Abstract
Accurate knowledge of retention time (RT) in liquid chromatography-based mass spectrometry data facilitates peptide identification, quantification, and multiplexing in targeted and discovery-based workflows. Retention time prediction is particularly important for peptide analysis in emerging data-independent acquisition (DIA) experiments such as SWATH-MS. The indexed RT approach, iRT, uses synthetic spiked-in peptide standards (SiRT) to set RT to a unit-less scale, allowing for normalization of peptide RT between different samples and chromatographic set-ups. The obligatory use of SiRTs can be costly and complicates comparisons and data integration if standards are not included in every sample. Reliance on SiRTs also prevents the inclusion of archived mass spectrometry data for generation of the peptide assay libraries central to targeted DIA-MS data analysis. We have identified a set of peptide sequences that are conserved across most eukaryotic species, termed Common internal Retention Time standards (CiRT). In a series of tests to support the appropriateness of the CiRT-based method, we show: (1) the CiRT peptides normalized RT in human, yeast, and mouse cell lysate derived peptide assay libraries and enabled merging of archived libraries for expanded DIA-MS quantitative applications; (2) CiRTs predicted RT in SWATH-MS data within a 2-min margin of error for the majority of peptides; and (3) normalization of RT using the CiRT peptides enabled the accurate SWATH-MS-based quantification of 340 synthetic isotopically labeled peptides that were spiked into either human or yeast cell lysate. To automate and facilitate the use of these CiRT peptide lists or other custom user-defined internal RT reference peptides in DIA workflows, an algorithm was designed to automatically select a high-quality subset of datapoints for robust linear alignment of RT for use. Implementations of this algorithm are available for the OpenSWATH and Skyline platforms. Thus, CiRT peptides can be used alone or as a complement to SiRTs for RT normalization across peptide spectral libraries and in quantitative DIA-MS studies.
Highlights
Identification of a Set of Conserved Eukaryotic Internal Retention Time Standards for Dataindependent Acquisition Mass Spectrometry*□S
We tested the capabilities of the Common internal Retention Time standards (CiRT)-reference peptides to normalize retention time (RT) in a total of five different data sets: Two unfractionated whole cell lysate samples generated from human and yeast cell lines; one data set comprised of 12 separate shotgun files generated from a basic reverse phase fractionation of mouse VSMC digest; and two additional data sets comprised of 11 OFFGEL fractions and 15 basic reverse phase fractions generated from human cell lysates that were accessed and downloaded from the PRIDE data repository
The CiRT peptides identified in this manuscript provide a method to normalize RT for peptide identification and quantification in SWATH data analysis across experiments performed on most eukaryotic species
Summary
Identification of a Set of Conserved Eukaryotic Internal Retention Time Standards for Dataindependent Acquisition Mass Spectrometry*□S. Accurate knowledge of retention time (RT) in liquid chromatography-based mass spectrometry data facilitates peptide identification, quantification, and multiplexing in targeted and discovery-based workflows. To automate and facilitate the use of these CiRT peptide lists or other custom user-defined internal RT reference peptides in DIA workflows, an algorithm was designed to automatically select a high-quality subset of datapoints for robust linear alignment of RT for use. Implementations of this algorithm are available for the OpenSWATH and Skyline platforms. This is of particular concern in comparative experiments where it is crucial that signals arising from identical peptides are appropriately aligned and quantified across samples
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