Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MSⁿ (Part 2: Application to isomeric mixtures)

Abstract

A complex mixture of isomeric neutral oligosaccharides from pooled human milk was analyzed by nano-electrospray ionization (ESI) in a quadrupole ion trap mass spectrometer (QIT-MS) in the negative ion mode. Since deprotonated molecules of neutral oligosaccharides follow distinct fragmentation rules, which have been elucidated by using model compounds (see [1]), spectra obtained from consecutive CID experiments allowed the differentiation of isomers out of this highly complex mixture. With this method new human milk oligosaccharides of previously unknown isomeric structures have been identified, e.g., the occurence of three isomeric fucosylated lacto-N-hexaoses could be determined precisely, which have not been described before: . Also, free oligosaccharides, which are present either extracellular or in cell matrices, are believed to have other than nutritional function. As the (bio)chemical basis of many life processes is known, both recognition of diseases and the development of medical treatment becomes possible. Also preventional health treatment gains importance; especially nutrition is known to be an essential factor. This leads to the development of nutritional additives with healing and precautional character (functional food).As revealed by MALDI/MS human milk contains neutral as well as acidic oligosaccharides of high molecular weight [3,4], of which the biological function is yet not known, but there is some evidence that these compounds have essential biological functions [5,6]. It is hypothesized that due to their structural similarity to the sugars in N-and O-glycans and their resistance against digestion by glycosidases specific for N-and Oglycans, human milk oligosaccharides are able to reach the colon. There they may be potential, water-soluble receptor analogues which prevent interactions of pathogenic bacteria, viruses, fungi, or effects of toxins. They also may have benificial effects for the development of a specific intestinal flora of breast-fead infants. As they are potential nonimmunological factors, human milk oligosaccharides are under discussion as supplements for infant formula. Therefore, knowledge of their specific biological activity and the crucial concentration is necessary.This demands analytical tools that provide structural elucidation of bioactive compounds and their active concentration. Today, there is no universal method for the characterization of oligosaccharides. Traditional methods for structural analysis of oligosaccharides are time and cost consuming. Even though NMR spectroscopy as well as X-ray crystallography have been successfully applied [7,8], both methods need high amounts of sample in high purity. Mass spectrometry is a useful and sensitive method to investigate samples of low absolute amount and concentration or in complex mixtures. The useful combination of classical enzymatic approach and mass spectrometry is not only time consuming and costly, it is also limited to samples, that

The role of mobile protons in negative ion CID of oligosaccharides

“…The methyl esterified Di4SDi4S°precursor°(Figure°4b)°produces°an°abundant C 3 ion but does not yield 0,2 X 3 and Y 3 ions. A-type product ions have been shown to arise from retro-aldol rearrangement of oligosaccharide ions in the negative mode° [16,°17].°This°mechanism°requires°an°open-ring reducing terminal aldehyde, and such structures are formed°by°C-type°fragmentation° [23][24][25].°The°0 ,2 A 3 ion formed from cross-ring cleavage to the internal GlcA residue°in°Figure°4b°is°therefore°likely°to°arise°from°the C 3 ion by retro-aldol rearrangement. The results show that replacement of the carboxyl proton with a methyl group alters the product ion pathways by disfavoring formation of B-and Y-type ions and favoring A-type cleavage in the internal GlcA residue.…”

“…Sialic acids and sulfate groups are eliminated readily in the positive mode, resulting in the loss of information [21]. In the negative mode, neutral glycans produce abundant C-type ions, and cross-ring cleavages are observed within reducing terminal residues and to internal 4-linked residues [22][23][24][25][26]. Three-linked residues undergo double C/Z (D-type) cleavage, providing useful information on glycan structure [23].…”

The influence of sialylation on glycan negative ion dissociation and energetics

“…Other exceptions arise for small structures in which few other fragmentation pathways exist to compete with X-type cross-ring cleavage (see, for example, Figure 5). Low-energy CID tandem MS of negative ions has proven to be useful for differentiating type 1 (-Gal␤1-3GlcNAc-) and type 2 (-Gal␤1-4GlcNAc-) chains and patterns of fucosylation in native milk oligosaccharides [10,30,31] and closed-ring glycosylamine labeled neutral oligosaccharides [32]. A D-type ion results from cleavage of the 1-and 3-substituents of the core Man residue.…”

Human Milk Oligosaccharides: Evolution, Structures and Bioselectivity as Substrates for Intestinal Bacteria

“…The analytical methods that are capable of separating and characterizing the various sugar compositions and structures of oligosaccharides in human milk include high-performance liquid chromatography (HPLC), high pH anion exchange chromatography (HPAEC), capillary electrophoresis and various mass spectrometry platforms (MS) [13][14][15][16][17][18][19][20][21]. These methods as currently used are technically cumbersome, incapable of producing large quantities of highly purified isolated molecules and, as a result, there is little information on many of the basic biological properties of this class of molecule.…”