Direct Injection Mass Spectrometry and iFishMass for the High-Throughput Analysis of Antibody Modifications

High-throughput analysis has become a critical component in chemical biology and analytical chemistry due to the large libraries of compounds that are screened every day for drug development. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein modifications, both chemically deposited and biological post-translational modifications (PTMs). With the advent of antibody drug conjugates (ADCs) and other novel protein-based conjugates, the demand for such an analysis has skyrocketed. Here, we present a new protocol that achieves quantitative data for modified peptides in approximately 30 s of MS acquisition time. This platform includes a direct injection MS approach coupled with new software named iFishMass to extract targeted signals from hundreds of runs. iFishMass automatically generates plots and statistics. This platform will enable a faster analysis of synthetic modifications installed on monoclonal antibodies to create ADCs, and it is potentially scalable to biological PTMs. Sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates, paving the way to an inexpensive but effective platform for high-throughput screening of conjugation sites on proteins.