Abstract
Reversed-phase HPLC is a leading tool for peptide fractionation in proteomics. Various combinations of stationary and mobile phases are employed depending on the purpose of the separation and the detection procedure. This diversity results in significant changes in separation selectivity, which are currently not well understood. Our ongoing work, focused on collection of peptide retention data and the development of sequence specific algorithms for peptide retention prediction, reveals these differences and helps define sets of conditions where predictive models can be considered independent from chromatographic platform used. We describe our observations on peptide separation selectivity variations for a wide range of RP-HPLC conditions: C18 sorbents of different pore sizes, using trifuoroacetic/formic/heptafluorobutyric acids as ion-pairing modifiers, RP separation at pH 10, and RP separation with an alternative perfluorinated chemistry of the bonded phase. Both the charge distribution within the peptide chain and the ion-pair formation mechanisms were found to be major factors in determining the peptide separation selectivity in RP-HPLC.
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Acknowledgements
This work was supported in part by grants from the Technology Transfer Office at the University of Manitoba and the Natural Sciences and Engineering Research Council of Canada (O.V.K.).
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Dwivedi, R.C., Spicer, V., Krokhin, O.V. (2011). Reversed-Phase HPLC of Peptides – A Valuable Sample Preparation Tool in Bottom-Up Proteomics: Separation Selectivity in Single and Multi-dimensional Separation Modes. In: Ivanov, A., Lazarev, A. (eds) Sample Preparation in Biological Mass Spectrometry. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-0828-0_48
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