Structural determination by negative-ion MALDI-QIT-TOFMSn after pyrene derivatization of variously fucosylated oligosaccharides with branched decaose cores from human milk

Junko Amano 1 Minako Osanai 1 Takahiro Orita 1 Daisuke Sugahara 1 Kenji Osumi 0 0 Laboratory of Glyco-organic Chemistry, The Noguchi Institute , 1-8-1, Kaga, Itabashi, Tokyo 173-0003 , Japan 1 Laboratory of Glycobiology c The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: - We prepared neutral oligosaccharide fraction from milk of a woman (blood type A, Leb+) by anion-exchange column chromatography after the removal of lipids and proteins. Further fractionation was performed by means of Aleuria aurantia lectin-Sepharose column chromatography and reverse-phase HPLC after labeling with a pyrene derivative. This pyrene labeling allowed identification by negative-MALDI-TOFMSn analysis of 22 oligosaccharides with decaose cores, among which 21 had novel structures. Negative ions could not be produced from neutral oligosaccharides without labeling on MALDI. Mono-, di-, tri-, and tetrafucosylated decaose fractions contained three, nine, six, and four isomers, respectively. Our method enables easy determination of fucosylated structures on the Nacetyllactosamine branches of these isomers. On negativeMSn the fragment ions included several A and D ions, from which fucosylation on the branches could be elucidated. Other characteristic ions were also detected. Y-type cleavage at the reducing side of -3GlcNAc indicated the occurrence of type 1 chain. Specific fragment ions were produced from H, Lea, and Lex antigens. Linkage-specific exoglycosidase digestion confirmed the structures. The results indicate that the diversity of the oligosaccharides is due to combinations of type 1 H, Lea, Lex, and Leb/Ley on branched decaose cores. In typical oligosaccharides, 6-branches always consist of type 2 chain, while 3-branches, such as and chains, are fucosylated type 1 chains. From the viewpoint of biosynthesis, the presence of fucosylation and type 1 chain may halt elongation of the N-acetyllactosamine and promote formation of branched structures. Human milk is known to contain oligosaccharides with larger size (from lactose to those larger than octadecasaccharides) and greater diversity as compared to bovine milk, which mainly con1To whom correspondence should be addressed: Tel: +81-3-3961-3255; Fax: +81-3-3964-5588; e-mail: tains small oligosaccharides, such as lactose and sialyllactose. Human milk oligosaccharides are derived from various core oligosaccharides by sialylation via 2-3 or 2-6 linkages and fucosylation via 1-2, 1-3 or 1-4 linkages. There is considerable evidence that virulent enteric bacteria and viruses initiate infection by binding to particular sugar chains of glycolipids and glycoproteins on the surface of their target cells (Sharon 1996). D Due to their structural mimicry of the sugar chains of glycopro- now teins on the mucous membrane, human milk oligosaccharides lao are considered to protect breast-fed infants against infections edd by blocking the adhesion of pathogens (Newburg et al. 2005; rfo Bode 2006). Therefore, milk oligosaccharides are expected to m be inhibitors of infection by these bacteria and viruses (Kobata ttph 2003). ://g The structures of many human milk oligosaccharides, which lcy have cores smaller than octaose, have been identified in detail .boo (Kobata et al. 1978; Haeuw-Fievre et al. 1993). These oligosac- fxo charides often express ABO blood group antigens and Le jrd antigens containing fucose. Lebrillas group detected 58 ruo foulicgoossyalcactehda.riSdeevseinn opfoot hleed1h0ummaonstmabilukn,daanndt 4o4ligoofstahcecshearwideeres .lrsano were fucosylated, accounting for approximately 46% of the /gb entire quantity (Nimomuevo et al. 2006). Recently, various gy kinds of mass spectrometry have been applied to elucidate the seu structures of milk oligosaccharides. Negative-ion electrospray tno mass spectrometry with collision-induced dissociation (CID) oN was proved to be useful for determination of the structures of e v m variously fucosylated oligosaccharides, and underivatized neu- eb tral oligosaccharides were identified even from mixtures on the ,r9 basis of the specific fragmentation behavior of deprotonated 20 molecules (Pfenninger et al. 2002). Many linkage-specific frag- 4 1 ment ions were obtained from linear and branched oligosaccharides using closed-ring chromophore labeling (Cheng and Her 2002). Chai et al. investigated various neutral oligosaccharides, including a monofucosylated lacto-N-decaose, by combined use of electrospray MS/MS and NMR spectroscopy (Chai et al. 2001, 2002, 2005; Kogelberg et al. 2004). They identified linkages by using GC-MS analysis of partially methylated alditol acetates and NMR spectroscopy. Both kinds of analysis require substantial amounts of samples. Instead, we applied linkage-specific enzyme digestion, e.g., with 2-fucosidase and 4-galactosidase (followed by 4-galactosyltransferase) to pyrene-labeled oligosaccharides, and measured MS of the reaction mixtures. Sub-picomole amounts are sufficient for this method. By using a combination of enzymatic modifications and MALDI-MSn, we succeeded in identifying the structures of individual isomers in mixtures of isomeric oligosaccharides which could not be originally separated by HPLC because the mass numbers of the isomers became different from each other. The combination of pyrene labeling and negative-ion MALDI-QIT-TOF MSn that we have established is a powerful tool for structural determination of neutral oligosaccharides because negative ions are not easily produced from neutral oligosaccharides in MALDI-MS. The key advantages of this method are high sensitivity and protection against loss of fucose. Furthermore, many fragment ions such as A-, D-, Y-, and C-type ions are observed, and determination of isomeric and branched structures is possible (see Amano et al. 2009). In the present study, we focused on oligosaccharides with a decaose core, the structures of which have not previously been well elucidated. Using our method, we were able to obtain detailed structures of variously fucosylated oligosaccharides from human milk. Results and discussion Fractionation of oligosaccharides A neutral oligosaccharide fraction, N-2, was considered to contain mainly decaose cores because fraction N-3 contained oligosaccharides with hexaose cores and octaose cores. The oligosaccharides in fraction N-2 were labeled with pyrene butanoic acid hydrazide (PBH) and subjected to Aleuria aurantia lectin (AAL)-Sepharose column chromatography to afford three fractions: AAL-1, 5 column volumes of washing; AAL-2, next 5 column volumes of washing; AAL-3, 5 column volumes of eluent with fucose. The percent molar ratios of oligosaccharides in the fractions AAL-1, AAL-2, and AAL-3 were 18.1, 62.5, and 19.4, respectively. It seems that most of these oligosaccharides are fucosylated and many of them contain more than two fucose residues because at least two fucose residues within an oligosaccharide are necessary for (...truncated)