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Molecular & Cellular Proteomics 5:2146-2157, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Unusual N-Glycan Structures in -Mannosidase II/IIx Double Null Embryos Identified by a Systematic Glycomics Approach Based on Two-dimensional LC Mapping and Matrix-dependent Selective Fragmentation Method in MALDI-TOF/TOF Mass Spectrometry^*,S

Megumi Hato, Hiroaki Nakagawa, Masaki Kurogochi, Tomoya O. Akama, Jamey D. Marth^¶, Michiko N. Fukuda^,|| and Shin-Ichiro Nishimura^,**

From the Laboratory of Advanced Chemical Biology, Graduate School of Advanced Life Science, Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, N21, W11, Sapporo 001-0021, Japan, The Burnham Institute, La Jolla, California 92037, and ^¶ Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California San Diego, La Jolla, California 92093

-Mannosidase IIx (MX) is an enzyme closely related to -mannosidase II (MII), a key enzyme in N-glycan biosynthesis that catalyzes the first step in conversion of hybrid- to complex-type N-glycans in Golgi apparatus. Recently we generated MII/MX double knock-out mice and found that double nulls completely lack the complex-type N-glycans (Akama, T. O., Nakagawa, H., Wong, N. K., Sutton-Smith, M., Dell, A., Morris, H. R., Nakayama, J., Nishimura, S.-I., Pai, A., Moremen, K. W., Marth, J. D., and Fukuda, M. N. (2006) Essential and mutually compensatory roles of -mannosidase II and -mannosidase IIx in N-glycan processing in vivo in mice. Proc. Natl. Acad. Sci. U. S. A. 103, 8983–8988). In the present study, we determined minor but unusual N-glycan structures found in MII/MX double knock-out mice. We identified such N-glycans by a systematic glycomics approach applying a two-dimensional LC mapping database and matrix-dependent selective fragmentation technique in MALDI-TOF/TOF MS, a highly sensitive and reliable technique that provides specific fragmentations enabling the determination of precise oligosaccharide structures including regioisomers (Kurogochi, M., and Nishimura, S.-I. (2004) Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry. Anal. Chem. 76, 6097–6101). Quantitative profiling of all N-glycan structures including minor components from MII/MX nulls, MII nulls, MX nulls, and wild-type mice at embryonic day 15.5 yielded a total of 37 species when structural heterogeneity was reduced by the removal of the sialic acids. Among six unusual N-glycan structures, two glycoforms were novel and were found only in MII/MX double nulls. We characterize such structure as pseudocomplex-type N-glycans. The present study demonstrated that use of the versatile matrix-dependent selective fragmentation method in MALDI-TOF/TOF MS greatly accelerates detailed structural analysis of a trace amount of N-glycans.

^** To whom correspondence may be addressed. Tel.: 81-11-706-9043; E-mail: shin{at}glyco.sci.hokudai.ac.jp

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