Bioorthogonal Peptide Enrichment from Complex Samples Using a Rink-Amide-Based Catch-and-Release Strategy

Abstract

Uptake and processing of antigens by antigen presenting cells (APCs) is a key step in the initiation of the adaptive immune response. Studying these processes is complex as the identification of low abundant exogenous antigens from complex cell extracts is difficult. Mass-spectrometry based proteomics – the ideal analysis tool in this case – requires methods to retrieve such molecules with high efficiency and low background. Here, we present a method for the selective and sensitive enrichment of antigenic peptides from APCs using click-antigens; antigenic proteins expressed with azidohomoalanine (Aha) in place of methionine residues. We here describe the capture of such antigens using a new covalent method namely, alkynyl functionalized PEG-based Rink amide resin, that enables capture of click-antigens via copper-catalyzed azide-alkyne [2 + 3] cycloaddition (CuAAC). The covalent nature of the thus formed linkage allows stringent washing to remove a-specific background material, prior to retrieval peptides by acid-mediated release. We successfully identified peptides from a tryptic digest of the full APC proteome containing femtomole amounts of Aha-labelled antigen, making this a promising approach for clean and selective enrichment of rare bioorthogonally modified peptides from complex mixtures.