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 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.

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.

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.

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 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.

Simple Method for De Novo Structural Determination of Underivatised Glucose Oligosaccharides.

Carbohydrates have various functions in biological systems. However, the structural analysis of carbohydrates remains challenging. Most of the commonly used methods involve derivatization of carbohydrates or can only identify part of the structure. Here, we report a de novo method for completely structural identification of underivatised oligosaccharides. This method, which can provide assignments of linkages, anomeric configurations, and branch locations, entails low-energy collision-induced dissociation (CID) of sodium ion adducts that enable the cleavage of selective chemical bonds, a logical procedure to identify structurally decisive fragment ions for subsequent CID, and the specially prepared disaccharide CID spectrum databases. This method was first applied to determine the structures of four underivatised glucose oligosaccharides. Then, high-performance liquid chromatography and a mass spectrometer with a built-in logical procedure were established to demonstrate the capability of the in situ CID spectrum measurement and structural determination of the oligosaccharides in chromatogram. This consolidation provides a simple, rapid, sensitive method for the structural determination of glucose oligosaccharides, and applications to oligosaccharides containing hexoses other than glucose can be made provided the corresponding disaccharide databases are available.

Simple Approach for De Novo Structural Identification of Mannose Trisaccharides.

Oligosaccharides have diverse functions in biological systems. However, the structural determination of oligosaccharides remains difficult and has created a bottleneck in carbohydrate research. In this study, a new approach for the de novo structural determination of underivatized oligosaccharides is demonstrated. A low-energy collision-induced dissociation (CID) of sodium ion adducts was used to facilitate the cleavage of desired chemical bonds during the dissociation. The selection of fragments for the subsequent CID was guided using a procedure that we built from the understanding of the saccharide dissociation mechanism. The linkages, anomeric configurations, and branch locations of oligosaccharides were determined by comparing the CID spectra of oligosaccharide with the fragmentation patterns based on the dissociation mechanism and our specially prepared disaccharide CID spectrum database. The usefulness of this method was demonstrated to determine the structures of several mannose trisaccharides. This method can also be applied in the structural determination of oligosaccharides larger than trisaccharides and containing hexose other than mannose if authentic standards are available. Graphical Abstract.

Formation of Metal-Related Ions in Matrix-Assisted Laser Desorption Ionization.

In a study of the metal-related ion generation mechanism in matrix-assisted laser desorption ionization (MALDI), crystals of matrix used in MALDI were grown from matrix- and salt-containing solutions. The intensities of metal ion and metal adducts of the matrix ion obtained from unwashed crystals were higher than those from crystals washed with deionized water, indicating that metal ions and metal adducts of the matrix ions are mainly generated from the surface of crystals. The contributions of preformed metal ions and metal adducts of the matrix ions inside the matrix crystals were minor. Metal adducts of the matrix and analyte ion intensities generated from a mixture of dried matrix, salt, and analyte powders were similar to or higher than those generated from the powder of dried droplet crystals, indicating that the contributions of the preformed metal adducts of the matrix and analyte ions were insignificant. Correlation between metal-related ion intensity fluctuation and protonated ion intensity fluctuation was observed, indicating that the generation mechanism of the metal-related ions is similar to that of the protonated ions. Because the thermally induced proton transfer model effectively describes the generation of the protonated ions, we suggest that metal-related ions are mainly generated from the salt dissolution in the matrix melted by the laser. Graphical Abstract ᅟ.

Fluorescence spectroscopy of UV-MALDI matrices and implications of ionization mechanisms.

