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 ß-anomeric configurations of furanose and pyranose forms. Conventional methods used to determine the structures of polysaccharides include hydrolysis of polysaccharides into oligosaccharides followed by identification of these oligosaccharides' structures individually through nuclear magnetic resonance spectroscopy (NMR). Although the sensitivity of mass spectrometry is much higher than that of NMR, conventional mass spectrometry provides only limited useful information on oligosaccharide structure determination, only the linkage positions of glycosidic bonds. In this study, we demonstrated a mass spectrometry method for the identification of linkage positions, anomeric configurations, and monosaccharide stereoisomers of intact oligosaccharides consisting of arabinose and xylose. We separated arabinose and xylose monosaccharides into α-furanose, ß-furanose, α-pyranose, and ß-pyranose forms through high-performance liquid chromatography and obtained the corresponding collision-induced dissociation mass spectra. Using these monosaccharide spectra and a flow chart consisting of the proper CID sequences derived from the dissociation mechanisms of pentose, a simple multi-stage tandem mass spectrometry method for structural identification of intact oligosaccharides consisting of arabinose and xylose was developed. The new mass spectrometry method provides a simple method for determining the structure of polysaccharides consisting of arabinose and xylose. The flow chart can be used in computer coding for automation, an ultimate goal for oligosaccharide structure determination.

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. Understanding the carbohydrate dissociation mechanism is important for obtaining the structural information from MS/MS. In this work, we studied the CID mechanism of galactose-N-acetylgalactosamine (Gal-GalNAc) and glucose-N-acetylglucosamine (Glc-GlcNAc) disaccharides with 1→3 and 1→4 linkages. For Gal-GalNAc disaccharides, the CID mass spectra of sodium ion adducts show significant difference between the α- and ß-anomers of GalNAc at the reducing end, while no difference in the CID mass spectra between two anomers of Glc-GlcNAc disaccharides was found. Quantum chemistry calculations show that for Gal-GalNAc disaccharides, the difference of the dissociation barriers between dehydration and glycosidic bond cleavage is significantly small in the ß-anomer compared to that in the α-anomer; while these differences are similar between the α- and ß-anomers of Glc-GlcNAc disaccharides. These differences can be attributed to the different orientations of hydroxyl and N-acetyl groups located at GalNAc and GlcNAc. The calculation results are consistent with the CID spectra of isotope labelled disaccharides. Our study provides an insight into the CID of 1→3 and 1→4 linked Gal-GalNAc and Glc-GlcNAc disaccharides. This information is useful for determining the anomeric configurations of GalNAc in oligosaccharides.

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

Collision-induced dissociation tandem mass spectrometry (CID-MSn) 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 (αFruf), ß-fructofuranose (ßFruf), α-fructopyranose (αFrup), and ß-fructopyranose (ßFrup). The four isomers can be separated by high-performance liquid chromatography (HPLC) and they show different mass spectra, indicating that CID-MSn 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. It has been demonstrated that the preference for the C-C cleavage over the competing isomerization of linear fructose is the main reason for the previously reported differences between the CID-MS spectra of aldohexoses and ketohexoses. In addition, the kinetic modeling helped to confirm the assignment of the different measured mass spectra to the different fructose isomers. The previously reported assignment based on the peak intensities in the HPLC chromatogram had left some open questions as the preference for the dehydration channels did not always follow trends previously observed for aldohexoses. Setting up the kinetic model further enabled us to directly compare the computational and experimental results, which indicated that the model can reproduce most trends in the differences between the dissociation pathways of the four cyclic fructose isomers.

