IonQuant Enables Accurate and Sensitive Label-Free Quantification with FDR-Controlled Match-Between-Runs

Missing values weaken the power of label-free quantitative proteomic experiments to uncover true quantitative differences between biological samples or experimental conditions. Match-between-runs (MBR) has become a common approach to mitigate the missing value problem, where peptides identified by tandem mass spectra in one run are transferred to another by inference based on m/z, charge state, retention time, and ion mobility when applicable. Though tolerances are used to ensure such transferred identifications are reasonably located and meet certain quality thresholds, little work has been done to evaluate the statistical confidence of MBR. Here, we present a mixture model-based approach to estimate the false discovery rate (FDR) of peptide and protein identification transfer, which we implement in the label-free quantification tool IonQuant. Using several benchmarking datasets generated on both Orbitrap and timsTOF mass spectrometers, we demonstrate superior performance of IonQuant with FDR-controlled MBR compared with MaxQuant (19-38 times faster; 6-18% more proteins quantified and with comparable or better accuracy). We further illustrate the performance of IonQuant and highlight the need for FDR-controlled MBR, in two single-cell proteomics experiments, including one acquired with the help of high-field asymmetric ion mobility spectrometry separation. Fully integrated in the FragPipe computational environment, IonQuant with FDR-controlled MBR enables fast and accurate peptide and protein quantification in label-free proteomics experiments.