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1.
J Proteome Res ; 23(3): 939-955, 2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-38364797

RESUMO

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.


Assuntos
Mama , Manose , Humanos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Bases de Dados Factuais , Polissacarídeos
2.
J Phys Chem A ; 128(19): 3812-3820, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38690855

RESUMO

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.

3.
Anal Chem ; 95(23): 8789-8797, 2023 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-37235553

RESUMO

N-linked glycosylation is one of the most important post-translational modifications of proteins. Current knowledge of multicellular eukaryote N-glycan biosynthesis suggests high mannose N-glycans are generated in the endoplasmic reticulum and Golgi apparatus through conserved biosynthetic pathways. According to conventional biosynthetic pathways, four Man7GlcNAc2 isomers, three Man6GlcNAc2 isomers, and one Man5GlcNAc2 isomer are generated during this process. In this study, we applied our latest mass spectrometry method, logically derived sequence tandem mass spectrometry (LODES/MSn), to re-examine high mannose N-glycans extracted from various multicellular eukaryotes which are not glycosylation mutants. LODES/MSn identified many high mannose N-glycan isomers previously unreported in plantae, animalia, cancer cells, and fungi. A database consisting of retention time and CID MSn mass spectra was constructed for all possible MannGlcNAc2 (n = 5, 6, 7) isomers that include the isomers by removing arbitrary numbers and positions of mannose from canonical N-glycan, Man9GlcNAc2. Many N-glycans in this database are not found in current N-glycan mass spectrum libraries. The database is useful for rapid high mannose N-glycan isomeric identification.


Assuntos
Eucariotos , Manose , Humanos , Manose/química , Eucariotos/metabolismo , Vias Biossintéticas , Polissacarídeos/química , Espectrometria de Massas em Tandem/métodos
4.
Analyst ; 148(8): 1712-1731, 2023 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-36929945

RESUMO

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.


Assuntos
Pentoses , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Arabinose , Xilose , Oligossacarídeos/análise , Polissacarídeos/química
5.
Phys Chem Chem Phys ; 25(33): 22179-22194, 2023 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-37565323

RESUMO

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.


Assuntos
Dissacarídeos , Espectrometria de Massas em Tandem , Dissacarídeos/química , Oligossacarídeos/química , Carboidratos , Glucose
6.
Rapid Commun Mass Spectrom ; 36(18): e9352, 2022 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-35830282

RESUMO

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. The decay of carbohydrates in ESI has not been investigated yet, and it may have interference in carbohydrate structural determination. METHODS: Commercial apparatus, including a high-performance liquid chromatography (HPLC), an ESI source, and a linear ion trap mass spectrometer, were used to investigate the fragmentation of several N-glycans during the ESI process. RESULTS: About 0.2%-3% of neutral N-glycans and more than 50% of N-glycans consisting of a sialic acid are dissociated into small N-glycans by ESI in-source decay in typical ESI operating conditions. The efficiencies of most dissociation channels increase as the temperature of ion transfer capillary increases, indicating that part of the energy deposited into the precursor ions for cracking is from the heated capillary. The cracking patterns of ESI in-source decay are slightly different from those of gaseous phase collision-induced dissociation. CONCLUSIONS: Large N-glycans are dissociated into small N-glycans in ESI in-source decay that may result in the interference of the structural identification of small N-glycans. Separation of large N-glycans from small N-glycans, for example, using HPLC, prior to ESI ionization is necessary to eliminate the interference. This is particularly important when N-glycans consist of sialic acid or large N-glycans have much higher concentration than that of small N-glycans in ESI solution.


Assuntos
Ácido N-Acetilneuramínico , Espectrometria de Massas por Ionização por Electrospray , Cromatografia Líquida de Alta Pressão , Polissacarídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
7.
Phys Chem Chem Phys ; 24(35): 20856-20866, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36043336

RESUMO

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.


Assuntos
Frutose , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão/métodos , Íons/química , Isomerismo , Sódio , Espectrometria de Massas em Tandem/métodos
8.
J Phys Chem A ; 126(47): 8799-8808, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36394324

RESUMO

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.


Assuntos
Isomaltose , Maltose , Humanos , Desidratação , Espectrometria de Massas em Tandem , Dissacarídeos , Glicosídeos , Glucose
9.
J Phys Chem A ; 126(9): 1486-1495, 2022 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-35212541

RESUMO

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.


Assuntos
Maltose , Espectrometria de Massas em Tandem , Celobiose , Glucose , Íons/química , Espectrometria de Massas em Tandem/métodos
10.
Glycoconj J ; 38(2): 177-189, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32062823

RESUMO

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.


