Separation of peptide isomers and conformers by ultra performance liquid chromatography

Peptide isomers are characterized by an identical brutto formula, so that their specific detection by LC-MS/MS requires an individual LC retention time and/or an individual MS/MS spectrum. Mixtures of various classes of peptide isomers were analyzed by reversed phase nano ultra high performance liquid chromatography (UPLC)-MS/MS. Gradient elution was performed with a water/acetonitrile/formic acid system. Using this solvent system and gradients of medium length (30 or 60 min), mixtures were analyzed composed of structural isomers of modified peptides, sequence isomers of unmodified peptides, peptide/isopeptide pairs, diastereomeric peptide pairs, and peptide conformers. The large majority of the peptide isomers analyzed could be completely separated due to the high resolving power of UPLC. For most isomers, the observed retention time differences significantly exceeded the corresponding baseline peak widths leading for several isomeric pairs to resolutions above 10. In addition, hints for a separation of peptide conformers were observed. Most of the peptides analyzed were of synthetic origin, so that their individual assignment in the UPLC-MS/MS runs was straightforward. The relative elution order of numerous sets of peptide isomers is documented and discussed. The study highlights the important benefits of a high chromatographic separation power for the specificity of LC-MS/MS in the field of analytical proteomics.