Normal-Phase Nanoscale Liquid Chromatography -Mass Spectrometry of Underivatized Oligosaccharides at Low-Femtomole Sensitivity Manfred Wuhrer,* Carolien A. M. Koeleman, Andre ´ M. Deelder, and Cornelis H. Hokke Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands We here describe the online liquid chromatography (LC) electrospray ionization mass spectrometry (MS) of un- derivatized 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 struc- tures. 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 oligosac- charides from biotechnologically produced glycoproteins as well as scarcely available biological samples. Mass spectrometric characterization of oligosaccharides is mainly performed by MALDI-TOF-MS of native or reductively aminated species. 1 To obtain more information by fragment ion analysis, oligosaccharides are generally analyzed by electrospray (ESI)-MS in underivatized form, 2 after reductive amination, 3-7 or after permethylation. 8-11 While most studies have been performed using direct infusion of oligosaccharide samples with an offline electrospray source, during the past few years, oligosaccharide fractionation by HPLC coupled online with ESI-MS has been introduced as a potent method for the characterization of complex glycan samples. 12-18 Only recently, downscaling of oligosaccharide online-LC-ESI-MS to the capillary scale (columns of 0.2-0.3-mm inner diameter) at flow rates of 2-5 µL/ min have led to a tremendous gain in sensitivity. 19,20 For underivatized oligosaccharides, online-LC-ESI-MS analyses have so far been performed using graphitized carbon chroma- tography. 15-21 The most generally applicable and best validated HPLC systems for analyzing oligosaccharides, however, are based on normal-phase chromatography after fluorescent labeling of glycans with 2-aminopyridine 22-26 or 2-aminobenzamide. 27-29 Normal- * To whom correspondence should be addressed. Tel: +31-71-526-5077. Fax: 31-71-526-6907. E-mail: m.wuhrer@ lumc.nl. (1) Harvey, D. J. Mass Spectrom. Rev. 1999 , 18, 349-450. (2) Bahr, U.; Pfenninger, A.; Karas, M.; Stahl, B. Anal. Chem. 1997 , 69, 4530- 4535. (3) Okamoto, M.; Takahashi, K.; Doi, T. Rapid Commun. Mass Spectrom. 1995 , 9, 641-643. (4) Yoshino, K.; Takao, T.; Murata, H.; Shimonishi, Y. Anal. Chem. 1995 , 67, 4028-4031. (5) Mo, W.; Sakamoto, H.; Nishikawa, A.; Kagi, N.; Langridge, J. I.; Shimonishi, Y.; Takao, T. Anal. Chem. 1999 , 71, 4100-4106. (6) Harvey, D. J. J. Am. Soc. Mass Spectrom. 2000 , 11, 900-915. (7) Harvey, D. J. Analyst 2000 , 125, 609-617. (8) Viseux, N.; de Hoffmann, E.; Domon, B. Anal. Chem. 1997 , 69, 3193- 3198. (9) Reinhold: V. N.; Reinhold: B. B.; Chan, S. Methods Enzymol. 1996 , 271, 377-402. (10) Sheeley, D. M.; Reinhold: V. N. Anal. Chem. 1998 , 70, 3053-3059. (11) Weiskopf, A. S.; Vouros, P.; Harvey, D. J. Anal. Chem. 1998 , 70, 4441- 4447. (12) Thomsson, K. A.; Karlsson, H.; Hansson, G. C. Anal. Chem. 2000 , 72, 4543- 4549. (13) Gennaro, L. A.; Harvey, D. J.; Vouros, P. Rapid Commun. Mass Spectrom. 2003 , 17, 1528-1534. (14) Schmid, D.; Behnke, B.; Metzger, J.; Kuhn, R. Biomed. Chromatogr. 2002 , 16, 151-156. (15) Itoh, S.; Kawasaki, N.; Ohta, M.; Hyuga, M.; Hyuga, S.; Hayakawa, T. J. Chromatogr., A 2002 , 968, 89-100. (16) Kawasaki, N.; Ohta, M.; Hyuga, S.; Hashimoto, O.; Hayakawa, T. Anal. Biochem. 1999 , 269, 297-303. (17) Kawasaki, N.; Ohta, M.; Hyuga, S.; Hyuga, M.; Hayakawa, T. Anal. Biochem. 2000 , 285, 82-91. (18) Thomsson, K. A.; Karlsson, N. G.; Hansson, G. C. J. Chromatogr., A 1999 , 854, 131-9. (19) Kawasaki, N.; Itoh, S.; Ohta, M.; Hayakawa, T. Anal. Biochem. 2003 , 316, 15-22. (20) Schulz, B. L.; Packer, N. H.; Karlsson, N. G. Anal. Chem. 2002 , 74, 6088- 6097. (21) Barroso, B.; Dijkstra, R.; Geerts, M.; Lagerwerf, F.; van Veelen, P.; de Ru, A. Rapid Commun. Mass Spectrom. 2002 , 16, 1320-1329. (22) Kuraya, N.; Hase, S. Anal. Biochem. 1996 , 233, 205-211. (23) Makino, Y.; Kuraya, N.; Omichi, K.; Hase, S. Anal. Biochem. 1996 , 238, 54-59. Anal. Chem. 2004, 76, 833-838 10.1021/ac034936c CCC: $27.50 © 2004 American Chemical Society Analytical Chemistry, Vol. 76, No. 3, February 1, 2004 833 Published on Web 12/30/2003