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

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.