Shotgun Characterization of the Circulating IgM Antigenome of an Infectious Pathogen by Immunocapture-LC–MS/MS from Dried Serum Spots

One of the main challenges in compiling the complete
collection
of protein antigens from pathogens for the selection of vaccine candidates
or intervention targets is to acquire a broad enough representation
of them to be recognized by the highly diversified immunoglobulin
repertoire in human populations. Dried serum spot sampling (DSS) retains
a large repertoire of circulating immunoglobulins from each individual
that can be representative of a population, according to the sample
size. In this work, shotgun proteomics of an infectious pathogen based
on DSS sampling coupled with IgM immunoprecipitation, liquid chromatography–mass
spectrometry (LC–MS/MS), and bioinformatic analyses was combined
to characterize the circulating IgM antigenome. Serum samples from
a malaria endemic region at different clinical statuses were studied
to optimize IgM binding efficiency and antibody leaching by varying
serum/immunomagnetic bead ratios and elution conditions. The method
was validated using Plasmodium falciparum extracts identifying 110 of its IgM-reactive antigens while minimizing
the presence of human proteins and antibodies. Furthermore, the IgM
antigen recognition profile differentiated between malaria-infected
and noninfected individuals at the time of sampling. We conclude that
a shotgun proteomics approach offers advantages in providing a high-throughput,
reliable, and clean way to identify IgM-recognized antigens from trace
amounts of serum. The mass spectrometry raw data and metadata have
been deposited with ProteomeXchange via MassIVE with the PXD identifier
PXD043800.