Matrix-assisted laser desorption ionization (MALDI) has been widely used in the mass analysis of biomolecules; however, there are a lot of debates about the ionization mechanisms. Previous studies have indicated that S1-S1 annihilation might be a key process in the generation of primary ions. This study investigates S1-S1 annihilation by examining the time-resolved fluorescence spectra of 12 matrices. No S1-S1 annihilation was observed in six of these matrices (3-hydroxy-picolinic acid, 6-aza-2-thiothymine, 2,4-dihydroxy-acetophenone, 2,6-dihydroxy-acetophenone, 2,4,6-trihydroxy-acetophenone, and ferulic acid). We observed two matrix molecules reacting in an electronically excited state (S1) in five of these matrices (2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxy-acetophenone, 2,3-dihydroxybenzoic acid, and 2,6-dihydroxybenzoic acid), and S1-S1 annihilation was a possible reaction. Among these five matrices, no S1-S1 annihilation was observed for 2,3-dihydroxybenzoic acid in typical peak power region of nanosecond laser pulses in MALDI, but a very small value of reaction rate constant was observed only in the high peak power region. The excited-state lifetime of sinapinic acid was too short to determine whether the molecules reacted in an electronically excited state. No correlation was observed between the ion generation efficiency of MALDI and S1-S1 annihilation. The results indicate that the proposal of S1-S1 annihilation is unnecessary in MALDI and energy pooling model for MALDI ionization mechanism has to be modified.

Does decarboxylation make 2,5-dihydroxybenzoic acid special in matrix-assisted laser desorption/ionization?

RATIONALE: Among the six positional isomers of dihydroxybenzoic acid (DHB), 2,5-DHB is a more favorable matrix for use in matrix-assisted laser desorption/ionization (MALDI) than the other isomers because of its high ion-generation efficiency at 337 and 355 nm. The generation of hydroquinone or p-benzoquinone through the decarboxylation of 2,5-DHB has been suggested to play a crucial role in the ion-generation efficiency of 2,5-DHB. METHODS: The mass spectra of desorbed neutrals generated from MALDI were measured using electron impact ionization (70 eV) and a quadrupole mass spectrometer and vacuum ultraviolet (118 nm) photoionization and a time-of-flight mass spectrometer. The mass spectra of desorbed ions generated from MALDI were investigated using a time-of-flight mass spectrometer. The dissociation barrier height and dissociation rate of decarboxylation were calculated by an ab initio method and RRKM theory. RESULTS: Decarboxylation of neutral 2,5-DHB and 2,5-DHB cations was not observed. Theoretical calculations indicated that decarboxylation of neutral 2,5-DHB and 2,5-DHB cations is too slow to occur. CONCLUSIONS: The high ion-generation efficiency of the 2,5-DHB matrix at 337 and 355 nm is not related to decarboxylation.

Direct analysis of 4-methylimidazole in foods using paper spray mass spectrometry.

Rapid qualitative and quantitative analysis of 4-methylimidazole (4-MEI) in caramel and beverage samples is demonstrated using the paper spray form of ambient ionization mass spectrometry. The minimum level of pure 4-MEI detectable using multiple reaction monitoring (MRM) in a triple quadrupole instrument was 3 pg µL⁻¹ in neat solvent and 5 pg µL⁻¹ in a matrix containing caramel. This method was used to analyze 11 caramel samples for 4-MEI. After implementing effective quality control protocols, average relative standard deviations for paper spray triple quadrupole MS were less than 15% and the linear dynamic range was three orders of magnitude. Results obtained on three different days by two different analyst groups agreed closely. An ion trap tandem MS method of approximate quantitative analysis is also described and it gave similar precision to the triple quadrupole experiment when fluctuations in ion currents were cancelled by simultaneously isolating the analyte and internal standard and fragmenting both in an ion trap simultaneous MRM experiment. As another alternative, a rapid qualitative screening method based on the use of high-resolution measurements instead of tandem mass spectrometry using an Orbitrap was also tested and found to give a detection limit of 100 pg µL⁻¹.

Alkylation effects on the energy transfer of highly vibrationally excited naphthalene.

