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 4 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.

Targeted Proteomics for Multiplexed Verification of Markers of Colorectal Tumorigenesis

“…Compared with in-solution trypsin digestion, serial proteolysis using Lys-C and trypsin increases the yield of fully cleaved peptides, thereby improving the accuracy and sensitivity of SRM protein quantification (35). Peptide solutions were desalted on SepPak C18 columns, lyophilized in a vacuum centrifuge, and resolubilized in 50 l (1:1 v/w) of a solution of 3% acetonitrile, 0.1% formic acid, and indexed Retention Time (iRT) 10ϫ Mix (Biognosys AG, Zurich, Switzerland) (36). The iRT mix was prepared in accordance with the manufacturer's protocol.…”

“…Peptides were analyzed on the TSQ Vantage in the SRM mode, which involves repeated cycling through a list of transitions, with predefined dwell-times on each transition (17). Retention times extracted from the SRM scans were used to calculate iRT values (36) for each peptide in Skyline. SRM scans were searched to confirm the near absence of endogenous transitions in the crude peptide mix.…”

“…Standard isotope-labeled peptides were used as internal controls during statistical analysis of our SRM data and for the measurement of relative changes in protein abundance across tissue samples. Each tissue peptide digest also contained a known concentration of a mixture of iRT reference peptides (36), which served as internal controls for monitoring instrument and technical variations during time-scheduled SRM measurements. Based on precursor and fragment ion intensities, two to four transitions were selected for each standard (heavy) and endogenous (light) peptide.…”

Autocrine and Paracrine Regulation of Keratinocyte Proliferation through a Novel Nrf2–IL-36γ Pathway

“…Mascot search results were converted to a spectral library using the BiblioSpec implementation in Skyline (15). In addition, we converted empirical retention times into iRT scores following the procedure described by Escher et al (16). For each target protein we selected three unique peptide precursors present in the spectral library and their most intense (top3) fragment ions from the y and b ion series.…”

A Skyline Plugin for Pathway-Centric Data Browsing

“…However, the iRT algorithm [18], using synthetic or common internal [19] retention time standard peptides, can provide a mechanism for inclusion of peptides from the external spectral library by estimating a predicted retention time window. This approach could be valuable in those instances where additional peptides are needed for a protein of interest, or when proteins of interest are not represented in the user's spectral library.…”