Targeted Analysis of >1500 Plasma Proteoforms via Individual Ion Mass Spectrometry

Abstract Plasma proteomics has emerged as a powerful approach for interrogating human biology, identifying disease-associated proteomic changes, and advancing precision diagnostics. In recent years, affinity-based plasma proteomics platforms with predefined protein panels, such as Olink and SomaScan, have emerged to complement bottom-up mass spectrometry (BUP). This study leverages a novel mass spectrometry platform to capture targeted proteoform information lost by mainline antibody-, aptamer-, and BUP-driven workflows. The Plasma Proteoform Assay (PPA) uses Individual Ion Mass Spectrometry (I2MS) to resolve mixtures of intact proteins presented by direct injection. Two proteoform panels, PPA 526 and PPA 1514, were defined from human plasma samples obtained from 81 individuals. The panels quantify 526 proteoforms derived from 59 genes and 1,514 proteoforms from 155 genes, respectively. Across both panels, the proportion of targeted proteoforms with CVs below 20% ranged from 59% to 80%. PPA was benchmarked in studies including 9 pooled plasma samples derived from subjects with hepatic cirrhosis (N = 30 individuals) and a cohort of resilient agers carrying a SERPINE1 (PAI-1) mutation (N = 27 individuals). PPA signatures distinguished SERPINE1 mutation carriers from affected individuals and provided sufficient resolution to discriminate among cirrhosis disease stages. In summary, we present PPA 526 and PPA 1514, the first scalable plasma proteoform panels capable of tracking hundreds to thousands of targets in a few minutes per sample.