Characterization of oligosaccharides of the lactosamine series derived from keratan sulfates by tandem mass spectrometry

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Eur. J. Mass Spectrom. 6, 193 - 203 (2000)

Characterization of oligosaccharides of the lactosamine series derived from keratan sulfates by tandem mass spectrometry

Masayuki Kubota
Department of Applied Physics and Chemistry, The University of Electro-Communications, Chofu, Tokyo , Japan
Keiichi Yoshida and Akira Tawada
Tokyo Research Institute, Seikagaku Corporation, Higashiyamatoshi, Tokyo 189, Japan
Mamoru Ohashi^*
Department of Materials Science, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa , Japan

ABSTRACT:

Positive- and negative-ion fast-atom bombardment tandem mass spectrometry with collision-induced dissociation (FAB-CID-MS/MS) has been used in the characterization of di-and tetra-saccharides of the lactosamine series from keratan sulfates. FAB-CID-MS/MS of Galb1-4GlcNAc (L1) exhibited strong fragment ions originating from ring cleavage at the reducing-terminal sugar moiety together with glycosidic bond-cleavage ions, whereas GlcNAcb1-3Gal (K1) showed strong glycosidic bond-cleavage ions but no ring-cleavage ions. A series of ring-cleavage fragment ions was observed with members of the L-series which have free hydroxyl groups at the C1 and C3 positions. CID-MS/MS spectra of the [M + Na SO₃]⁺ ion (m/z 406) from L2 and the [M + Na 2SO₃]⁺ ion (m/z 406) from L4 were almost identical with the CID-MS/MS spectrum of the [M + Na]⁺ ion (m/z 406) from L1, which indicated that the sugar skeletons of L2 and L4 are the same as that of L1. On the other hand, the CID-MS/MS spectrum of the [M + Na SO₃]⁺ ion (m/z 508) from L4 did not resemble that of the [M + Na]⁺ ion (m/z 508) from L2. The former showed peaks that were additional to the peaks in the latter. Since these extra peaks were accounted for on the basis of the structure of L3 [Galb1(6S)-4GlcNAc, S = sulfate], the in-source loss of sulfate groups by ester exchange upon FAB ionization takes place in a dual manner; one reaction at the non-reducing-terminal sugar to give L2 and the other at the reducing-terminal sugar to give L3. The CID-MS/MS spectra were characteristic for the tetrasaccharides L1-L1, L2-L2 and L4-L4 while in-source fragmentation confirms the component disaccharides of each tetrasaccharide. The structure of a tetrasaccharide trisulfate was confirmed as L2L4 and not L4L2 by CID-MS/MS. Negative-ion FAB-CID-MS/MS spectra of the sulfated di-and tetra-saccharides showed a pattern similar to that of the positive-ion spectra. Subtraction of the CID-MS/MS spectrum of the [M H] ion of L2 [Galb1-4GlcNAc(6S)] from that of the [M H SO₃] ion of L4 [Gal(6S)b1-4GlcNAc(6S)] gave several specific ions whose origins were nicely explained on the basis of the structure of L3. The structure of a pentasaccharide consisting of N-acetylneuraminic acid and a tetrasaccharide trisulfate was confirmed, on the basis of FAB-CID-MS/MS, as NeuNAca2-6L2-L4.

Keywords: sulfated disaccharide, sulfated tetrasaccharide, keratan sulfate, FAB, MS/MS, CID, lactosamine

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