Zoltan Szabo1, Junhua Wang2, Yury Agroskin1, Jim Thayer1, Julian Saba2,3, Rosa Viner2, Andreas Huhmer2, Jeff Rohrer1, Liu Yan1, Kannan Srinivasan1, Dietmar Reusch4 and Christopher A. Pohl1
We report on a novel workflow that combines fast release of N-linked glycans and their analysis by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD) coupled to a Q Exactive mass spectrometer. Ion chromatography supports simultaneous separation and detection of neutral and sialylated (charged) glycans without the need for derivatization. The chromatographic resolution of glycans is based on the number of sialic acid units, branch and positional isomers and the presence/absence of core or outer arm fucose. This work exemplifies the importance of denaturation prior to digestion. The reproducibility of peak area distribution of glycans released from denatured monoclonal antibody (mAb) was excellent and within 5% for glycans present at ≥1% of the total glycan pool. MS2 spectra with diagnostic fragments allow for highly reliable annotation of glycan species. High resolution separation of hu-AGP glycans resulted in 53 peaks illustrating the excellent resolving power of HPAE for charged glycan species. The method described here supports highly informative glycan analysis without introducing labeling bias.
1Thermo Fisher Scientific, Sunnyvale, CA, USA
2Thermo Fisher Scientific, San Jose, CA, USA
3Thermo Fisher Scientific, Mississauga, ON, Canada
4Roche Diagnostics GmbH, Penzberg, Germany
United States
