Streamlined Development of Targeted Mass Spectrometry‐Based Method Combining Scout‐MRM and a Web‐Based Tool Indexed with Scout Peptides

MS‐based targeted proteomics is a relevant technology for sensitive and robust relative or absolute quantification of proteins biomarker candidates in complex human biofluids or tissue extracts. Performing a multiplex assay imposes time scheduling of peptide monitoring only around their expected retention time that needs to be defined with synthetic peptide. Time‐scheduled monitoring is clearly a constraint that precludes from straightforward assay transfer between biological matrices or distinct experimental setup. Any unexpected retention time (RT) shift challenges assay robustness and its implementation for large‐scale analysis. Recently, Scout‐multiple reaction monitoring that fully releases multiplexed targeted acquisition from RT scheduling by successively monitoring complex transition groups triggered with sentinel molecules called Scout has been introduced. It is herein documented how Peptide Selector database and tool streamlines the building of a multiplexed method thanks to RT indexation relative to Scout peptides. This case study deals with surrogate peptides of biomarker candidates related to drug‐induced liver and vascular injury, running such on‐line built method (eight Scouts triggering the monitoring of a total of 692 transitions) enables 100% recovery of a panel of 93 spiked‐in heavy labeled standards, despite significant RT shifts between serum, plasma, or urine. This result illustrates the simplicity of automatically building and deploying robust proteomics targeted assay.