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

Abstract

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.

Figure 1

iRT is a normalized retention time (RT) scale, independent of the chromatographic setup. (a) Schematic illustration of how the iRT is empirically determined for the case of LC-MRM. In practice, 11 reference peptides (iRT-peptides) are used and the transformation is based on a linear fit; for simplicity, in this illustration only two peptides are shown. A target peptide and the iRT-peptides are measured using liquid chromatography coupled to MRM (LC-MRM). The retention time of the target peptide (RT_x) is normalized and hereby transformed into iRT using the coordinates of the iRT-peptides. Simplified, the iRT of the target peptide is expressed relative to the RTs of the iRT-peptides. iRT can be stored as a single stable number and transferred to any chromatographic setup using the iRT-peptides. (b) One calibration run is used to convert iRT to a new chromatographic setup. Depicted are three possible chromatographic setups with gradients of different length. (c) In order to increase the number of peptides measured in a single LC-MRM run, the measurement can be scheduled to the time point where the peptide elutes. The window width should be chosen such that the difference between anticipated and empirical RT never exceeds the window size. Hence, the more accurate the anticipated RT is, the smaller the RT window can be chosen. (d) LC-MRM measurement of the 11 iRT-peptides to define the iRT-C18 scale.

Figure 2

Accuracy of iRT and SSRCalc based retention time (RT) prediction based on the measurement of 148 peptides in a 90min linear gradient. (a) SSRCalc predicts the measured (empirical) RT with a good correlation of 0.92. (b) iRT values determined in an independent experiment in a 30min linear gradient predict RTs with a very high correlation (0.997) and low variance. (c) The absolute difference between empirical and anticipated retention time abs (ΔRT) is shown for predictions based on iRT and SSRCalc. iRT predictions are more accurate which allows choosing shorter RT windows without missing any targeted protein.

Figure 3

Quantification accuracy of 150 precursors (148 peptides) as a function of the retention time (RT) window size when using scheduled MRM. A log2 intensity ratio of 2.32 (5-fold difference) was expected based on the sample preparation. Generally, quantification was most accurate when using the smallest retention time windows of two minutes.

Figure 4

RT shifts induced by different amounts of protein loaded on the column can be compensated using on-the-fly RT calibration. Grey lines represent peptide retention times when varying the sample load on the column. Black crosses indicate peptides that would have been missed without using on-the-fly RT calibration. With 2μg protein loaded on the column 111, 122, and 49 peptides would elute outside the retention time window and hence be missed in the three technical replicates. The earliest eluting peptide was missed once even using on-the-fly RT calibration in the second run of the 1μg load.