Mass Spectrometric Characterization of Glycated β-Lactoglobulin Peptides Derived from Galacto-oligosaccharides Surviving the In Vitro Gastrointestinal Digestion

A mass spectrometric study has been carried out to elucidate the structures of glycated peptides obtained after in vitro gastrointestinal digestion of bovine β-lactoglobulin (β-LG) glycated with prebiotic galacto-oligosaccharides (GOS). The digests of both native and glycated β-LG were analyzed by MALDI-MS, LC-ESI-MS, and LC-ESI-MS/MS. MALDI-MS profiles showed marked differences mainly related to the lower intensity of ions corresponding to the digest of glycated β-LG. Overall, 58 and 23 unglycated peptides covering 97% and 63% of the mature β-LG sequence could be identified in the digests of native and glycated samples, respectively. The LC-ESI-MS analyses corroborated the MALDI-MS results regarding the unglycated peptides but they also enabled an extensive investigation into the digest of glycated β-LG. Thus, a total of 19 peptides glycated with GOS from two to seven hexose units could be identified. The tandem mass spectra of glycated peptides were mostly characterized by two neutral losses of 1026/1056, 864/894, 702/732, 540/570, 378/408, and 216/246 u, corresponding to the formation of the furylium ion and its subsequent “CHOH” loss, indicative of the peptide glycation with hepta-, hexa-, penta-, tetra-, tri-, and disaccharides, respectively. Also, other minor ionic species containing the furylium ring linked to different galactose units could be also detected, showing the diversity of the fragmentation pattern of peptides glycated with larger size carbohydrates. Finally, the putative GOS glycation sites could be determined at the NH2-terminal Leu residue and at Lys residues located in positions 14, 47, 75, 77, 83, 91, 100, 135, and 138.