We have identified a set of peptide sequences that are conserved across most eukaryotic species, termed Common internal Retention Time standards (CiRT).
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. The obligatory use of SiRTs can be costly and complicates comparisons and data integration if standards are not included in every sample. 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. Retention time prediction is particularly important for peptide analysis in emerging data-independent acquisition (DIA) experiments such as SWATH-MS. 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.
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