title: Identification of a Set of Conserved Eukaryotic Internal Retention Time Standards for Data-independent Acquisition Mass Spectrometry.
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.
authors.affiliation: ‡‡Advanced Clinical Biosystems Research Institute, The Heart Institute, and Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California;
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Parker
authors.affiliation: §Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland; ¶PhD Program in Systems Biology, University of Zurich and ETH Zurich, Zurich, Switzerland;
authors.forename: Hannes
authors.initials: H
authors.lastname: Rost
authors.affiliation: §Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland; ¶PhD Program in Systems Biology, University of Zurich and ETH Zurich, Zurich, Switzerland;
authors.forename: George
authors.initials: G
authors.lastname: Rosenberger
authors.affiliation: §Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland;
authors.forename: Ben C
authors.initials: BC
authors.lastname: Collins
authors.affiliation: ‖S3IT, University of Zurich;
authors.forename: Lars
authors.initials: L
authors.lastname: Malmström
authors.affiliation: **Stanford University;
authors.forename: Dario
authors.initials: D
authors.lastname: Amodei
authors.affiliation: ‡‡Advanced Clinical Biosystems Research Institute, The Heart Institute, and Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California;
authors.forename: Vidya
authors.initials: V
authors.lastname: Venkatraman
authors.affiliation: ‡‡Advanced Clinical Biosystems Research Institute, The Heart Institute, and Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California;
authors.forename: Koen
authors.initials: K
authors.lastname: Raedschelders
authors.affiliation: From the ‡Department of Medicine, Johns Hopkins University, Baltimore Maryland; ‡‡Advanced Clinical Biosystems Research Institute, The Heart Institute, and Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California;
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Van Eyk
authors.affiliation: §Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland; §§Faculty of Science, University of Zurich, Zurich, Switzerland aebersold@imsb.biol.ethz.ch.
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
mesh_terms: D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards*; D051379:Mice; D019151:Peptide Library; D010455:Peptides / Q000032:analysis*; D040901:Proteomics / Q000379:methods / Q000592:standards*; D013997:Time Factors; D015003:Yeasts
chemical_list: D019151:Peptide Library; D010455:Peptides