Controllable Enzymatic Synthesis of Natural Asymmetric Human Milk Oligosaccharides.

Among human milk oligosaccharides (HMOs), linear HMOs are synthesized through mature but varied routes. Although branched HMOs can be synthesized by chemical, enzymatic, or chemoenzymatic methods, these methods cannot be easily applied to the synthesis of asymmetric multiantennary oligosaccharides. Herein, we developed a controllable method to synthesize asymmetric biantennary HMOs.

Versatile Strategy for the Chemoenzymatic Synthesis of Branched Human Milk Oligosaccharides Containing the Lacto-N-Biose Motif.

Human milk oligosaccharides (HMOs) exhibit prebiotic, antimicrobial, and immunomodulatory properties and confer significant benefits to infants. Branched HMOs are constructed through diverse glycosidic linkages and prominently feature the lacto-N-biose (LNB, Gal-β1,3-GlcNAc) motif with fucose and/or sialic acid modifications, displaying structural complexity that surpasses that of N- and O-glycans. However, synthesizing comprehensive libraries of branched HMO is challenging due to this complexity.

High Abundance of Unusual High Mannose -Glycans Found in Beans.

High mannose -glycans extracted from eight different beans (black bean, soybean, pea, white kidney bean, pinto bean, mung bean, white hyacinth bean, and red bean) were studied using the state-of-the-art mass spectrometry method logically derived sequence tandem mass spectrometry (LODES/MS). These beans show very similar -glycan isomer profiles: one isomer of ManGlcNAc and ManGlcNAc, two isomers of ManGlcNAc, three isomers of ManGlcNAc, and five isomers of ManGlcNAc were found. Isomers not predicted by current -glycan biosynthetic pathways were found in all beans, indicating the possibility of alternative biosynthetic pathways in these plants.

Collision-Induced Dissociations of Linear Hexose and Disaccharides with Linear Hexose at the Reducing End.

Characterization of carbohydrate structures using mass spectrometry is a challenging task. Understanding the dissociation mechanisms of carbohydrates in the gas phase is crucial for characterizing these structures through tandem mass spectrometry. In this study, we investigated the collision-induced dissociation (CID) of glucose, galactose, and mannose in their linear forms, as well as the linear forms of hexose at the reducing end of 1-6 linked disaccharides, using quantum chemistry calculations and tandem mass spectrometry.

Structural determination of fructooligosaccharides and raffinose family oligosaccharides using logically derived sequence tandem mass spectrometry.

Fructooligosaccharides (FOS) and raffinose family oligosaccharides (RFOs) are two highly abundant water-soluble carbohydrates in plants. The typical procedures for the FOS and RFO structural determination using mass spectrometry involve permethylation, followed by the hydrolysis of the permethylated oligosaccharides into monosaccharides, and then the identification of linkage positions using GC mass spectrometry. However, the determination of linkage position sequence is not straightforward, thus this method is limited to small oligosaccharides or oligosaccharides with simple linkages.

Collision-Induced Dissociation of Fucose and Identification of Anomericity.

Structural determination of carbohydrates using mass spectrometry remains challenging, particularly, the differentiation of anomeric configurations. In this work, we studied the collision-induced dissociation (CID) mechanisms of sodiated α- and β-l-fucose using an experimental method and quantum chemistry calculations. The calculations show that α-l-fucose is more likely to undergo dehydration due to the fact that O1 and O2 are on the same side of the sugar ring.

Chromatograms and Mass Spectra of High-Mannose and Paucimannose -Glycans for Rapid Isomeric Identifications.

-Linked glycosylation is one of the most essential post-translational modifications of proteins. However, -glycan structural determination remains challenging because of the small differences in structures between isomers. In this study, we constructed a database containing collision-induced dissociation MS mass spectra and chromatograms of high-performance liquid chromatography for the rapid identification of high-mannose and paucimannose -glycan isomers.

