期刊
JOURNAL OF CHROMATOGRAPHY A
卷 1383, 期 -, 页码 88-95出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2015.01.028
关键词
Quantitative N-glycan profiling; Isotope coded labeling; PGC separation; Mass spectrometry
资金
- University of Vienna
Glycan reductive isotope labeling (GRIL) using C-12(6)-/C-13(6)-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. (C) 2015 Elsevier B.V. All rights reserved.
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