Targeted Quantification of Proteoforms in Complex Samples by Proteoform Reaction Monitoring

Existing mass spectrometric assays used for sensitive and specific measurements of target proteins across multiple samples, such as selected/multiple reaction monitoring (SRM/MRM) or parallel reaction monitoring (PRM), are peptide-based methods for bottom-up proteomics. Here, we describe an approach based on the principle of PRM for the measurement of intact proteoforms by targeted top-down proteomics termed Proteoform Reaction Monitoring (PfRM). We explore the ability of our method to circumvent traditional limitations of top-down proteomics such as sensitivity and reproducibility. We also introduce a new software, Proteoform Finder, specifically designed for easy analysis of PfRM data. PfRM was initially benchmarked by quantifying three standard proteins. Linearity of the assay was shown over almost three orders of magnitude in the femtomole range. We later applied our multiplexed PfRM assay to complex samples to quantify biomarker candidates in peripheral blood mononuclear cells (PBMCs) from liver transplanted patients, demonstrating its possible translational applications. These results demonstrate that PfRM has the potential to contribute to the accurate quantification of protein biomarkers for diagnostic purposes and to improve understanding of disease etiology at the proteoform level.