Reversed-Phase HPLC and Hyphenated Analytical Strategies for Peptidomics

Peptide study and analysis widely involve liquid chromatography. Among the different strategies available, reversed-phase liquid chromatography (RP-HPLC) is one of the methods of choice to separate species in a nontargeted approach. The compounds are sorted according to their hydrophobicity, even though the experimental order of elution could change according to the nature of the mobile phase and the stationary phase. In our work, we have developed protocols to resolve hundred of peptidic species. To overcome the limitations of peak capacity of RP-HPLC alone, it has been coupled downstream to tandem mass spectrometry using two different ionization modes. To overcome the limitations of peak capacity of RP-HPLC MS/MS, it has been coupled upstream to strong cation exchange liquid chromatography. Multidimensional analysis allows for a deeper description of a sample because the limit of detection is often due to a lack of dynamic range of the detection itself rather than due to a lack of sensitivity. In this chapter, different protocols are presented. They should be considered as examples that could be used as starting point for new protocols optimization. Even if RP-HPLC is a universal peptide separation method, it should be optimized according to the specific characteristics of the peptide(s) of interest.