Peptide Shifter: Enhancing Separation Reproducibility Using Correlated Expression Profiles

Chromatographic protein and peptide separation technologies enable comprehensive proteomic analysis of plasma and other complex biological samples by mass spectrometry. However, as the number of separations and/or fractions increases, so does the number of peptides split across fraction boundaries. Irreproducibility of peptide chromatographic separation results in peptides on or near the boundary moving partially or entirely into adjacent fractions. Peptide shifting across fraction boundaries increases the variability of measured peptide abundance, and so there is a trade-off between proteomic comprehensiveness using separation technologies and accurate quantitative proteomic measurements. In this paper, a method for detecting and correcting split peptides, called Peptide Shifter, is introduced and evaluated. An essential component of Peptide Shifter is a global peptide expression profile analysis that allows the inference of the underlying peptide shift pattern without the use of peptide labeling or internal standards. A controlled proteomic analysis of plasma samples demonstrates a 34% decrease in peptide intensity variability after the application of Peptide Shifter.