title: Click-MS: Tagless Protein Enrichment Using Bioorthogonal Chemistry for Quantitative Proteomics.
abstract: Epitope-tagging is an effective tool to facilitate protein enrichment from crude cell extracts. Traditionally, N- or C-terminal fused tags are employed, which, however, can perturb protein function. Unnatural amino acids (UAAs) harboring small reactive handles can be site-specifically incorporated into proteins, thus serving as a potential alternative for conventional protein tags. Here, we introduce Click-MS, which combines the power of site-specific UAA incorporation, bioorthogonal chemistry, and quantitative mass spectrometry-based proteomics to specifically enrich a single protein of interest from crude mammalian cell extracts. By genetic encoding of p-azido-l-phenylalanine, the protein of interest can be selectively captured using copper-free click chemistry. We use Click-MS to enrich proteins that function in different cellular compartments, and we identify protein-protein interactions, showing the great potential of Click-MS for interaction proteomics workflows.
authors.affiliation: Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen , Nijmegen, The Netherlands.
authors.forename: Arne H
authors.initials: AH
authors.lastname: Smits
authors.affiliation: Department of Bio-organic Chemistry, Faculty of Science, Institute for Molecules and Materials, Radboud University Nijmegen , Nijmegen, The Netherlands.
authors.forename: Annika
authors.initials: A
authors.lastname: Borrmann
authors.affiliation: Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen , Nijmegen, The Netherlands.|Department of Bio-organic Chemistry, Faculty of Science, Institute for Molecules and Materials, Radboud University Nijmegen , Nijmegen, The Netherlands.
authors.forename: Mark
authors.initials: M
authors.lastname: Roosjen
authors.affiliation: Department of Bio-organic Chemistry, Faculty of Science, Institute for Molecules and Materials, Radboud University Nijmegen , Nijmegen, The Netherlands.
authors.forename: Jan C M
authors.initials: JC
authors.lastname: van Hest
authors.affiliation: Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen , Nijmegen, The Netherlands.
authors.forename: Michiel
authors.initials: M
authors.lastname: Vermeulen
mesh_terms: D001386:Azides / Q000737:chemistry*; D057930:Click Chemistry / Q000379:methods*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D050794:STAT1 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001386:Azides; D004268:DNA-Binding Proteins; C115369:MBD3 protein, human; D050794:STAT1 Transcription Factor; C027162:4-azidophenylalanine; D010649:Phenylalanine