Sensitive untargeted identification of short hydrophilic peptides by high performance liquid chromatography on porous graphitic carbon coupled to high resolution mass spectrometry.

The combination of an efficient chromatographic separation with post-column addition of a supercharging agent was evaluated for the determination of small peptides. The procedure takes advantage of porous graphitic carbon (PGC) ability in retaining very polar and ionic molecules to overstep the poor retention of small peptides on conventional reversed phase (RP) columns. The method was developed specifically for the most hydrophilic di-, tri- and tetrapeptides, which are not identified in ordinary peptidomics experiments. In addition to retention mechanisms acting on conventional RP, the method exploited the charge induced interactions generated by the charges on the peptides with the polarizable surface of PGC. This results in efficient retention of very short and highly polar peptides using classical RP mobile phases. The effects of varying mobile phase composition (organic solvent and ion-pairing additives) as well as column temperature have been thoroughly investigated using short peptide standards. Under optimized conditions (water and acetonitrile/tetrahydrofuran 99:1 (v/v), both with 0.15% trifluoroacetic acid, as phase A and B, respectively, 0.5 mL min flowrate at 50 °C) the effect of post-column addition of 3-nitrobenzylic alcohol was also investigated allowing effective coupling of the chromatographic system with high resolution mass spectrometry. Finally, an untargeted approach for peptide identification was pursued, based on precursor identification in database with all possible combinations of the 20 natural amino acids and fragmentation spectra matching to in silico generated spectra. The method was then applied to investigation of the short endogenous peptides in human serum from healthy individuals resulting in the identification of 30 short peptides.