Peptide Retention Time Calibration Mixtures enable assessing chromatography and MS instrument performance. More importantly, they are paramount for normalizing results for variation in retention times and peak intensities between runs. Commercial mixes are expensive and cannot be added retrospectively in the case of datasets that were not spiked in the first place. It is, therefore, desirable to identify endogenous peptides that can be used for that purpose. Parker et al (2015) reported a set of peptide sequences that are conserved across eukaryotic species which they termed Common internal Retention Time standards (CiRT). We identified the limitation of CiRT which, being mostly derived from cytoskeletal and ribosomal proteins, are not detectable in blood plasma. Plasma is the most commonly used sample in both human biomedical research and for clinical veterinary applications and both animal and translational biomedical research, would benefit from the availability of calibration peptides. We have, therefore, conducted a bioinformatics study to develop a novel set of endogenous peptides which could serve the same purpose as CiRT but which are specifically present in sufficient abundance in plasma of multiple species including human.
Initially, we selected a list of 81 peptides from the in-house study on cross-species plasma proteome. We then compared this set of identified peptides with human and mouse plasma repository downloaded from PeptideAtlas. To further confirm the ubiquitous occurrence of peptides across a larger set of mammals we downloaded 12 species reference proteome from UniProt Knowledgebase. This resulted in the identification of a set of 13 peptides and called them as Plasma internal retention Time calibration peptides (PiRT). We have validated this new set of peptides by extracting them from plasma samples collected from 5 different and distantly-related species (human, mouse, cattle, sheep, giraffe) and showing that they can be used for retention time calibration.