The energy transfer of highly vibrationally excited isomers of dimethylnaphthalene and 2-ethylnaphthalene in collisions with krypton were investigated using crossed molecular beam/time-of-flight mass spectrometer/time-sliced velocity map ion imaging techniques at a collision energy of approximately 300 cm(-1). Angular-resolved energy-transfer distribution functions were obtained directly from the images of inelastic scattering. The results show that alkyl-substituted naphthalenes transfer more vibrational energy to translational energy than unsubstituted naphthalene. Alkylation enhances the V→T energy transfer in the range -ΔE(d)=-100~-1500 cm(-1) by approximately a factor of 2. However, the maximum values of V→T energy transfer for alkyl-substituted naphthalenes are about 1500~2000 cm(-1), which is similar to that of naphthalene. The lack of rotation-like wide-angle motion of the aromatic ring and no enhancement in very large V→T energy transfer, like supercollisions, indicates that very large V→T energy transfer requires special vibrational motions. This transfer cannot be achieved by the low-frequency vibrational motions of alkyl groups.

Energy transfer of highly vibrationally excited phenanthrene and diphenylacetylene.

The energy transfer between Kr atoms and highly vibrationally excited, rotationally cold phenanthrene and diphenylacetylene in the triplet state was investigated using crossed-beam/time-of-flight mass spectrometer/time-sliced velocity map ion imaging techniques. Compared to the energy transfer between naphthalene and Kr, energy transfer between phenanthrene and Kr shows a larger cross-section for vibrational to translational (V → T) energy transfer, a smaller cross-section for translational to vibrational and rotational (T → VR) energy transfer, and more energy transferred from vibration to translation. These differences are further enlarged in the comparison between naphthalene and diphenylacetylene. In addition, less complex formation and significant increases in the large V → T energy transfer probabilities, termed supercollisions in diphenylacetylene and Kr collisions were observed. The differences in the energy transfer between these highly vibrationally excited molecules are attributed to the low-frequency vibrational modes, especially those vibrations with rotation-like wide-angle motions.

Energy transfer of highly vibrationally excited biphenyl.

The energy transfer between Kr atoms and highly vibrationally excited, rotationally cold biphenyl in the triplet state was investigated using crossed-beam/time-of-flight mass spectrometer/time-sliced velocity map ion imaging techniques. Compared to the energy transfer of naphthalene, energy transfer of biphenyl shows more forward scattering, less complex formation, larger cross section for vibrational to translational (V→T) energy transfer, smaller cross section for translational to vibrational and rotational (T→VR) energy transfer, larger total collisional cross section, and more energy transferred from vibration to translation. Significant increase in the large V→T energy transfer probabilities, termed supercollisions, was observed. The difference in the energy transfer of highly vibrationally excited molecules between rotationally cold naphthalene and rotationally cold biphenyl is very similar to the difference in the energy transfer of highly vibrationally excited molecules between rotationally cold naphthalene and rotationally hot naphthalene. The low-frequency vibrational modes with out-of-plane motion and rotationlike wide-angle motion are attributed to make the energy transfer of biphenyl different from that of naphthalene.

Energy transfer of highly vibrationally excited 2-methylnaphthalene: Methylation effects.

The methylation effects in the energy transfer between Kr atoms and highly vibrationally excited 2-methylnaphthalene in the triplet state were investigated using crossed-beam/time-sliced velocity-map ion imaging at a translational collision energy of approximately 520 cm(-1). Comparison of the energy transfer between naphthalene and 2-methylnaphthalene shows that the difference in total collisional cross section and the difference in energy transfer probability density functions are small. The ratio of the total cross sections is sigma(naphthalene): sigma(methylnaphthalene)=1.08+/-0.05:1. The energy transfer probability density function shows that naphthalene has a little larger probability at small T-->VR energy transfer, DeltaE(u)<300 cm(-1), and 2-methylnaphthalene has a little larger probability at large V-->T energy transfer, -800 cm(-1)

Energy transfer of highly vibrationally excited naphthalene. III. Rotational effects.