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. Understanding the dissociation mechanisms is crucial for MS/MS-based structure determination. Herein, we investigate the collision-induced dissociation mechanism of ß-cellobiose and ß-maltose sodium adducts using quantum chemical calculations and experimental measurements. Four dissociation channels are studied. Dehydration mainly occurs through the transfer of an H atom to O1 of the sugar at the reducing end, followed by a C1-O1 bond cleavage; cross-ring dissociation starts with a ring-opening reaction, which occurs through the transfer of an H atom from O1 to O5 of the sugar at the reducing end. These two dissociation channels are analogous to that of glucose monosaccharide. The third channel, generation of B1 and Y1 ions, occurs through the transfer of an H atom from O3 (cellobiose) or O2 (maltose) to O1 of the sugar at the nonreducing end, followed by a glycosidic bond cleavage. The fourth channel, C1-Z1 fragmentation, has two mechanisms: (1) the transfer of an H atom from O3 or O2 to O4 of the sugar at the reducing end to generate C ions in the ring form and (2) the transfer of an H atom from O3 of the sugar at the reducing end to O5 of the sugar at the nonreducing end to produce C ions in the linear form. The results of calculations are supported by experimental collision-induced dissociation spectral measurements.

Improved Fixation Stability of a Dedicated Rib Fixation System in Flail Chest: A Retrospective Study.

Background and Objectives: Flail chest typically results from major trauma to the thoracic cage and is accompanied by multiple rib fractures. It has been well documented that surgical fixation of rib fractures can decrease both morbidity and mortality rates. This study aimed to evaluate the effectiveness of a dedicated APS Rib Fixation System, which features a pre-contoured design based on anatomical rib data of the Asian population. Materials and Methods: We reviewed 43 consecutive patients, who underwent surgical stabilization for flail chest with the traditional Mini bone plate (n = 20), APS plate (n = 13), or Mini + APS (n = 10). Demographic and injury variables were documented. We used X-ray radiography to determine plate fractures and screw dislocations after surgical fixation. Results: No statistical differences were noted in the demographic or injury variables. APS plates demonstrated fewer cases of plate fractures and screw dislocations than Mini plates (OR = 0.091, p = 0.008). Conclusions: The pre-contoured design of the APS plate demonstrated a superior rib implant failure rate as compared to the traditional Mini bone plate. Our study indicates that the APS plate may serve as an effective surgical tool for the treatment of flail chest.

Logically derived sequence tandem mass spectrometry for structural determination of Galactose oligosaccharides.

Mass spectrometry has high sensitivity and is widely used in the identification of molecular structures, however, the structural determination of oligosaccharides through mass spectrometry is still challenging. A novel method, namely the logically derived sequence (LODES) tandem mass spectrometry (MSn), for the structural determination of underivatized oligosaccharides was developed. This method, which is based on the dissociation mechanisms, involves sequential low-energy collision-induced dissociation (CID) of sodium ion adducts, a logical sequence for identifying the structurally decisive product ions for subsequent CID, and a specially prepared disaccharide CID spectrum database. In this work, we reported the assignment of the specially prepared galactose disaccharide CID spectra. We used galactose trisaccharides and tetrasaccharides as examples to demonstrate LODES/MSn is a general method that can be used for the structural determination of hexose oligosaccharides. LODES/MSn has the potential to be extended to oligosaccharides containing other monosaccharides provided the dissociation mechanisms are understood and the corresponding disaccharide database is available.

De novo 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/MSn), 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. The CID sequences are derived from carbohydrate dissociation mechanisms. LODES/MSn does not require oligosaccharide standards or the prior knowledge of the rules and principles of biosynthetic pathways; thus LODES/MSn is particularly useful for the investigation of undiscovered oligosaccharides. We demonstrated that the structure of core oligosaccharides in glycosphingolipids can be identified from more than 500 000 isomers using LODES/MSn. The same method can be applied for determining the structures of other oligosaccharides, such as N-, and O-glycans, and free oligosaccharides in milk.

