Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics

Abstract

We present protease specificity profiling based on quantitative proteomics in combination with proteome-derived peptide libraries. Peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines before exposure to a protease under investigation. After incubation with a test protease, treated and control libraries are differentially isotope-labeled using cost-effective reductive dimethylation. Upon analysis by liquid chromatography-tandem mass spectrometry, cleavage products of the test protease appear as semi-specific peptides that are enriched for the corresponding isotope label. We validate our workflow with two proteases with well-characterized specificity profiles: trypsin and caspase-3. We provide the first specificity profile of a protease encoded by a human endogenous retrovirus and for chlamydial protease-like activity factor (CPAF). For CPAF, we also highlight the structural basis of negative subsite cooperativity between subsites S1 and S2'. For A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4, -5, and -15, we show a canonical preference profile, including glutamate in P1 and glycine in P3'. In total, we report nearly 4000 cleavage sites for seven proteases. Our protocol is fast, avoids enrichment or synthesis steps, and enables probing for lysine selectivity as well as subsite cooperativity. Due to its simplicity, we anticipate usability by most proteomic laboratories.

Fig. 1.

Overview of workflow. Peptide libraries are prepared by specific endoproteolytic digestion of complex proteomes, such as cell lysates. Following inactivation of the digestion protease and clean-up, the peptide library is split in two parts. One aliquot is incubated with the protease under investigation; the second aliquot serves as a control. After incubation with the test protease, the peptide library aliquots are differentially labeled using stable isotope compounds, such as dimethylation of primary amines with either light (C¹²H₂O) or heavy (C¹³D₂O) formaldehyde. Equal amounts of the labeled protease-treated and control library are mixed and analyzed by LC-MS/MS. Cleavage events lead to semi-specific peptides enriched in the sample treated with the test protease. The matching prime or nonprime sequences are derived bioinformatically by database searches, similar to the original PICS strategy (5).

Fig. 2.

(A) Specificity profiling of trypsin using a GluC peptide library. The protease:library ratio was 1:500 (wt/wt). Incubation at pH 8.0 occurred for either 1 h or 16 h at 37 °C. The histograms show the fold-change value distribution (log₂ of label ratios for trypsin/control) of the semi-specific peptides. Semi-specific peptides with a more than eightfold enrichment (log2 fold-change value > 3) for the protease-treated sample (here: trypsin) are considered to represent specific cleavage events mediated by the test protease. These were used for reconstruction of the substrate cleavage sites, which were aligned and summarized as heat maps clearly showing the expected stringent trypsin specificity with arginine and lysine in P1. (B) Specificity profiling of human caspase-3. The protease:library ratio was 1:300 (wt/wt). Incubation at pH 7.4 occurred for 3 h at 37 °C. The specificity heatmap is in line with canonical description of caspase-3 specificity, with the exception of the P3 position. However, caspase-3 affinity for aliphatic residues in P3 and a preference for DLVD over the DEVD sequence present in common synthetic caspase-3 substrates have also been reported by others (51). (C) Specificity profiling of human endogenous retrovirus HERV-K(HML-2) protease. The protease:library ratio was 1:100 (wt/wt). Incubation at pH 5.0 occurred for 16 h at 37 °C. P1 constitutes the major specificity determinant with a preference for aromatic residues.

Fig. 3.

(A) Specificity profiling of chlamydial protease-like activity factor. The protease:library ratio was 1:500 (wt/wt). Incubation at pH 7.5 occurred for either 1 h or 16 h at 37 °C. P1 constitutes the major specificity determinant with a mixed preference for either small (alanine, glycine) or aliphatic (methionine) residues. (B and C) Negative correlation between P1 methionine and P2′ isoleucine. (D) Structural modeling of the peptide sequences VM↓VA or VM↓VI (“↓” indicates the cleavage site) in the active site of CPAF (“Ser” denotes active site serine 499).

Fig. 4.

Specificity profiling of proteases of the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family. The protease:library ratio was 1:50 (wt/wt). Incubation at pH 7.5 occurred for 16 h at 37 °C. P1 constitutes the major specificity determinant with a canonical preference for glutamate.