Middle-Down 193-nm Ultraviolet Photodissociation for Unambiguous Antibody Identification and its Implications for Immunoproteomic Analysis.

Autor:	Cotham VC; Department of Chemistry, ‡Center for Systems and Synthetic Biology, §Department of Biomedical Engineering, ∥Department of Chemical Engineering, ⊥Institute for Cellular and Molecular Biology, #Department of Molecular Biosciences, The University of Texas at Austin , Austin, Texas 78712, United States., Horton AP; Department of Chemistry, ‡Center for Systems and Synthetic Biology, §Department of Biomedical Engineering, ∥Department of Chemical Engineering, ⊥Institute for Cellular and Molecular Biology, #Department of Molecular Biosciences, The University of Texas at Austin , Austin, Texas 78712, United States., Lee J; Department of Chemistry, ‡Center for Systems and Synthetic Biology, §Department of Biomedical Engineering, ∥Department of Chemical Engineering, ⊥Institute for Cellular and Molecular Biology, #Department of Molecular Biosciences, The University of Texas at Austin , Austin, Texas 78712, United States., Georgiou G; Department of Chemistry, ‡Center for Systems and Synthetic Biology, §Department of Biomedical Engineering, ∥Department of Chemical Engineering, ⊥Institute for Cellular and Molecular Biology, #Department of Molecular Biosciences, The University of Texas at Austin , Austin, Texas 78712, United States., Brodbelt JS; Department of Chemistry, ‡Center for Systems and Synthetic Biology, §Department of Biomedical Engineering, ∥Department of Chemical Engineering, ⊥Institute for Cellular and Molecular Biology, #Department of Molecular Biosciences, The University of Texas at Austin , Austin, Texas 78712, United States.
Jazyk:	angličtina
Zdroj:	Analytical chemistry [Anal Chem] 2017 Jun 20; Vol. 89 (12), pp. 6498-6504. Date of Electronic Publication: 2017 May 26.
DOI:	10.1021/acs.analchem.7b00564
Abstrakt:	Mass spectrometry (MS) has emerged as a powerful tool within the growing field of immunoproteomics, which aims to understand antibody-mediated immunity at the molecular-level based on the direct determination of serological antibody repertoire. To date, these methods have relied on the use of high-resolution bottom-up proteomic strategies that require effective sampling and characterization of low abundance peptides derived from the antigen-binding domains of polyclonal antibody mixtures. Herein, we describe a method that uses restricted Lys-C enzymatic digestion to increase the average mass of proteolytic IgG peptides (≥4.5 kDa) and produce peptides which uniquely derive from single antibody species. This enhances the capacity to discriminate between very similar antibodies present within polyclonal mixtures. Furthermore, our use of 193-nm ultraviolet photodissociation (UVPD) improves spectral coverage of the antibody sequence relative to conventional collision- and electron-based fragmentation methods. We apply these methods to both a monoclonal and an antibody mixture. By identifying from a database search of approximately 15 000 antibody sequences those which compose the mixture, we demonstrate the analytical potential of middle-down UVPD for MS-based serological repertoire analysis.
Databáze:	MEDLINE
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