Using iRT, a normalized retention time for more targeted measurement of peptides

• Published in: Proteomics (Weinheim) Vol. 12; no. 8; pp. 1111 - 1121
• Main Authors: Escher, Claudia, Reiter, Lukas, MacLean, Brendan, Ossola, Reto, Herzog, Franz, Chilton, John, MacCoss, Michael J., Rinner, Oliver
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 01.04.2012; Wiley Subscription Services, Inc
• Subjects: Amino Acid Sequence; Calibration; HeLa Cells; High-Throughput Screening Assays - standards; Humans; Ions; Laboratories; Leptospira interrogans - chemistry; Mass Spectrometry - instrumentation; Mass Spectrometry - methods; Mass Spectrometry - standards; Molecular Sequence Data; Multiplexing; Optimization; Peptides; Peptides - analysis; Peptides - chemical synthesis; Proteomics - instrumentation; Proteomics - methods; Proteomics - standards; Quantitative analysis; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Software; Technology; Time Factors
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.201100463

Multiple reaction monitoring (MRM) has recently become the method of choice for targeted quantitative measurement of proteins using mass spectrometry. The method, however, is limited in the number of peptides that can be measured in one run. This number can be markedly increased by scheduling the acquisition if the accurate retention time (RT) of each peptide is known. Here we present iRT, an empirically derived dimensionless peptide‐specific value that allows for highly accurate RT prediction. The iRT of a peptide is a fixed number relative to a standard set of reference iRT‐peptides that can be transferred across laboratories and chromatographic systems. We show that iRT facilitates the setup of multiplexed experiments with acquisition windows more than four times smaller compared to in silico RT predictions resulting in improved quantification accuracy. iRTs can be determined by any laboratory and shared transparently. The iRT concept has been implemented in Skyline, the most widely used software for MRM experiments.