Antibody-Coupled Magnetic Beads Can Be Reused in Immuno-MRM Assays To Reduce Cost and Extend Antibody Supply

Immunoaffinity
enrichment of peptides coupled to targeted, multiple
reaction monitoring mass spectrometry (immuno-MRM) enables precise
quantification of peptides. Affinity-purified polyclonal antibodies
are routinely used as affinity reagents in immuno-MRM assays, but
they are not renewable, limiting the number of experiments that can
be performed. In this technical note, we describe a workflow to regenerate
anti-peptide polyclonal antibodies coupled to magnetic beads for enrichments
in multiplex immuno-MRM assays. A multiplexed panel of 44 antibodies
(targeting 60 peptides) is used to show that peptide analytes can
be effectively stripped off of antibodies using acid washing without
compromising assay performance. The performance of the multiplexed
panel (determined by correlation, agreement, and precision of reused
assays) is reproducible (R2 between 0.81
and 0.99) and consistent (median CVs 8–15%) for at least 10
times of washing and reuse. Application of this workflow to immuno-MRM
studies greatly reduces per sample assay cost and increases the number
of samples that can be interrogated with a limited supply of polyclonal
antibody reagent. This allows more characterization for promising
and desirable targets prior to committing funds and efforts to conversion
to a renewable monoclonal antibody.