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

Victoria C. Cotham, Andrew P. Horton, Jiwon Lee, George Georgiou, Jennifer S. Brodbelt

    Research output: Contribution to journalArticlepeer-review

    11 Scopus citations

    Abstract

    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.

    Original languageEnglish (US)
    Pages (from-to)6498-6504
    Number of pages7
    JournalAnalytical Chemistry
    Volume89
    Issue number12
    DOIs
    StatePublished - Jun 20 2017

    ASJC Scopus subject areas

    • Analytical Chemistry

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