The collision-induced dissociation mechanism of sodiated Hex-HexNAc disaccharides.

Determining carbohydrate structures, such as their compositions, linkage positions, and in particular the anomers and stereoisomers, is a great challenge. Isomers of different anomers or stereoisomers have the same sequences of chemical bonds, but have different orientations of some chemical bonds which are difficult to be distinguished by mass spectrometry. Collision-induced dissociation (CID) tandem mass spectroscopy (MS/MS) is a widely used technique for characterizing carbohydrate structures.

Identification of the High Mannose -Glycan Isomers Undescribed by Conventional Multicellular Eukaryotic Biosynthetic Pathways.

-linked glycosylation is one of the most important post-translational modifications of proteins. Current knowledge of multicellular eukaryote -glycan biosynthesis suggests high mannose -glycans are generated in the endoplasmic reticulum and Golgi apparatus through conserved biosynthetic pathways. According to conventional biosynthetic pathways, four ManGlcNAc isomers, three ManGlcNAc isomers, and one ManGlcNAc isomer are generated during this process.

A simple tandem mass spectrometry method for structural identification of pentose oligosaccharides.

Differentiation of stereoisomers that are only dissimilar in the orientation of chemical bonds in space by mass spectrometry remains challenging. Structural determination of carbohydrates by mass spectrometry is difficult, mainly due to the large number of stereoisomers in carbohydrates. Arabinose and xylose are pentose stereoisomers typically present in plant polysaccharides and exist in α- and β-anomeric configurations of furanose and pyranose forms.

Collision-Induced Dissociation of α-Isomaltose and α-Maltose.

Determination of carbohydrate structures remains a considerable challenge. Collision-induced dissociation (CID) tandem mass spectroscopy (MS/MS) is widely used for carbohydrate structure determination. Structural information derived from MS/MS relies on an understanding of the carbohydrate dissociation mechanism.

Identification of side-reaction products generated during the ammonia-catalyzed release of N-glycans.

N-linked glycosylation is one of the most important post translational modification of proteins. Various analytical techniques are used for the structural identification of the N-glycans released from proteins through various enzymatic and chemical methods. Although very few side-reaction products are generated during the enzymatic release of N-glycans, this method is expensive and suitable only for small quantities of samples.

The Good, the Bad, and the Ugly Memories of Carbohydrate Fragments in Collision-Induced Dissociation.

Collision-induced dissociation (CID) tandem mass spectrometry is commonly used for carbohydrate structural determinations. In the CID tandem mass spectrometry approach, carbohydrates are dissociated into fragments, and this is followed by the structural identification of fragments through subsequent CID. The success of the structural analysis depends on the structural correlation of fragments before and after dissociation, that is, structural memory of fragments.

Collision-induced dissociation of Na-tagged ketohexoses: experimental and computational studies on fructose.

Collision-induced dissociation tandem mass spectrometry (CID-MS) and computational investigation at the MP2/6-311+G(d,p) level of theory have been employed to study Na-tagged fructose, an example of a ketohexose featuring four cyclic isomers: α-fructofuranose (αFru), β-fructofuranose (βFru), α-fructopyranose (αFru), and β-fructopyranose (βFru). The four isomers can be separated by high-performance liquid chromatography (HPLC) and they show different mass spectra, indicating that CID-MS can distinguish the different fructose forms. Based on a simulation using a micro-kinetic model, we have obtained an overview of the mechanisms for the different dissociation pathways.

Electrospray ionization in-source decay of N-glycans and the effects on N-glycan structural identification.

Rational: Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are soft ionization techniques commonly used in mass spectrometry. Although in-source and post-source decays of MALDI have been investigated extensively, the analogous decays of ESI have received little attention. Previous studies regarding the analogous decays of ESI focus on the dissociation of multiply charged proteins and peptides.

Unusual free oligosaccharides in human bovine and caprine milk.

