title: Selective 351 nm photodissociation of cysteine-containing peptides for discrimination of antigen-binding regions of IgG fragments in bottom-up liquid chromatography-tandem mass spectrometry workflows.
abstract: Despite tremendous inroads in the development of more sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies for mass spectrometry-based proteomics, there remains a significant need for enhancing the selectivity of MS/MS-based workflows for streamlined analysis of complex biological mixtures. Here, a novel LC-MS/MS platform based on 351 nm ultraviolet photodissociation (UVPD) is presented for the selective analysis of cysteine-peptide subsets in complex protein digests. Cysteine-selective UVPD is mediated through the site-specific conjugation of reduced cysteine residues with a 351 nm active chromogenic Alexa Fluor 350 (AF350) maleimide tag. Only peptides containing the AF350 chromophore undergo photodissociation into extensive arrays of b- and y-type fragment ions, thus providing a facile means for differentiating cysteine-peptide targets from convoluting peptide backgrounds. With the use of this approach in addition to strategic proteolysis, the selective analysis of diagnostic heavy-chain complementarity determining regions (CDRs) of single-chain antibody (scAb) fragments is demonstrated.
authors: Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station A5300, Austin, TX 78712, USA.
authors: Victoria C
authors: VC
authors: Cotham
authors: Yariv
authors: Y
authors: Wine
authors: Jennifer S
authors: JS
authors: Brodbelt
mesh_terms: D000595:Amino Acid Sequence; D000941:Antigens / Q000276:immunology / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D022801:Complementarity Determining Regions / Q000032:analysis*; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D055668:Photochemical Processes; D040901:Proteomics; D017386:Sequence Homology, Amino Acid; D057127:Single-Chain Antibodies / Q000276:immunology / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D053719:Tandem Mass Spectrometry / Q000379:methods*; D057188:Workflow
chemical_list: D000941:Antigens; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G; D010446:Peptide Fragments; D057127:Single-Chain Antibodies; D003545:Cysteine