Journal
ANALYTICAL CHEMISTRY
Volume 89, Issue 12, Pages 6498-6504Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.7b00564
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Funding
- NSF [CHE1402753]
- Welch Foundation [F1155]
- NSF Graduate Research Fellowship [DGE-110007]
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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 antibodies present within polyclonal mixtures. Furthermore, our use of 193-species. This enhances the capacity to discriminate between very similar nm ultraviolet photo dissociation (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.
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