The rotational effects in the energy transfer between Kr atoms and highly vibrationally excited naphthalene in the triplet state were investigated using crossed-beam/time-sliced velocity map ion imaging at various translational collision energies. As the initial rotational temperature changes from less than 10 to approximately 350 K, the ratio of vibrational to translational (V-->T) energy transfer cross section to translational to vibrational/rotational (T-->VR) energy transfer cross section increases, but the probability of forming a complex during the collisions decreases. Significant increases in the large V-->T energy transfer probabilities, termed supercollisions, at high initial rotational temperature were observed.

Energy transfer of highly vibrationally excited naphthalene. II. Vibrational energy dependence and isotope and mass effects.

The vibrational energy dependence, H and D atom isotope effects, and the mass effects in the energy transfer between rare gas atoms and highly vibrationally excited naphthalene in the triplet state were investigated using crossed-beam/time-sliced velocity-map ion imaging at various translational collision energies. Increase of vibrational energy from 16 194 to 18 922 cm(-1) does not make a significant difference in energy transfer. The energy transfer properties also remain the same when H atoms in naphthalene are replaced by D atoms, indicating that the high vibrational frequency modes do not play important roles in energy transfer. They are not important in supercollisions either. However, as the Kr atoms are replaced by Xe atoms, the shapes of energy transfer probability density functions change. The probabilities for large translation to vibration/rotation energy transfer (T-->VR) and large vibration to translation energy transfer (V-->T) decrease. High energy tails in the backward scatterings disappear, and the probability for very large vibration to translation energy transfer such as supercollisions also decreases.

Energy transfer of highly vibrationally excited naphthalene. I. Translational collision energy dependence.

Energy transfer between highly vibrationally excited naphthalene and Kr atom in a series of translational collision energies (108-847 cm(-1)) was studied separately using a crossed-beam apparatus along with time-sliced velocity map ion imaging techniques. Highly vibrationally excited naphthalene in the triplet state (vibrational energy: 16,194 cm(-1); electronic energy: 21,400 cm(-1)) was formed via the rapid intersystem crossing of naphthalene initially excited to the S(2) state by 266 nm photons. The collisional energy transfer probability density functions were measured directly from the scattering results of highly vibrationally excited naphthalene. At low collision energies a short-lived naphthalene-Kr complex was observed, resulting in small amounts of translational to vibrational-rotational (T-->VR) energy transfer. The complex formation probability decreases as the collision energy increases. T-->VR energy transfer was found to be quite efficient at all collision energies. In some instances, nearly all of the translational energy is transferred to vibrational-rotational energy. On the other hand, only a small fraction of vibrational energy is converted to translational energy. The translational energy gained from vibrational energy extend to large energy transfer (up to 3000 cm(-1)) as the collision energy increases to 847 cm(-1). Substantial amounts of large V-->T energy transfer were observed in the forward and backward directions at large collision energies.

Energy transfer of highly vibrationally excited azulene. III. Collisions between azulene and argon.

The energy transfer dynamics between highly vibrationally excited azulene molecules (37 582 cm(-1) internal energy) and Ar atoms in a series of collision energies (200, 492, 747, and 983 cm(-1)) was studied using a crossed-beam apparatus along with time-sliced velocity map ion imaging techniques. The angular resolved collisional energy-transfer probability distribution functions were measured directly from the scattering results of highly vibrationally excited azulene. Direct T-VR energy transfer was found to be quite efficient. In some instances, nearly all of the translational energy is transferred to vibrational/rotational energy. On the other hand, only a small fraction of vibrational energy is converted to translational energy (V-T). Significant amount of energy transfer from vibration to translation was observed at large collision energies in backward and sideway directions. The ratios of total cross sections between T-VR and V-T increases as collision energy increases. Formation of azulene-argon complexes during the collision was observed at low enough collision energies. The complexes make only minor contributions to the measured translational to vibrational/rotational (T-VR) energy transfer.