Relative quantitation of glycosylation variants by stable isotope labeling of enzymatically released N-glycans using...

Glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline was used for relative quantitation of N-glycans. In a first step, the labeling method by reductive amination was optimized for this reagent. It could be demonstrated that selecting aniline as limiting reactant and using the reductant in excess is critical for achieving high derivatization yields (over 95 %) and good reproducibility (relative standard deviations ∼1-5 % for major and ∼5-10 % for minor N-glycans). In a second step, zwitterionic-hydrophilic interaction liquid chromatography in capillary columns coupled to electrospray mass spectrometry with time-of-flight analyzer (μZIC-HILIC-ESI-TOF-MS) was applied for the analysis of labeled N-glycans released from intact glycoproteins. Ovalbumin, bovine α1-acid-glycoprotein and bovine fetuin were used as test glycoproteins to establish and evaluate the methodology. Excellent separation of isomeric N-glycans and reproducible quantitation via the extracted ion chromatograms indicate a great potential of the proposed methodology for glycoproteomic analysis and for reliable relative quantitation of glycosylation variants in biological samples.