Free oligosaccharides are abundant macronutrients in milk and involved in prebiotic functions and antiadhesive binding of viruses and pathogenic bacteria to colonocytes. Despite the importance of these oligosaccharides, structural determination of oligosaccharides is challenging, and milk oligosaccharide biosynthetic pathways remain unclear. Oligosaccharide structures are conventionally determined using a combination of chemical reactions, exoglycosidase digestion, nuclear magnetic resonance spectroscopy, and mass spectrometry.

Collision-Induced Dissociation of Cellobiose and Maltose.

Structure determination is a longstanding bottleneck of carbohydrate research. Tandem mass spectrometry (MS/MS) is one of the most widely used methods for carbohydrate structure determination. However, the effectiveness of MS/MS depends on how the precursor structures are derived from the observed fragments.

Structural Determination of Polysaccharides Lichenin Using Logically Derived Sequence Tandem Mass Spectrometry.

A new mass spectrometry method, logically derived sequence (LODES) tandem mass spectrometry (MS), was applied to determine the primary structure of polysaccharide lichenin. Conventional polysaccharide structural analysis requires complex processes, including derivation, permethylation, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectrometry. Many of these processes can be replaced by LODES/MS.

structural determination of oligosaccharide isomers in glycosphingolipids using logically derived sequence tandem mass spectrometry.

Despite the importance of carbohydrates in biological systems, structural determination of carbohydrates remains difficult because of the large number of isomers. In this study, a new mass spectrometry method, namely logically derived sequence tandem mass spectrometry (LODES/MS), was developed to characterize oligosaccharide structures. In this approach, sequential collision-induced dissociation (CID) of oligosaccharides is performed in an ion trap mass spectrometer to identify the linkage position, anomeric configuration, and stereoisomers of each monosaccharide in the oligosaccharides.

Identification of Anomericity and Linkage of Arabinose and Ribose through Collision-Induced Dissociation.

Arabinose and ribose are two common pentoses that exist in both furanose and pyranose forms in plant and bacteria oligosaccharides. In this study, each pentose isomer, namely α-furanose, β-furanose, α-pyranose, and β-pyranose, was first separated through high-performance liquid chromatography followed by an investigation of collision-induced dissociation in an ion trap mass spectrometer. The major dissociation channels, dehydration and cross-ring dissociation, were analyzed by using high-level quantum chemistry calculations and transition state theory.

Collision-induced dissociation of xylose and its applications in linkage and anomericity identification.

Collision-induced dissociation (CID) of α-xylose and β-xylose were studied using mass spectrometry and quantum chemistry calculations. Three dissociation channels, namely loss of metal ions, dehydration, and cross-ring dissociation were found. The major dissociation channel of sodium adducts is the loss of sodium ions, and the minor dissociation channels are dehydration and cross-ring dissociation.

Fluorescence quantum yields of matrices used in ultraviolet matrix-assisted laser desorption/ionization.

Rationale: Ultraviolet matrix-assisted laser desorption/ionization (MALDI) is among the most popular soft ionization methods in mass spectrometry. Several theoretical models have been proposed to explain the primary ion generation in MALDI. These models require knowledge of various matrix molecular parameters for simulation.

Temperature Dependence of Desorbed Ions and Neutrals and Ionization Mechanism of Matrix-Assisted Laser Desorption/Ionization.

Two separate temperature-dependent experiments were performed to investigate the ionization mechanism of ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) of matrix 2,5-dihydroxybenzoic acid (2,5-DHB). First, the angular resolved intensity and velocity distributions of neutrals desorbed from the 2,5-DHB solid sample through UV laser (355 nm) pulse irradiation were measured using a rotating quadrupole mass spectrometer. Second, the desorbed neutrals, at an angle normal to the surface, and the desorbed ions were simultaneously detected for each laser shot using the quadrupole mass spectrometer and a time-of-flight mass spectrometer, respectively.