Separation of multiphosphorylated cyclopeptides and their positional isomers by hydrophilic interaction liquid...

Peptides are efficient models used in different fields such as toxicology to study the interactions of several contaminants at the molecular scale, requiring the development of bio-analytical strategies. In this context, Hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS) was used to separate synthetic multiphosphorylated cyclopeptides and their positional isomers at physiological pH. We assessed (i) the selectivity of eleven HILIC columns, from different manufacturers and packed with diverse polar sorbents, and (ii) the effect of mobile phase composition on the separation selectivity. The best selectivity and baseline resolution were achieved with the columns grafted by neutral sorbents amide and diol. Furthermore, we investigated the HILIC retention mechanism of these peptides by examining the effect of the number of phosphorylated residues in the peptide scaffold on their retention. The peptide behavior followed the classical hydrophilic partitioning mechanism exclusively on amide and diol columns. This trend was not fully respected on bare and hybrid silica due to the attractive/repulsive interactions of the deprotonated surface silanol groups with the Arginine or Glutamate residues in the peptide scaffold according to the peptide sequence. The position of the phosphorylated amino acid in the peptide backbone also showed to have an impact on the retention, making possible the separation of positional isomers of these multiphosphorylated cyclic peptides using HILIC.