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.
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