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

Collision-induced dissociation (CID) of α-xylose and ß-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. By contrast, dehydration and cross-ring dissociation are the major dissociation channels of lithium adducts, and the corresponding dissociation mechanisms can be used to determine the anomericity and linkages of xylose in oligosaccharides. These mechanisms include (1) the dehydration branching ratio can be used to differentiate the anomericity of xylose and xylose in oligosaccharides because α-xylose has a larger branching ratio of dehydration than ß-xylose, (2) various cross-ring dissociation reactions can be used to identify linkage positions. The oligosaccharide with xylose at the reducing end is predicted to undergo 0,2X, 0,3X, and 0,2A cross-ring dissociation for the 1 → 2, 1 → 3, and 1 → 4 linkages, respectively. Application of these mechanisms to determine the anomericity and linkage positions of xylobiose was demonstrated.

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, ß-furanose, α-pyranose, and ß-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. The branching ratio of major dissociation channels was governed by two geometrical features: one being the cis or trans configuration of O1 and O2 atoms determining dehydration preferability and the other being the number of hydroxyl groups on the same side of the ring as the O1 atom determining the preferability of cross-ring dissociation. The relative branching ratios of the major channels were used to identify anomericity and the linkages of arabinose and ribose. Arabinose in the ß-configuration and ribose in the α-configuration are predicted to have larger relative dehydration branching ratios than arabinose in the α-configuration and ribose in the ß-configuration, respectively. Arabinose and ribose at the reducing end of oligosaccharides with 1 → 2 (pyranose and furanose), 1 → 3 (pyranose and furanose), 1 → 4 (pyranose only), and 1 → 5 (furanose only) linkages are predicted to undergo 0,2X, 0,3X, 0,2A, and 0,2A/0,3A cross-ring dissociation, respectively. Application of the dissociation mechanism to the disaccharide linkage determination is demonstrated.

Epigallocatechin-3-Gallate-Loaded Liposomes Favor Anti-Inflammation of Microglia Cells and Promote Neuroprotection.

Microglia-mediated neuroinflammation is recognized to mainly contribute to the progression of neurodegenerative diseases. Epigallocatechin-3-gallate (EGCG), known as a natural antioxidant in green tea, can inhibit microglia-mediated inflammation and protect neurons but has disadvantages such as high instability and low bioavailability. We developed an EGCG liposomal formulation to improve its bioavailability and evaluated the neuroprotective activity in in vitro and in vivo neuroinflammation models. EGCG-loaded liposomes have been prepared from phosphatidylcholine (PC) or phosphatidylserine (PS) coated with or without vitamin E (VE) by hydration and membrane extrusion method. The anti-inflammatory effect has been evaluated against lipopolysaccharide (LPS)-induced BV-2 microglial cells activation and the inflammation in the substantia nigra of Sprague Dawley rats. In the cellular inflammation model, murine BV-2 microglial cells changed their morphology from normal spheroid to activated spindle shape after 24 h of induction of LPS. In the in vitro free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, EGCG scavenged 80% of DPPH within 3 min. EGCG-loaded liposomes could be phagocytized by BV-2 cells after 1 h of cell culture from cell uptake experiments. EGCG-loaded liposomes improved the production of BV-2 microglia-derived nitric oxide and TNF-α following LPS. In the in vivo Parkinsonian syndrome rat model, simultaneous intra-nigral injection of EGCG-loaded liposomes attenuated LPS-induced pro-inflammatory cytokines and restored motor impairment. We demonstrated that EGCG-loaded liposomes exert a neuroprotective effect by modulating microglia activation. EGCG extracted from green tea and loaded liposomes could be a valuable candidate for disease-modifying therapy for Parkinson's disease (PD).

Automatic Full Glycan Structural Determination through Logically Derived Sequence Tandem Mass Spectrometry.

