title: Comparative analysis of cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics.
abstract: The advances in bioorthogonal ligation methods have provided new opportunities for proteomic analysis of newly synthesized proteins, posttranslational modifications, and specific enzyme families using azide/alkyne-functionalized chemical reporters and activity-based probes. Efficient enrichment and elution of azide/alkyne-labeled proteins with selectively cleavable affinity tags are essential for protein identification and quantification applications. Here, we report the synthesis and comparative analysis of Na₂S₂O₄-cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics. We demonstrated that ortho-hydroxyl substituent is required for efficient azobenzene-bond cleavage and show that these cleavable affinity tags can be used to identify newly synthesized proteins in bacteria targeted by amino acid chemical reporters as well as their sites of modification on endogenously expressed proteins. The azobenzene-based affinity tags are compatible with in-gel, in-solution, and on-bead enrichment strategies and should afford useful tools for diverse bioorthogonal proteomic applications.
authors.affiliation: Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Yu-Ying
authors.initials: YY
authors.lastname: Yang
authors.forename: Markus
authors.initials: M
authors.lastname: Grammel
authors.forename: Anuradha S
authors.initials: AS
authors.lastname: Raghavan
authors.forename: Guillaume
authors.initials: G
authors.lastname: Charron
authors.forename: Howard C
authors.initials: HC
authors.lastname: Hang
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry*; D000480:Alkynes / Q000737:chemistry*; D001386:Azides / Q000737:chemistry; D001391:Azo Compounds / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D002210:Caprylates / Q000737:chemistry*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000737:chemistry; D004227:Dithionite / Q000737:chemistry; D013058:Mass Spectrometry; D009646:Norleucine / Q000737:chemistry*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics; D012486:Salmonella typhimurium / Q000378:metabolism
chemical_list: C554399:2-aminooctynoic acid; D000345:Affinity Labels; D000480:Alkynes; D001386:Azides; D001391:Azo Compounds; D001426:Bacterial Proteins; D002210:Caprylates; D020543:Proteome; D004227:Dithionite; D003300:Copper; D009646:Norleucine; C009850:azobenzene