Applying mini-bore HPAEC-MS/MS for the characterization and quantification of Fc N-glycans from heterogeneously glycosylated IgGs.
High-performance anion-exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD) is a highly sensitive method for the analysis of oligosaccharides without the need for prior derivatization. However, the method suffers from the lack of chemical information with peak assignments based on the retention times of authentic standards or known peaks of reference materials. Here we applied HPAEC coupled on-line with electrospray ion trap mass spectrometry (HPAEC-MS) using a prototype mini-bore (1mm I.D.) CarboPac PA200 column and challenged the analytical separation based method for the structural assignment of heterogeneous mixtures of N-glycans derived from immunoglobulin G from human plasma, glyco-engineered CHO cells, and Sp2/0 mouse myeloma cells. Compared to an analytical scale 3mm I.D. column, the mini-bore column demonstrated a superior performance with up to 8-fold improved limit of detection for specific N-glycans determined by PAD. Quantitative evaluation by extracted ion current chromatograms revealed detection limits in the 50-100 femtomole range using ion trap MS operated in positive ionization mode. In our hands HPAEC-MS/MS allowed the detection and quantification of even low abundant glycan species including biantennary complex-type, high mannose, hybrid and hybrid bisected structures. In comparison to the detection of N-glycans as lithiated or sodiated adducts, we obtained a 65-fold improved signal-to-noise ratio with protonated ions only. Relative quantitative evaluation by single ion current chromatograms was successfully applied and demonstrated an excellent performance with respect to selectivity in the relative quantification of heterogeneous samples of N-glycans compared to HPAEC-PAD and HILIC-UPLC of 2-AB labelled N-glycans.
Pharma Technical Development Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany.
Maria
M
Maier
Pharma Technical Development Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany.
Dietmar
D
Reusch
Center for Proteomics and Metabolomics, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
Cees
C
Bruggink
Pharma Technical Development Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany.
Patrick
P
Bulau
Center for Proteomics and Metabolomics, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
Manfred
M
Wuhrer
Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany. Electronic address: michael.molhoj@roche.com.
Michael
M
Mølhøj
D000818:Animals; D000838:Anions; D016466:CHO Cells; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000032:analysis*; D057230:Limit of Detection; D051379:Mice; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis*; D012996:Solutions; D053719:Tandem Mass Spectrometry / Q000379:methods*; D006023:Glycoproteins
D000838:Anions; D007074:Immunoglobulin G; D019158:N-Acetylneuraminic Acid; D011134:Polysaccharides; D012996:Solutions; C046179:glycosylated IgG; D006023:Glycoproteins
HPAEC-MS/MS; IgG; Mass spectrometry; N-Glycan; Oligosaccharides; Pulsed amperometric detection; Sialyltransferase