Glycans have diverse functions and play vital roles in many biological systems, such as influenza, vaccines, and cancer biomarkers. However, full structural identification of glycans remains challenging. The glycan structure was conventionally determined by chemical methods or NMR spectroscopy, which require a large amount of sample and are not readily applicable for glycans extracted from biological samples. Although it has high sensitivity and is widely used for structural determination of molecules, current mass spectrometry can only reveal parts of the glycan structure. Herein, the full structures of glycans, including diastereomers, the anomericity of each monosaccharide, and the linkage position of each glycosidic bond, which can be determined by using tandem mass spectrometry guided by a logically derived sequence (LODES), are shown. This new method provides de novo oligosaccharide structural identification with high sensitivity and has been applied to automatic in situ structural determination of oligosaccharides eluted by means of HPLC. It is shown that the structure of a given trisaccharide from a trisaccharide mixture and bovine milk were determined from nearly 3000 isomers by using 6-7 logically selected collision-induced dissociation spectra. The entire procedure for mass spectrometry measurement guided by LODES can be programmed in a computer for automatic full glycan structure identification.

De novo structural determination of mannose oligosaccharides by using a logically derived sequence for tandem mass spectrometry.

Carbohydrates play important roles in biological recognition processes. However, determining the structures of carbohydrates remains challenging because of their complexity. A simple tandem mass spectrometry-based method for determining the structure of underivatized mannose tetrasaccharides was demonstrated. This method employed the multistage low-energy collision-induced dissociation (CID) of sodium adducts in an ion trap, a logically derived sequence (LODES) from the dissociation mechanism for deciding the sequence of CID, and a specially prepared disaccharide spectrum database. Through this method, the linkages, anomeric configurations, and branch locations of carbohydrates could be determined without the prior assumption of possible structures. We validated this method by blind test of all the commercial available mannose tetrasaccharides. We showed that the structure of a given tetrasaccharide can be determined from 928 isomers by using only three to six appropriately selected CID mass spectra according to the proposed procedure. This method is simple and rapid and has the potential to be applied to other hexoses and oligosaccharides larger than tetrasaccharides. The CID procedures can be built in a computer-controlled mass spectrometer for automatic structural determination of underivatized oligosaccharides. Graphical abstract.

Unexpected Dissociation Mechanism of Sodiated N-Acetylglucosamine and N-Acetylgalactosamine.

The mechanism for the collision-induced dissociation (CID) of two sodiated N-acetylhexosamines (HexNAc), N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc), was studied using quantum-chemistry calculations and resonance excitation in a low-pressure linear ion trap. Experimental results show that the major dissociation channel of the isotope labeled [1-18O, D5]-HexNAc is the dehydration by eliminating HDO, where OD comes from the OD group at C3. Dissociation channels of minor importance include the 0,2A cross-ring dissociation. No difference has been observed between the CID spectra of the α- and ß-anomers of the same HexNAc. At variance, the CID spectra of GlcNAc and GalNAc showed some differences, which can be used to distinguish the two structures. It was observed in CID experiments involving disaccharides with a HexNAc at the nonreducing end that a ß-HexNAc shows a larger dissociation branching ratio for the glycosidic bond cleavage than the α-anomer. This finding can be exploited for the rapid identification of the anomeric configuration at the glycosidic bond of HexNAc-R' (R' = sugar) structures. The experimental observations indicating that the dissociation mechanisms of HexNAcs are significantly different from those of hexoses were explained by quantum-chemistry calculations. Calculations show that ring opening is the major channel for HexNAcs in a ring form. After ring opening, dehydration shows the lowest barrier. In contrast, the glycosidic bond cleavage becomes the major channel for HexNAcs at the nonreducing end of a disaccharide. This reaction has a lower barrier for ß-HexNAcs as compared with the barrier of the corresponding α-anomers, consistent with the higher branching ratio for ß-HexNAcs observed in experiment.

Toward Closing the Gap between Hexoses and N-Acetlyhexosamines: Experimental and Computational Studies on the Collision-Induced Dissociation of Hexosamines.

