title: Quantitative isomer-specific N-glycan fingerprinting using isotope coded labeling and high performance liquid chromatography-electrospray ionization-mass spectrometry with graphitic carbon stationary phase.
abstract: Glycan reductive isotope labeling (GRIL) using (12)C6-/(13)C6-aniline as labeling reagent is reported with the aim of quantitative N-glycan fingerprinting. Porous graphitized carbon (PGC) as stationary phase in capillary scale HPLC coupled to electrospray mass spectrometry with time of flight analyzer was applied for the determination of labeled N-glycans released from glycoproteins. The main benefit of using stable isotope-coding in the context of comparative glycomics lies in the improved accuracy and precision of the quantitative analysis in combined samples and in the potential of correcting for structure-dependent incomplete enzymatic release of oligosaccharides when comparing identical target proteins. The method was validated with respect to mobile phase parameters, reproducibility, accuracy, linearity and limit of detection/quantification (LOD/LOQ) using test glycoproteins. It is shown that the developed method is capable of determining relative amounts of N-glycans (including isomers) comparing two samples in one single HPLC-MS run. The analytical potential and usefulness of GRIL in combination with PGC-ESI-TOF-MS is demonstrated comparing glycosylation in human monoclonal antibodies produced in Chinese hamster ovary cells (CHO) and hybridoma cell lines.
authors: Institute of Analytical Chemistry, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria.
authors: Claudia
authors: C
authors: Michael
authors: Institute of Analytical Chemistry, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria. Electronic address: andreas.rizzi@univie.ac.at.
authors: Andreas M
authors: AM
authors: Rizzi
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation; D006224:Cricetinae; D003412:Cricetulus; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006108:Graphite / Q000737:chemistry; D006801:Humans; D007536:Isomerism; D007553:Isotope Labeling; D009844:Oligosaccharides / Q000032:analysis* / Q000302:isolation & purification; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000814:Aniline Compounds; D006023:Glycoproteins; D009844:Oligosaccharides; D006108:Graphite; C023650:aniline
keywords: Isotope coded labeling; Mass spectrometry; PGC separation; Quantitative N-glycan profiling