Inference and quantification of peptidoforms in large sample cohorts by SWATH-MS

The consistent detection and quantification of protein post-translational modifications (PTMs) across sample cohorts is an essential prerequisite for the functional analysis of biological processes. Data-independent acquisition (DIA), a bottom-up mass spectrometry based proteomic strategy, exemplified by SWATH-MS, provides complete precursor and fragment ion information of a sample and thus, in principle, the information to identify peptidoforms, the modified variants of a peptide. However, due to the convoluted structure of DIA data sets the confident and systematic identification and quantification of peptidoforms has remained challenging. Here we present IPF (Inference of PeptidoForms), a fully automated algorithm that uses spectral libraries to query, validate and quantify peptidoforms in DIA data sets. The method was developed on data acquired by SWATH-MS and benchmarked using a synthetic phosphopeptide reference data set and phosphopeptide-enriched samples. The data indicate that IPF reduced false site-localization by more than 7-fold in comparison to previous approaches, while recovering 85.4% of the true signals. IPF was applied to detect and quantify peptidoforms carrying ten different types of PTMs in DIA data acquired from more than 200 samples of undepleted blood plasma of a human twin cohort. The data approportioned, for the first time, the contribution of heritable, environmental and longitudinal effects on the observed quantitative variability of specific modifications in blood plasma of a human population.