Motivated by the fundamental difference in the reactivity of hexoses and N-acetylhexosamines under collision-induced dissociation (CID) mass spectrometry conditions, we have investigated the CID of two hexosamines, glucosamine (GlcN) and galactosamine (GalN), experimentally and computationally. Both hexosamines undergo ring-opening and then dissociate via the 0,2A and the 0,3A (0,3X) cross-ring cleavage channels. The preference for the ring-opening is similar to the behavior of N-acetylhexosamines and explains why the two anomers of the same sugar give the same mass spectrum. While the spectrum for GlcN is dominated by the 0,2A signal, the signal intensities for both 0,2A and the 0,3A (0,3X) dissociation channels are comparable for GalN, which allows GlcN and GalN to be distinguished easily. Calculations at MP2 level of theory indicate that this is related to the differences in the relative barrier heights for the 0,2A and the 0,3A (0,3X) cross-ring cleavage channels. This, in return, reflects the circumstance that the 0,2A cross-ring cleavage barriers are different for the two sugars, while the barriers of all other dissociation channels are comparable. While the mechanisms of the cross-ring dissociation channels of hexoses are well described using the retro-aldol mechanism in the literature, this study proposes a new mechanism for the 0,3A (0,3X) cross-ring cleavage of hexosamines that involves the formation of an epoxy intermediate or a zwitterionic intermediate.

Collision-induced dissociation of sodiated glucose, galactose, and mannose, and the identification of anomeric configurations.

Collision-induced dissociation of sodiated α-glucose, ß-glucose, α-galactose, ß-galactose, α-mannose, and ß-mannose was studied using electronic structure calculations and resonance excitation in a low-pressure linear ion trap. We made an extensive search of conformers and transition states in calculations to ensure the transition state with the lowest barrier height for each dissociation channel could be located. The major dissociation channels, in addition to desodiation, are cross-ring dissociation and dehydration. Cross-ring dissociation starts with H atom transfer from the O1 atom to the O0 atom, followed by the cleavage of the C1-O0 bond. Dehydration of the anomer with O1 and O2 atoms in the cis configuration involves the transfer of an H atom from the O2 atom to the O1 atom, followed by the cleavage of the C1-O1 bond. In contrast, dehydration of the anomer with O1 and O2 atoms in the trans configuration mainly occurs through H atom transfer from the O3 or O2 atom to the O1 atom for glucose, from the O3 or O4 atom to the O1 atom for galactose, and from the O4 or O2 atom to the O1 atom for mannose, followed by the cleavage of the C1-O1 bond. The dehydration barrier heights are lower than those of cross-ring dissociation for cis anomers, but higher than those of cross-ring dissociation for trans anomers. The relative barrier heights from calculations are consistent with the experimental measurements of branching ratios. Both computational and experimental results show that the branching ratio of dehydration can be generalized as a simple rule for rapidly identifying the anomeric configurations of these monosaccharides.

Dichroic Behavior of Gold Nanoparticles Synthesized in Aqueous Solution with Insufficient Reducing Agent.

The spherical gold nanoparticles (AuNPs) typically are red in solution. However, in this study, the dichroic and spherical AuNPs were synthesized using a modified seeding growth method under reducing agent insufficiency in an aqueous solution. This particular AuNP solution is orange in reflected light and red in transmitted light. The reflectance curves confirm that the dichroic AuNPs are different from the classic AuNPs. With particle assembling, the AuNP solution is fainter orange in reflected light, but purple in transmitted light when the color of classic spherical AuNP solution is purple in both lights. Furthermore, the aggregated-nanogold solutions were added to HAuCl4 solutions with the addition of an insufficient amount reducing agent. The solution changed from faint orange to bright orange in reflected light and from purple to blue in transmitted light. It indicates that the gold assembling under a reducing agent insufficiency, not the shape of AuNP, causes the dichroic phenomenon. To the best of our knowing, this is the first study to report how the AuNP is synthesized, not the shape, affects the color of the AuNP.

Does low-energy collision-induced dissociation of lithiated and sodiated carbohydrates always occur at anomeric carbon of the reducing end?

