Sequencing and Quantifying IgG Fragments and Antigen-Binding Regions by Mass Spectrometry

In cancer and autoimmune diseases, immunoglobulins with a specific
molecular signature that could potentially be used as diagnostic or
prognostic markers are released into body fluids. An immunomics approach
based on this phenomenon relies on the ability to identify the specific
amino acid sequences of the complementarity-determining regions (CDR)
of these immunoglobulins, which in turn depends on the level of accuracy,
resolution, and sensitivity that can be achieved by advanced mass
spectrometry. Reproducible isolation and sequencing of antibody fragments
(e.g., Fab) by high-resolution mass spectrometry (MS) from seven healthy
donors revealed 43 217 MS signals: 225 could be associated
with CDR1 peptides, 513 with CDR2 peptides, and 19 with CDR3 peptides.
Seventeen percent of the 43 217 MS signals did not overlap
between the seven donors. The Fab isolation method used is reproducible
and fast, with a high yield. It provides only one Fab sample fraction
for subsequent characterization by high-resolution MS. In 17% and
4% of these seven healthy donors, qualitative (presence/absence) and
quantitative (intensity) differences in Fab fragments could be demonstrated,
respectively. From these results, we conclude that the identification
of a CDR signature as biomarker for autoimmune diseases and cancer
without prior knowledge of the antigen is feasible.