Protease specificity profiling by tandem mass spectrometry using proteome-derived peptide libraries

Oliver Schilling, Ulrich Auf Dem Keller, Christopher M. Overall

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

Abstract

Protease specificity profiling using proteome-derived, database-searchable peptide libraries is a novel approach to define the active site specificity of proteolytic enzymes we call PICS (Proteomic Identification of protease Cleavage Sites). Proteome-derived peptide libraries are generated by trypsin, GluC, orchymotrypsin digestion of biologically relevant proteomes, such as cytosolic lysates, to generate three separate libraries that each differ from the others in their C-terminal amino acid residues according to the protease specificity. Primary amines of all peptides are then chemically protected so that after incubation with a test protease, the neo-N-termini of the prime-side cleavage products with exposed -αamines can be specifically biotinylated, enriched, and identified by liquid chromatography-tandem mass spectrometry. The corresponding nonprime-side sequences are derived bioinformatically. Suited for all protease classes except carboxyproteases and those aminoproteases and dipeptidases requiring a free -αamine for cleavage, PICS simultaneously profiles the specificity of prime and nonprime positions and directly determines scissile peptide bonds of up to hundreds of cleavage site sequences in a single experiment. This wealth of sequence specificity information also allows for the investigation of subsite cooperativity. Herein we describe a simplified procedure to produce PICS peptide libraries, the methods to perform a PICS assay, and a new method of data analysis.

Original languageEnglish
Title of host publicationGel-Free Proteomics: Methods and Protocols
Volume753
Publication date2011
Pages257-272
Chapter17
ISBN (Print)9781617791475
DOIs
Publication statusPublished - 2011
Externally publishedYes
SeriesMethods in Molecular Biology
Volume753
ISSN1064-3745

Keywords

  • Active site specificity
  • Peptide library
  • Protease profiling
  • Protease specificity
  • Subsite cooperativity

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