Stable Isotope Labels Girard’s Reagent P For Relative Quantitation Of Reductive Glycans By Electrospray Ionization Mass Spectrometry

Over fifty percent of proteins undergo a post-translational glycosylation modification. Many diseases are related to alteration of glycans attached to proteins, such as CDGs, infection and cancer. Therefore, abnormal expression of glycans could be considered as potential biomarkers.Recent years, modern biological mass spectrometry has performed a detail analysis of carbohydrate molecules. Stable isotope labeling coupled with mass spectrometry analysis show promise in detecting these important biological changes. A novel strategy for glycan relative quantitation using a stable isotope-labeled Girard’s reagent P has been suggested. Absence of multiple complex adduct ion peaks such as [M+Na]+and [M+K]+in MS profile presents an important advantage of the method. Presence of only [M]+ion peaks in both MALDI-and ESI-MS drastically simplifies the mass-spectra profiles and facilitates the subsequent numerical calculations. Simultaneously, the detection sensitivity is enhanced. In this study, according to the differences of relative intensity between the do-and d5-GP derivatives in MS profile, the relative quantitation of the glycan can be calculated. Reliability and sensitivity of the method was tested and checked for the model lactose sample. The suggested method has been then validated for N-glycans released from chicken ovalbumin and ribonuclease B by Peptide N-glycosidase F. The results exhibit good linearity and high reproducibility within a10-fold concentration range. The method has been also tested for the analysis of N-glycans from bovine and porcine serum IgG, human milk oligosaccharides of different decades as well as human serum glycoproteins.