.

Figure 13.1

An overview of the subtiligase N-terminal labeling method. (A) Proteins with free N-termini in a mixture are selectively tagged using the engineered enzyme, subtiligase. Whole protein samples are incubated with subtiligase and the peptide ester containing a biotin tag. After enzymatic labeling, free N-termini are captured on avidin beads. Proteins are digested by trypsin. The final N-terminal peptide is released from beads via TEV protease cleavage and identified by mass spectrometry. (B) The current peptide ester contains an ester subtiligase acylation site, Abu-tag for positive mass spectrometry identification, a TEV protease site and a biotin label. The peptide ester can be further modified for specific experimental needs.

Figure 13.2

A schematic difference between Forward and Reverse experiments. Forward experiments use samples from intact biological systems, either perturbed or unperturbed that is then harvested, lysed, and labeled. Reverse experiments involve exogenous addition of protease to whole cell or tissue lysate of interest followed by labeling.

Figure 13.3

Monitoring apoptosis and proteomic distribution of cleavage substrates. (A) Measuring cell viability and caspase activation. It is important to monitor apoptosis versus time after exposure to drug, as the rate of apoptosis can vary substantially depending on the drug. Caspase activity appears before cell viability decreases. (B) Comparison of caspase substrates identified versus broad range of baseline protein abundance. Protein abundance estimated derived from PaxDB. Extensive distribution overlap indicates that subtiligase-based N-terminomics leads to broad coverage across 6-logs of abundance in the proteome. Figure adapted from Crawford et al. (2013) with permission of the authors.

Figure 13.4

Selected reaction monitoring (SRM) for proteolytic substrate quantification. (A) Schematic diagram of triple quadrupole (Q1–Q2–Q3) mass spectrometer used for SRM. ESI, electrospray ionization, representing ionized peptides eluted from liquid chromatography column into the mass spectrometer. (B) Example data from SRM monitoring caspase-cleaved peptide from ATF4 protein during bortezomib-induced apoptosis. Each individual trace represents a parent-fragment ion pair (transition). Coelution of multiple transitions from the same peptide confirms peptide identity. Peak area can be used for quantification, with kinetic parameters derived based on change across the time course.

Figure 13.5

Monitoring kinetics of recombinant caspase cleavage. (A) SRM transitions show increase in intensity across time course after caspase addition. (B) Peptide intensities are fit to pseudo-first-order kinetic equations to determine kinetic efficiency (k_cat/K_m) for each substrate. (C and D) Rank order of catalytic efficiencies for substrates of caspases-3 and -7 span at least two orders of magnitude. Caspase-3 plot indicates substrates with rapid, medium, and slow cleavage. Figure adapted from Agard et al. (2012) with permission of the authors.

Figure 13.6

Plasma N-terminomics. Proteins identified with free N-termini in plasma demonstrate over six-order of magnitude range of abundance, demonstrating ability of subtiligase labeling to track low-abundance plasma proteins. Figure adapted from Wildes and Wells (2010) with permission of the authors.

Figure 13.7

Sequence features of identified N-termini. Aggregate plots across all N-termini shown using IceLogo, where amino acids favored at a given site are above the baseline while those disfavored are below (Colaert, Helsens, Martens, Vandekerckhove, & Gevaert, 2009). Cleavage occurs between position P1 (left of lightning bolt) and P1′. (A) Across all peptides identified in Forward Discovery experiments in apoptotic cells, Asp at P1 is highly enriched. (B) Focusing on only peptides shown in (A) with Asp at P1, a signature of caspase cleavage, we identify the canonical D-E-V-D cleavage motif for caspases from P4 to P1 site. (C and D) This motif is also conserved in N-termini identified in cell lysate incubated with recombinant caspase-3 during Reverse Discovery experiments.