In-Depth Specificity Profiling of Endopeptidases Using Dedicated Mix-and-Split Synthetic Peptide Libraries and Mass Spectrometry

Abstract

Proteases comprise the class of enzymes that catalyzes the hydrolysis of peptide bonds, thereby playing a pivotal role in many aspects of life. The amino acids surrounding the scissile bond determine the susceptibility toward protease-mediated hydrolysis. A detailed understanding of the cleavage specificity of a protease can lead to the identification of its endogenous substrates, while it is also essential for the design of inhibitors. Although many methods for protease activity and specificity profiling exist, none of these combine the advantages of combinatorial synthetic libraries, i.e., high diversity, equimolar concentration, custom design regarding peptide length, and randomization, with the sensitivity and detection power of mass spectrometry. Here, we developed such a method and applied it to study a group of bacterial metalloproteases that have the unique specificity to cleave between two prolines, i.e., Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-side specificity of PPEP-1 and PPEP-2, but also revealed some new unexpected peptide substrates. Moreover, we have characterized a new PPEP (PPEP-3) that has a prime-side specificity that is very different from that of the other two PPEPs. Importantly, the approach that we present in this study is generic and can be extended to investigate the specificity of other proteases.

Figure 1

Overview of the PPEPs used in this study. (A) The three PPEPs that are used in this study and their respective origins and substrate specificity. For PPEP-1 and 2 the cleavage specificity is based on the endogenous substrates. For PPEP-3, no substrates have been described yet. (B) The genomic architecture of the PPEPs and their substrates. For PPEP-1, the gene encoding the substrate CD2831 is adjacent to PPEP-1. The gene encoding the second substrate (CD3246) is positioned elsewhere in the genome. The genes for PPEP-2 and its substrate VMSP are located adjacent to each other. For PPEP-3, no adjacent genes contain the consensus PPEP cleavage motif (i.e., PPXP). (C) Crystal (PPEP-1 and PPEP-2) and predicted (PPEP-3) structures.^, PPEP-3 structure was predicted using the Alphafold algorithm.

Figure 2

Design of the synthetic combinatorial peptide library and workflow to determine the activity and prime-side specificity of a Pro-Pro endopeptidase (PPEP). The library was designed to contain an XXPPXX motif, with X representing any residue (X ≠ Cys). At the N-terminus, peptides were modified with a biotin, allowing removal of uncleaved peptides and N-terminal product peptides after incubation of the library with a protease, i.e., PPEP. At the C-terminus, a peptide tail (GGLEEF) was added in order for the C-terminal cleavage products to be compatible with LC-MS/MS analysis. This stretch of amino acids was also chosen based on a previously recorded MS/MS spectrum, showing favorable fragmentation characteristics (Figure S1). First, the library was precleaned on avidin beads to remove nonbiotinylated peptides. Then, the library was incubated with a PPEP. The scissile bond is indicated by the arrow. Following this, biotinylated peptides (noncleaved peptides and N-terminal product peptides) were captured on a streptavidin column. The flow-through, containing nonbiotinylated C-terminal product peptides (PXXGGLEEF) were then analyzed by LC-MS/MS, after which the prime-side specificity could be determined. Ahx: 1-aminohexanoic acid.

Figure 3

MALDI-FT-ICR MS analysis of PPEP-1 product peptides using two different combinatorial sublibraries. The P3 = Val and P3 = Lys sublibraries were incubated with PPEP-1 for 3 h. Following depletion of biotinylated peptides, nonbiotinylated product peptides (PXXGGLEEF) were analyzed using MALDI-FT-ICR MS. The two indicated sodiated species are from the PPPGGLEEG and P(I/L)PGGLEEF/(PP(I/L)GGLEEF peptides, respectively.

Figure 4

Top 10 most highly abundant 9-mer product peptides of PPEP-1, -2, and -3 reveal differences in prime-side specificity. The full combinatorial peptide library was incubated with recombinant PPEP-1, PPEP-2, or PPEP-3. Product peptides were analyzed using LC-MS/MS. Abundances were determined by summing the intensities of singly and doubly charged peptides. Discrimination between PXP and PPX peptides relied on both inspection of fragmentation spectra and C18 column separation (Figures S3 and S4). The 10 most highly abundant 9-mer product peptides formed by PPEP-1 (A), PPEP-2 (B), and PPEP-3 (C) and their abundances are represented as bars. A cleavage motif was constructed based on the relative intensities of the products peptides. The sequence on the X-axis represents the P1′-P3′ residues of the PXXGGLEEF product peptides.

Figure 5

Extracted ion chromatograms of PXP(GGLEEF)/PPX(GGLEEF) product peptides after incubation with PPEPs reveal prime-side specificity profiles. The full combinatorial peptide library was incubated with each of the PPEPs for 3 h. A nontreated control was included to identify the amount of background peptides. After analysis of the product peptides using LC-MS/MS, EICs were constructed for all possible PXP/PPX product peptides (in total 19, both 1+ and 2+ m/z values were used). Discrimination between PXP and PPX peptides relied on both inspection of fragmentation spectra and separation on a C18 column (Figures S3 and S4). If product peptides were not separated on the column, lines indicate the relative abundances of the nonseparated peptides. Mass tolerance was set to 10 ppm.

Figure 6

Time course of PPEP-1 mediated cleavage of synthetic FRET-quenched peptides with permutations at the P2′ position. The PPEP-1 substrate peptide VNP↓PVP was permutated to generate FRET-quenched peptides (Lys_Dabcyl-EVNPPXPD-Glu_Edans) containing any of the standard 20 amino acids at the P2′ position. These peptides were incubated with PPEP-1 and fluorescence was measured during 1 h. Peptides are sorted from the top left to bottom right based on their cleavage efficiency.

Figure 7

Time course of cleavage of synthetic FRET peptides by PPEP-1, PPEP-2, and PPEP-3. (A) Cleavage of PPEP-1 (Lys_Dabcyl-EVNP↓PVPD-Glu_Edans) and PPEP-2 (Lys_Dabcyl-EPLP↓PVPD-Glu_Edans) substrate peptides, and their P2′ = Pro variants, by PPEP-1, PPEP-2, and PPEP-3. (B) Cleavage of peptides containing cleavage motifs from putative G. thermodenitrificans PPEP-3 substrates by PPEP-3. Only the core sequences (P3-P3′) of the individual FRET-quenched peptides are indicated.