Deep profiling of protease substrate specificity enabled by dual random and scanned human proteome substrate phage libraries

Abstract

Proteolysis is a major posttranslational regulator of biology inside and outside of cells. Broad identification of optimal cleavage sites and natural substrates of proteases is critical for drug discovery and to understand protease biology. Here, we present a method that employs two genetically encoded substrate phage display libraries coupled with next generation sequencing (SPD-NGS) that allows up to 10,000-fold deeper sequence coverage of the typical six- to eight-residue protease cleavage sites compared to state-of-the-art synthetic peptide libraries or proteomics. We applied SPD-NGS to two classes of proteases, the intracellular caspases, and the ectodomains of the sheddases, ADAMs 10 and 17. The first library (Lib 10AA) allowed us to identify 104 to 105 unique cleavage sites over a 1,000-fold dynamic range of NGS counts and produced consensus and optimal cleavage motifs based position-specific scoring matrices. A second SPD-NGS library (Lib hP), which displayed virtually the entire human proteome tiled in contiguous 49 amino acid sequences with 25 amino acid overlaps, enabled us to identify candidate human proteome sequences. We identified up to 104 natural linear cut sites, depending on the protease, and captured most of the examples previously identified by proteomics and predicted 10- to 100-fold more. Structural bioinformatics was used to facilitate the identification of candidate natural protein substrates. SPD-NGS is rapid, reproducible, simple to perform and analyze, inexpensive, and renewable, with unprecedented depth of coverage for substrate sequences, and is an important tool for protease biologists interested in protease specificity for specific assays and inhibitors and to facilitate identification of natural protein substrates.

Citrullination of a phage-displayed human peptidome library reveals the fine specificities of rheumatoid arthritis-associated autoantibodies

“…The use of PTM PhIP-Seq is broadly applicable to studies of diverse PTMs, potentially adaptable to studies of glycosylation [49], carbamylation [50], phosphorylation [51], deamidation [52], and other modifications [6]. PTMs of the phage-displayed human peptidome may also be used to profile PTM-dependent protease substrate preference via the proteome-wide SEPARATE assay [53,54]. PTM PhIP-Seq is therefore a powerful addition to the programmable phage display toolkit with broad utility for genome-wide proteomic analyses.…”

Activatable Zymography Probes Enable In Situ Localization of Protease Dysregulation in Cancer

“…In contrast, further optimization is needed before such cleavage-based methods can be reliably leveraged as a tool to dissect protease biology with single enzyme specificity. High-throughput screens of phage-displayed substrate libraries (40) or peptides enriched with unnatural amino acids (41,42) may yield protease substrates with dramatically improved specificities.…”

Citrullination of a phage displayed human peptidome library reveals the fine specificities of rheumatoid arthritis-associated autoantibodies

“…6 PTMs of the phage displayed human peptidome may also be used to profile protease activity via the SEPARATE assay. 45,46 PTM PhIP-Seq is therefore a powerful addition to the programmable phage display toolkit with broad utility for genome-wide proteomic analyses.…”