Identification of proteolytic products and natural protein N-termini by Terminal Amine Isotopic Labeling of Substrates (TAILS)

Alain Doucet, Oded Kleifeld, Jayachandran Kizhakkedathu, Christopher M Overall

Research output: Chapter in Book/Report/Conference proceedingChapter (Book)Researchpeer-review

23 Citations (Scopus)

Abstract

Determining the sequence of protein N-termini and their modifications functionally annotates proteins since translation isoforms, posttranslational modifications, and proteolytic truncations direct localization, activity, and the half-life of most proteins. Here we present in detail the steps required to perform our recently described approach we call Terminal Amine Isotopic Labeling of Substrates (TAILS), a combined N-terminomics and protease substrate discovery degradomics platform for the simultaneous quantitative and global analysis of the N-terminome and proteolysis in one MS/MS experiment. By a 3-day procedure with flexible alpha- and varepsilon-amine labeling and blocking options, TAILS removes internal tryptic and C-terminal peptides by binding to a dendritic polyglycerol aldehyde polymer. Therefore, by negative selection, this enriches for both the N-terminal-labeled peptides and all forms of naturally blocked N-terminal peptides. In addition to providing valuable proteome annotation, the simultaneous analysis of the original mature N-terminal peptides enables these peptides to be used for higher confidence protein substrate identification by two or more different and unique peptides. Second, the analysis of the N-terminal peptides forms a statistical classifier to determine valid isotope ratio cutoffs in order to identify with high-confidence protease-generated neo-N-terminal peptides. Third, quantifying the loss of acetylated or cyclized N-terminal peptides that have been cleaved extends overall substrate coverage. Hence, TAILS allows for the global analysis of the N-terminome and determination of cleavage site motifs and substrates for protease including those with unknown or broad specificity.
Original languageEnglish
Title of host publicationMethods in Molecular Biology - Gel-Free Proteomices
EditorsK Gevaert, J Vandekerckhove
Place of PublicationUSA
PublisherHumana Press
Pages273 - 287
Number of pages15
Edition1st
ISBN (Print)9781617791475
DOIs
Publication statusPublished - 2011
Externally publishedYes

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