Highly multiplexed targeted proteomics using precise control of peptide retention time

Large‐scale proteomics applications using SRM analysis on triple quadrupole mass spectrometers present new challenges to LC‐MS/MS experimental design. Despite the automation of building large‐scale LC‐SRM methods, the increased numbers of targeted peptides can compromise the balance between sensitivity and selectivity. To facilitate large target numbers, time‐scheduled SRM transition acquisition is performed. Previously published results have demonstrated incorporation of a well‐characterized set of synthetic peptides enabled chromatographic characterization of the elution profile for most endogenous peptides. We have extended this application of peptide trainer kits to not only build SRM methods but to facilitate real‐time elution profile characterization that enables automated adjustment of the scheduled detection windows. Incorporation of dynamic retention time adjustments better facilitate targeted assays lasting several days without the need for constant supervision. This paper provides an overview of how the dynamic retention correction approach identifies and corrects for commonly observed LC variations. This adjustment dramatically improves robustness in targeted discovery experiments as well as routine quantification experiments.