Global substrate specificity profiling of post-translational modifying enzymes

Abstract

Enzymes that modify the proteome, referred to as post-translational modifying (PTM) enzymes, are central regulators of cellular signaling. Determining the substrate specificity of PTM enzymes is a critical step in unraveling their biological functions both in normal physiological processes and in disease states. Advances in peptide chemistry over the last century have enabled the rapid generation of peptide libraries for querying substrate recognition by PTM enzymes. In this article, we highlight various peptide-based approaches for analysis of PTM enzyme substrate specificity. We focus on the application of these technologies to proteases and also discuss specific examples in which they have been used to uncover the substrate specificity of other types of PTM enzymes, such as kinases. In particular, we highlight our multiplex substrate profiling by mass spectrometry (MSP-MS) assay, which uses a rationally designed, physicochemically diverse library of tetradecapeptides. We show how this method has been applied to PTM enzymes to uncover biological function, and guide substrate and inhibitor design. We also briefly discuss how this technique can be combined with other methods to gain a systems-level understanding of PTM enzyme regulation and function.

Keywords: kinases; mass spectrometry; peptide libraries; peptide synthesis; post-translation modifying enzymes; proteases; substrate specificity.

Figure 1

Construction of positional scanning‐synthetic combinatorial libraries for analysis of prime side specificity. (A) During solid‐phase peptide synthesis, 7‐amino‐4‐methylcoumarin (AMC)‐based positional scanning‐synthetic combinatorial libraries are generally conjugated to the solid support via the side chain of the P1 amino acid, while 7‐amino‐4‐carbamoylmethylcoumarin (ACC)‐based libraries are conjugated directly through the fluorophore. (B) The four P_n sublibraries each contain 20 distinct pools of substrates, where one amino acid is fixed at P_n position and the remaining positions (X) contain an equimolar mixture of amino acids.

Figure 2

The ecotin peptide (purple) binds to the active site of granzyme B in a linear conformation.64 The catalytic triad is shown in red. Recognition of the peptide, IEPD (written P4‐P1), is dominated by the S4 and S1 pockets (circled in yellow).

Figure 3

MSP‐MS workflow for protease specificity determination. A recombinant protease, patient sample, conditioned media, or other complex, protease‐containing biological sample is added to the MSP‐MS peptide library. Aliquots are removed at specific time points and peptide cleavage is assessed through LC‐MS/MS analysis. Cleavage‐site identification can be used to construct a sequence logo representation of the global substrate specificity. Cleavage product quantification enables the kinetic analysis of individual substrate cleavage events.

Figure 4

Application of the MSP‐MS library to other PTM enzymes. (A) A general scheme for the specificity analysis of PTM enzymes using the MSP‐MS assay. PTM enzymes are incubated with the peptide library and modification is detected through LC‐MS/MS analysis. (B) The kinase CDK2/cyclin A (shown in blue) recognizes a peptide substrate (depicted as green sticks) in a linear conformation.89 The active site lysine residue is colored red. (C) Similarly, the histone acetyltransferase HAT1 also binds a linear substrate.88