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

N-Linked glycosylation is one of the most essential post-translational modifications of proteins. However, N-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 MSn mass spectra and chromatograms of high-performance liquid chromatography for the rapid identification of high-mannose and paucimannose N-glycan isomers. These N-glycans include isomers by breaking of arbitrary numbers of glycosidic bonds at arbitrary positions of canonical Man9GlcNAc2 N-glycans. In addition, some GlcMannGlcNAc2 N-glycan isomers were included in the database. This database is particularly useful for the identification of the N-glycans not in conventional N-glycan standards. This study demonstrated the application of the database to structural assignment for high-mannose N-glycans extracted from bovine whey proteins, soybean proteins, human mammary epithelial cells, and human breast carcinoma cells. We found many N-glycans that are not expected to be generated by conventional biosynthetic pathways of multicellular eukaryotes.

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 ß-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. In contrast, ß-l-fucose is more prone to the ring-opening reaction because more OH groups are on the same side of the sugar ring as O1. These differences suggest a higher preference for the dehydration reaction in sodiated α-l-fucose but a lower preference for ring-opening compared to that of ß-l-fucose. The calculation results, which are used to assign the CID mass spectra of α- and ß-l-fucose separated by high-performance liquid chromatography, are supported by the fucose produced from the CID of disaccharides Fuc-ß-(1 → 3)-GlcNAc and Fuc-α-(1 → 4)-GlcNAc. This study demonstrates that the correlation of cis- and trans-configurations of O1 and O2 to the relative branching ratios of dehydration and cross-ring dissociation in CID, observed in aldohexose and ketohexose in the pyranose form, can be extended to deoxyhexoses for anomericity 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 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. Among various hexose disaccharides, the major dissociation channels (dehydration, glycosidic bond cleavage, and cross-ring dissociation) of 1→2-, 1→3-, and 1→4-linked disaccharide sodium ion adducts can be explained by the dissociation mechanism derived from hexose monosaccharides. However, 1→6-linked disaccharides, which have low branching ratios for dehydration and glycosidic bond cleavage, cannot be explained by the same dissociation mechanism. In this study, we performed high-level quantum chemistry calculations to examine the CID mechanism of the α-isomaltose sodium ion adduct, a 1→6-linked glucose disaccharide. For comparison, we examined the CID dissociation mechanism of the α-maltose sodium ion adduct, a 1→4-linked glucose-disaccharide. Calculations revealed that although α-isomaltose and α-maltose had similar dissociation mechanisms, energy differences between the lowest transition states of various dissociation channels led to different CID fragmentation patterns. The dissociation barriers of dehydration and glycosidic bond cleavage were similar for the two disaccharides, but the cross-ring dissociation, which has the lowest dissociation barrier, exhibited differences in barriers between the disaccharides. The cross-ring dissociation barrier for α-maltose was only slightly lower than those of dehydration and glycosidic bond cleavage. However, the cross-ring dissociation barrier for α-isomaltose was substantially lower than those of dehydration and glycosidic bond cleavage. In addition, most of the α-isomaltose conformers that led to dehydration also led to cross-ring dissociation, resulting in suppression of dehydration by cross-ring dissociation. The findings can explain the low branching ratios for dehydration and glycosidic bond cleavage observed in α-isomaltose CID spectra.

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.

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.

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.

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.

Anhydrous octyl-glucoside phase transition from lamellar to isotropic induced by electric and magnetic fields.

A static deuterium nuclear magnetic resonance (2HNMR) technique (magnetic field, B = 7.05 T) was employed to monitor the thermotropic lamellar phase of the anhydrous 1:1 mixture sample of octyl-b-D-glucoside (ßOG) and that of partially deuterium labelled at the alpha position on the chain, i.e.,ßOG-d2 In the absence of an electric field, the 2H NMR spectrum of the mixture gives a typical quadrupolar doublet representing the aligned lamellar phase. Upon heating to beyond the clearing temperature at 112 °C, this splitting converts to a single line expected for an isotropic phase. Simultaneous application of magnetic and electric fields (E = 0.4 MV/m) at 85 °C in the lamellar phase, whose direction was set to be parallel or perpendicular to the magnetic field, resulted in the change of the doublet into a single line and this recovers to the initial doublet with time for both experimental geometries. This implies E- and B-field-induced phase transitions from the lamellar to an isotropic phase and a recovery to the lamellar phase again with time. Moreover, these phase transformations are accompanied by a transient current. A similar observation was made in a computational study when an electric field was applied to a water cluster system. Increasing the field strength distorts the water cluster and weakens its hydrogen bonds leading to a structural breakdown beyond a threshold field-strength. Therefore, we suggest the observed field-induced transition is likely due to a structure change of the ßOG lamellar assembly caused by the field effect and not due to Joule heating.

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. Fragments that completely lose the memory of their original structures cannot be used for structural analysis. By contrast, fragments with extremely strong correlations between the structures before and after fragmentation retain the information on their original structures as well as have memories of their precursors' entire structures. The CID spectra of these fragments depend on their own structures and on the remaining parts of the precursor structures, making structural analysis impractical. For effective structural analysis, the fragments produced from a precursor must have good structural memory, meaning that the structures of these fragments retain their original structure, and they must not be strongly affected by the remaining parts of the precursors. In this study, we found that most of the carbohydrate fragments produced by low-energy CID have good memory in terms of linkage position and anomericity. Fragments with ugly memory, where fragment structures change with the remaining parts of the precursors, can be attributed to C ion formation in a linear form. Fragments with ugly memory can be changed to have good memory by preventing linear C ion generation by using an alternative CID sequence, or the fragments of ugly memory can become useful in structural analysis when the contribution of linear C ions in fragmentation patterns is understood.

Molecular dynamics study of anhydrous lamellar structures of synthetic glycolipids: effects of chain branching and disaccharide headgroup.

Glycolipids form materials of considerable potential for a wide range of surfactant and thin film applications. Understanding the effect of glycolipid covalent structure on the properties of their thermotropic and lyotropic assemblies is a key step toward rational design of new glycolipid-based materials. Here, we perform molecular dynamics simulations of anhydrous bilayers of dodecyl ß-maltoside, dodecyl ß-cellobioside, dodecyl ß-isomaltoside, and a C(12)C(10) branched ß-maltoside. Specifically, we examine the consequences of chain branching and headgroup identity on the structure and dynamics of the lamellar assemblies. Chain branching of the glycolipid leads to measurable differences in the dimensions and interactions of the lamellar assembly, as well as a more fluid-like hydrophobic chain region. Substitution of the maltosyl headgroup of ßMal-C(12) by an isomaltosyl moiety leads to a significant decrease in bilayer spacing as well as a markedly altered pattern of inter-headgroup hydrogen bonding. The distinctive simulated structures of the two regioisomers provide insight into the difference of ~90 °C in their observed clearing temperatures. For all four simulated glycolipid systems, with the exception of the sn-2 chain of the branched maltoside, the alkyl chains are ordered and exhibit a distinct tilt, consistent with recent crystallographic analysis of a branched chain Guerbet glycoside. These insights into structure-property relationships from simulation provide an important molecular basis for future design of synthetic glycolipid materials.