Two-dimensional hydrophilic interaction chromatography coupling anion-exchange and hydrophilic interaction columns for separation of 2-pyridylamino derivatives of neutral and sialylated N-glycans.
A novel chromatographic approach coupling anion-exchange (diethylaminoethylene) and hydrophilic-interaction (amide or zwitterionic type) columns was developed for the separating of 2-pyridylamino derivatives of N-glycans (PA-N-glycans). This is a kind of on-line, two-dimensional (2D) separation approach in hydrophilic-interaction chromatography (called the 2D-HILIC method), analogous to that of coupling cation- (or anion-, or mixed ion-) exchange and reversed-phase columns in hydrophobic interaction (reversed-phase) chromatography. The efficiency of the 2D-HILIC method was tested with biantennary neutral and sialylated PA-N-glycan standards by properly combining linear gradient elutions of water-acetonitrile and spiked-salt (ammonium acetate) elutions. The retention time RSDs of all the peaks in three sequential runs of a 100 min cycle are less than 0.52%, which indicates a reasonably good repeatability of the 2D-HILIC method. Then, the method was applied to a complex mixture of PA-N-glycans from human serum proteins. It was demonstrated that the neutral PA-N-glycans and mono-, di-, tri-, and tetrasialylated PA-N-glycans are able to be eluted in turn according to the number of sialic acids in an automated (programmed) single run.
Deguchi
Kisaburo
K
Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan. deguchi@glyco.sci.hokudai.ac.jp
Keira
Takuro
T
Yamada
Kuriko
K
Ito
Hiroki
H
Takegawa
Yasuhiro
Y
Nakagawa
Hiroaki
H
Nishimura
Shin-Ichiro
S
D000631:Aminopyridines / Q000737:chemistry*; D000837:Anion Exchange Resins / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D013058:Mass Spectrometry; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification*
D000631:Aminopyridines; D000837:Anion Exchange Resins; D011134:Polysaccharides