A new mass spectrometric process to generate biologically relevant, database searchable, peptide libraries to screen sequence specificity of post-translational modifications like proteolysis and phosphorylation

Advantages

• CLIP-PICS generates database-searchable peptide libraries that represent biological sequence diversity
• CLIP-PICS can be used to determine sequence specificity of post-translational modifications (proteolysis, phosphorylation) and receptor binding. CLIP-PICS enables application of LC-MS/MS as a high-throughput, high-content readout for the screen
• Identification of individual peptidic sequences results in a detailed specificity “landscape” that surpasses the information content of conventional, pooled sequencing approaches.
• For proteolysis, CLIP-PICS determine sequences amino- and carboxy terminal to the scissile peptide simultaneously in the same experiment. CLIP-PICS is suitable for all mechanistic classes of proteases (aspartic, serine, cysteine and metalloprotease).
• CLIP-PICS is a facile technique without the need for synthetic chemistry and is based on commercially available reagents.
• CLIP-PICS is a rapid one-step biochemical, high through-put, high-content platform technique.

Technology Details

Over 550 proteases exist in man, playing a pivotal role in all physiological and pathological processes.  Understanding the cleavage site motifs of these proteases is essential for their characterization.  Researchers at The University of British Columbia have developed a new and improved mass spectrometry process to determine protease cleavage sites. This process has been named Cellular Library of Peptide Substrates-Proteomic Identification of Protease Cleavage Specificity or CLIP-PICS. The process employs various biochemical steps to chemically modify and manipulate the natural proteome in order to allow for the precise selection of cleavage sites for the proteases in question.

• The natural proteome could consist of, but is not limited to cell conditioned medium, cells lysates, tissue extracts and body fluids etc.
• The natural proteome is fragmented into oligopeptides using a variety of endoproteases thus creating a complementary and extensive peptide library from the same biological source.
• The natural oligopeptides are biochemically modified and purified such that neo-termini  (carboxy and amino) can be captured and released from the mixture
• Isolated and tagged cleavage products are sequenced by tandem mass spectrometry. MS/MS analysis of the neo-amino and carboxy termini yields information regarding prime and non-prime substrate residues, respectively.
• This workflow can easily be modified for other types of screening applications, eg phosphorylation.