A bead-based cleavage method for large-scale identification of protease substrates

Abstract

Proteolysis is a major form of post translational modification which occurs when a protease cleaves peptide bonds in a target protein to modify its activity. Tracking protease substrates is indispensable for understanding its cellular functions. However, it is difficult to directly identify protease substrates because the end products of proteolysis, the cleaved protein fragments, must be identified among the pool of cellular proteins. Here we present a bead-based cleavage approach using immobilized proteome as the screening library to identify protease substrates. This method enables efficient separation of proteolyzed proteins from background protein mixture. Using caspase-3 as the model protease, we have identified 1159 high confident substrates, among which, strikingly, 43.9% of substrates undergo degradation during apoptosis. The huge number of substrates and positive support of in vivo evidence indicate that the BBC method is a powerful tool for protease substrates identification.

Figure 1. Screening of protease substrates by BBC method.

(A) Workflow of the bead-based cleavage (BBC) method for high-throughput screening of protease substrates. (B) Log₂Ratio distributions of all quantified peptides (H/L = caspase-3 treated/untreated). (C) The percentages of peptides derived from substrate proteins listed in CASBAH database for different log2Ratio values. (D) The dependence of the percentages of peptides derived from substrate proteins in CASBAH database on the peptide count numbers with log₂Ratio ≥ 1.8. (E) Overlap between substrates identified by BBC method and known substrates in CASBAH database. (F) Percentage of known substrates for identified hot and warm substrates.

Figure 2. Substrates degraded during apoptosis.

(A) Western blot analysis of in vitro cleavage of PARP, Nek9, Npl4 by caspase-3 with/without inhibitors. PARP as the known substrate of caspase-3 was used as a positive control, and GAPDH was used as the internal control. (B) Comparison of in vitro substrates identified by BBC method with degraded proteins during apoptosis identified by PROTOMAP and Subtiligase methods; (C) Percentage of substrates in “Hot” and “Warm” dataset found to be degraded during apoptosis identified by PROTOMAP and Subtiligase methods.