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

Abstract

We prepared neutral oligosaccharide fraction from milk of a woman (blood type A, Le(b+)) 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-TOFMS(n) 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 N-acetyllactosamine branches of these isomers. On negative-MS(n) 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, Le(a), and Le(x) 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, Le(a), Le(x), and Le(b)/Le(y) on branched decaose cores. In typical oligosaccharides, 6-branches always consist of type 2 chain, while 3-branches, such as beta and gamma 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.

Determination of Sialyl and Neutral Oligosaccharide Levels in Transition and Mature Milks of Samoan Women, Using Anthranilic Derivatization Followed by Reverse Phase High Performance Liquid Chromatography

“…The chromatogram displayed at least 14 peaks, but only six of these could be identified due to the lack of available standards. Although human milk and colostrum contain at least 114 oligosaccharides, 7,8) most of these, excluding those determined in this study, in which 1 liter of milk or colostrum were used, are present in only trace amounts, as indicated by the near absence of their peaks during HPLC. The oligosaccharides previously studied by other researchers were similar to those in the present study; their methods involved determination of peak areas in chromatograms using capillary electrophoresis, HPLC, or HPEAC-PAD of these oligosaccharides or their derivatives.…”

“…6) More than 130 oligosaccharides have been detected, and the structures of 114 have been characterized. 7,8) These oligosaccharides are classified into neutral and acidic. The former group contains fucosylated HMO as abundant constituents, while the latter are almost all sialylated.…”

Structural Characterization by Multistage Mass Spectrometry (MSn) of Human Milk Glycans Recognized by Human Rotaviruses

“…The energies used in an ion trap are relatively low and slow, conditions that allow time for electron rearrangement and eliminations that expose reproducible details of former structure, a feature that is not available with higher CA energies. Multiple approaches to characterize and catalogue HMGs have been attempted (7,8), and excellent reviews of compiled HMG structures are available (7, 9 -11).…”

Challenges in Separations of Proteins and Small Biomolecules and the Role of Modern Mass Spectroscopy Tools for Solving Them, as Well as Bypassing Them, in Structural Analytical Studies of Complex Biomolecular Mixtures

“…Although LC-MS combined with bioinformatic tools represents a state-of-the-art screening tool, state-of-the-art MALDI MS methods are more appropriate for detailed structural characterization of previously uncharacterized biomolecules. For example, nanoHPLC Chip/TOF MS approach is excellent for rapid-throughput screening of HMO samples for identification of HMO structures present in the database [7,8], but negative ion mode MALDI MS of derivatized HMOs, in combination with use of specific exoglysidases is necessary for structural characterization of novel HMO structures [84].…”