Electrospray Ionization and Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of Permethylated Oligosaccharides Touradj Solouki, † Bruce B. Reinhold, ‡ Catherine E. Costello, ‡ Matthew O’Malley, Shenheng Guan, § and Alan G. Marshall* ,§ Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Florida State University, Tallahassee, Florida 32310 Mass spectra of fragments of permethylated oligosaccha- rides are analyzed by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Sustained off- resonance irradiation (SORI) collision-induced dissocia- tion (CID), quadrupolar axialization, multiple stages of isolation and dissociation (MS n ), and ion remeasurement are exploited for carbohydrate structural analyses. That SORI CID internal energies are adequate for linkage analysis of a permethylated glucose oligomer is demon- strated by identifying ring-opened fragment ions from MALDI-generated mass-isolated and collisionally acti- vated ions. Ion remeasurement and axialization tech- niques enhance the sensitivity of ion fragmentation analy- sis. Multiple stages of isolation and dissociation of ion fragments (MS n ) provide for structural analysis of an electrospray-ionized permethylated lacto-N-fucopentaose isomer (LNFP II). Compared to MS 2 spectra taken with a triple quadrupole, FT-ICR MS n (n > 2) provides more extensive characterization of the parent molecular struc- ture than is available from a single stage of ion isolation and dissociation (MS 2 ). Biological carbohydrates constitute a diverse group of polymers playing various roles in cellular processes, ranging from energy storage (glycogen) or structural support (cellulose, chitin) to signaling, adhesion, and protein modifications. 1,2 Biological carbohydrates range from simple monosaccharides to megadalton homopolymers, from precisely structured signaling oligosaccha- rides to complicated mixtures of carbohydrate-modified proteins and lipids. The study of carbohydrate biology, or glycobiology, has seen enormous growth in the past decade, and with this growth comes an increasing emphasis on analytical tools capable of molecular resolution into structure/ function relationships. The principal difficulty in structural identification of carbohy- drate oligomers is the large number of isomers arising both from variation in linkage positions between monomer residues and from the possibility of multiple linkages to a single residue (branching), e.g., more than 10 12 structures for a reducing hexasaccharide! 3 Hence, for a straight chain oligomer, structural identification requires the sequence of glycosyl linkage types, i.e., the linkage position and anomeric configuration, as well as the sequence of monomer residues analogous to amino acid (protein) or nucleic acid (DNA, RNA) oligomers. Multiple linkages allow for multiple connection topologies, and biological carbohydrates generally exhibit complicated branching structures as well as linear oligo- mers. Minor differences in linkage types or branching topology may have only subtle effects on the molecule’s overall physical properties (hydrophobicity, hydrodynamic radius, or solution ionization) but may have profound effects on the local molecular geometry and, hence, on the biological activity. Along with the structural isomers that arise through the variation in the types and locations of glycosidic bonds, individual monosaccharides such as glucose, galactose, and mannose are, themselves, struc- tural isomers. For many of the carbohydrates derived from eukaryotic cells, the number of different monosaccharides is not very large; for prokaryotic cells, the monosaccharide diversity itself can pose a severe challenge. 4 Carbohydrates are often modified by sulfates or phosphates or otherwise acylated, and both the nature and position of the modification on the residue and the position of the modified residue within the oligomer may need to be determined. Further, many of the structurally interesting biological carbohydrates are glycoconjugates, e.g., glycoproteins, glycolipids, or, in the case of glycophosphatidyl inositol anchors, glycolipoproteins. Aspects of this association (degree of glyco- sylation, chemical nature of the linked lipids) are generally sought along with details of the carbohydrate structure. Conjugation also presents an additional dimension of structural isomers, in terms of the specific location of a carbohydrate on a glycoprotein. The combination of conjugation and isomeric structural complexity has prevented the development of standard sequencing methodologies † Present address: IIT Research Institute, 10 West 35th St., Chicago, IL 60616. ‡ Mass Spectrometry Resource, Boston University School of Medicine, Boston, MA 02118. § Member of the Department of Chemistry, Florida State University, Tal- lahassee, FL 32310. (1) Karlsson, K.-A. Annu. Rev. Biochem. 1989 , 58, 309-350. (2) Varki, A. Glycobiology 1993 , 3, 97-130. (3) Laine, R. A. Glycobiology 1994 , 4, 759-767. (4) Reinhold, B. B.; Hauer, C. R.; Plummer, T. H.; Reinhold, V. N. J. Biol. Chem. 1995 , 270, 13197-13203. Anal. Chem. 1998, 70, 857-864 S0003-2700(97)00562-3 CCC: $15.00 © 1998 American Chemical Society Analytical Chemistry, Vol. 70, No. 5, March 1, 1998 857 Published on Web 01/27/1998