Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.

Bruker Daltonics GmbH and Co KG Bremen 28359 Germany

Bruker Daltonics Inc Billerica Massachusetts 01821 United States

Bruker S R O District Brno City 61900 Czech Republic

Department of Cancer Biology and Blais Proteomics Center Dana Farber Cancer Institute Boston Massachusetts 02215 United States

Department of Chemical and Systems Biology and ChEM H Stanford University School of Medicine Stanford California 94305 United States

Department of Oncologic Pathology Dana Farber Cancer Institute Boston Massachusetts 02215 United States

Department of Pathology Brigham and Women's Hospital and Harvard Medical School Boston Massachusetts 02115 United States

Department of Pharmacology School of Medicine University of North Carolina Chapel Hill North Carolina 27599 United States

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