RESUMO
Identification of the N terminus of proteins in complex samples has been accelerated in recent years by the increased power of mass spectrometers, bioinformatic tools and biochemical methods. We developed Terminal Amine Isotopic Labeling of Substrates (TAILS), a powerful method for the isolation, identification and quantification of N terminal peptides comprising the N terminome from proteome in vitro analyses, cultured cells and tissues. TAILS depletes internal tryptic peptides so enriching for the natural and cleaved neo N-terminal peptides present in complex proteome samples. To do so N-terminal peptides are blocked at the protein level by isotopically labeled amine reactive reagents, then digested with trypsin. TAILS relies on a water-soluble 100-kDa highly-branched polyglycerol polymer (HPG-ALD) developed in our laboratories to bind and deplete the sample of tryptic peptides. Polymer-bound internal tryptic peptides are separated from the blocked N-terminal peptides by spin filters. Here we describe a revised TAILS protocol that uses a newly developed 800-kDa HPG-ALD polymer that allows precipitation of the polymer post-depletion. Precipitation has multiple advantages over spin filters including maximal N-terminal peptide recovery and faster high-throughput N-terminome identification and quantification in any sample type.