Normal-phase nanoscale liquid chromatography-mass spectrometry of underivatized oligosaccharides at low-femtomole sensitivity.
We here describe the online liquid chromatography (LC) electrospray ionization mass spectrometry (MS) of underivatized glycans using a nanoscale normal-phase amide column at a flow rate of 300 nL/min. Retention on the amide column is based on polar interactions of the oligosaccharide hydroxyl groups with the stationary phase, and thus, the retention time predictably increases with elongation of the oligosaccharide chain. The system is characterized by its high chromatographic resolution, which routinely allows the separation of isobaric structures. Separation of oligosaccharide mixtures over a 1-h range permits the detailed characterization of the different species by multiple ion selection and fragmentation steps using ion trap MS. The here presented miniaturization of the online-LC system to the nanoscale in combination with ion trap MS allows the detection of oligosaccharide species in a mixture at low-femtomole sensitivity. Online normal-phase nano-LC-MS of complex oligosaccharide mixtures further facilitates the sensitive and detailed structural analysis of oligosaccharides by overcoming the need for cumbersome and time-consuming derivatization procedures such as reductive amination for labeling with hydrophobic fluorophores or labeling with tritium. The method should be useful for the sensitive and quick analysis of glycosylation patterns and individual oligosaccharides from biotechnologically produced glycoproteins as well as scarcely available biological samples.
Wuhrer
Manfred
M
Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. m.wuhrer@lumc.nl
Koeleman
Carolien A M
CA
Deelder
André M
AM
Hokke
Cornelis H
CH
D000577:Amides / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D036103:Nanotechnology; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000577:Amides; D009844:Oligosaccharides