RATIONALE: Collision-induced dissociation (CID) tandem mass spectrometry is one of the major methods in the structural determination of carbohydrates. Previous experimental studies and theoretical investigation of lithiated and sodiated underivatized carbohydrates seem to indicate that dehydration reactions and cross-ring dissociation of low-energy CID mainly occur at the anomeric carbon of the reducing end. However, these studies only investigated a few carbohydrates. METHODS: ESI-MS/MS spectra of [M + Li]+ and [M + Na]+ ions of several 18 O1-labeled monosaccharides and disaccharides at O1 of the reducing end were studied using a linear ion trap mass spectrometer. RESULTS: Dissociations from the losses of both labeled and unlabeled neutral fragments were observed. The branching ratios of dissociations from the losses of unlabeled neutrals for dehydration reactions are larger than that for cross-ring dissociation; lithiated carbohydrates are larger than sodiated carbohydrates, and 1-4 linkages of disaccharides are larger than the other linkages. For some lithiated carbohydrates, dehydration reactions from the losses of unlabeled neutrals have larger branching ratios than that from the losses of labeled neutrals. The fragments from the losses of unlabeled neutrals investigated using MS3 showed that the losses of unlabeled H2 O mainly occur at the reducing monomer for sodiated carbohydrates, but the losses of unlabeled C2 H4 O2 for lithiated carbohydrates can occur at both reducing and nonreducing monomers. The ratio of B1 and Y1 ions to C1 and Z1 ions of disaccharides is related to the cis or trans configuration of the O1 and O2 atoms in the nonreducing monomer. The results are explained by the properties of transition states of dissociation channels. CONCLUSIONS: Our data shows that dehydration reactions and cross ring dissociation do not always occur at the anomeric carbon atom of the reducing monomer.

Collision-induced dissociation of sodiated glucose and identification of anomeric configuration.

Collision-induced dissociation (CID) of sodiated glucose was investigated using electronic structure calculations and resonance excitation in a low-pressure linear ion trap. The major dissociation channels in addition to desodiation are dehydration and C2H4O2 elimination reactions which the barrier heights are near to or lower than the sodiation energy of glucose. Dehydration reaction involves the transfer of the H atom from the O2 atom to the O1 atom, followed by the cleavage of the C1-O1 bond. Notably, α-glucose has a dehydration barrier lower than that of ß-glucose. This difference results in the larger branching ratio of dehydration reactions involving α-glucose, which provides a simple and fast method for identifying the anomeric configurations of glucose. The C2H4O2 elimination starts from the H atom transfer from the O1 atom to the O0 atom, followed by the cleavage of the C1-O0 bond. These results were further confirmed by experimental study using 18O-isotope-labeled compounds. Both the experimental data and theoretical calculations suggest that the dehydration reaction and cross-ring dissociation of sodiated carbohydrates mainly occur at the reducing end during low-energy CID.

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. By contrast, chemical methods can be used for large quantities of samples; however, various side-reaction products are generated when chemical methods are used. Recently, the ammonia-catalyzed release of N-glycans from proteins has been reported to be associated with no typical side reactions. In the present study, we discovered a new side reaction: the epimerization of N-acetylglucosamine present at the reducing end of N-glycans to N-acetylmannosamine. The product of this side reaction interfered with the structural identification N-glycans. We propose a simple method that can help identify this artifact N-glycan isomer and eliminate the aforementioned interference. This simple method widens the applicability of ammonia-catalyzed reactions for N-glycan release from proteins, and is also suitable for N-glycans released using any other alkaline solutions.

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. Most reported free oligosaccharides are highly abundant and have lactose at the reducing end, and current oligosaccharide biosynthetic pathways in human milk are proposed based on these oligosaccharides. In this study, a new mass spectrometry technique, which can identify linkages, anomericities, and stereoisomers, was applied to determine the structures of free oligosaccharides in human, bovine, and caprine milk. Oligosaccharides that do not follow the current biosynthetic pathways and are not synthesized by any discovered enzymes were found, indicating the existence of undiscovered biosynthetic pathways and enzymes.