Prediction of short peptides composition by RP-HPLC coupled to ESI mass spectrometry

The combination of reversed-phase chromatography and electrospray mass spectrometry was used to predict the amino acid composition of low molar weight peptides present in a complex mixture of rapeseed protein hydrolysates. First, amino acid retention times on a hydrophobic stationary phase were evaluated to determine hydrophobicity coefficients for each amino acid and then global hydrophobicity coefficients were established for standard peptides. In this way, a correlation between peptide hydrophobicity coefficients and retention time was established. Then, a C language program was developed to calculate all potential amino acid combinations from the mass of a peptide (molar mass < 1000 Da) and determine theoretical hydrophobicity coefficients corresponding to these combinations. Comparison between the theoretical retention time determined by the model and the experimental retention time resulted in the establishment of a sorted potential composition table. This methodology has been applied to two different rapeseed protein hydrolysates peptides that have been purified and sequenced.