Assuntos
Galactose/química , Oligossacarídeos/química , Espectrometria de Massas em Tandem/métodos , Configuração de Carboidratos , Oligossacarídeos/análise , Espectrometria de Massas por Ionização por Electrospray
11.
Rapid Commun Mass Spectrom ; 35 Suppl 1: e8382, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30623523

RESUMO

RATIONALE: Matrix-assisted ionization (MAI) mass spectrometry does not require voltages, a laser beam, or added heat to initiate ionization, but it is strongly dependent on the choice of matrix and the vacuum conditions. High charge state distributions of nonvolatile analyte ions produced by MAI suggest that the ionization mechanism may be similar to that of electrospray ionization (ESI), but different from matrix-assisted laser desorption/ionization (MALDI). While significant information is available for MAI using mass spectrometers operating at atmospheric and intermediate pressure, little is known about the mechanism at high vacuum. METHODS: Eleven MAI matrices were studied on a high-vacuum time-of-flight (TOF) mass spectrometer using a 266 nm pulsed laser beam under otherwise typical MALDI conditions. Detailed comparisons with the commonly used MALDI matrices and theoretical prediction were made for 3-nitrobenzonitrile (3-NBN), which is the only MAI matrix that works well in high vacuum when irradiated with a laser. RESULTS: Screening of MAI matrices with good absorption at 266 nm but with various degrees of volatility and laser energies suggests that volatility and absorption at the laser wavelength may be necessary, but not sufficient, criteria to explain the formation of multiply charged analyte ions. 3-NBN produces intact, highly charged ions of nonvolatile analytes in high-vacuum TOF with the use of a laser, demonstrating that ESI-like ions can be produced in high vacuum. Theoretical calculations and mass spectra suggest that thermally induced proton transfer, which is the major ionization mechanism in MALDI, is not important with the 3-NBN matrix at 266 nm laser wavelength. 3-NBN:analyte crystal morphology is, however, important in ion generation in high vacuum. CONCLUSIONS: The 3-NBN MAI matrix produces intact, highly charged ions of nonvolatile compounds in high-vacuum TOF mass spectrometers with the aid of ablation and/or heating by laser irradiation, and shows a different ionization mechanism from that of typical MALDI matrices.

12.
Analyst ; 146(23): 7345-7357, 2021 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-34766961

RESUMO

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.


Assuntos
Glicoesfingolipídeos , Espectrometria de Massas em Tandem , Isomerismo , Oligossacarídeos , Polissacarídeos
13.
Phys Chem Chem Phys ; 23(5): 3485-3495, 2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33511385

RESUMO

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.

14.
J Phys Chem A ; 125(28): 6109-6121, 2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34256570

RESUMO

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.

15.
Rapid Commun Mass Spectrom ; 34(18): e8846, 2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-32469439

RESUMO

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. One such parameter is the fluorescence quantum yield. However, the fluorescence quantum yield reported in previous studies remains controversial. METHODS: In this study, we used a commercial and a homemade integrating sphere to measure the absorption and fluorescence quantum yields of several commonly used matrices, including 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid (2,4-DHB), 2,5-dihydroxybenzoic acid (2,5-DHB), 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, 2,4,6-trihydroxyacetophenone, and ferulic acid. RESULTS: The fluorescence quantum yields of these matrices were determined to be low (<0.08) at low laser fluences and decreased as the laser fluence increased. The fluorescence quantum yields at the typical laser fluence for MALDI are below 0.04 (2,4-DHB and 2,5-DHB) and 0.01 (the other matrices). Shot-to-shot fluctuations of fluorescence intensity and absorption are not directly related to the fluctuation of ions. Possible mechanisms for the decrease in the fluorescence quantum yield as the laser fluence increased were discussed. CONCLUSIONS: The fluorescence quantum yields of these commonly used matrices are much lower than those reported in previous studies. Although fluorescence quantum yield is an important parameter and it is crucial to obtain an accurate value for theoretical models in simulations, the use of fluorescence quantum yield alone is not a sufficient parameter to justify these models.

16.
Chembiochem ; 20(18): 2351-2359, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31016827

RESUMO

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.


Assuntos
Polissacarídeos/análise , Polissacarídeos/química , Animais , Sequência de Carboidratos , Leite/química , Oligossacarídeos/análise , Oligossacarídeos/química , Espectrometria de Massas em Tandem/métodos
17.
Anal Bioanal Chem ; 411(15): 3241-3255, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31020368

RESUMO

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.


Assuntos
Manose/química , Oligossacarídeos/química , Espectrometria de Massas em Tandem/métodos , Configuração de Carboidratos , Sequência de Carboidratos , Isomerismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/economia
18.
J Phys Chem A ; 123(16): 3441-3453, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30945547

RESUMO

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.

19.
J Phys Chem A ; 123(31): 6683-6700, 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31294558

RESUMO

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.

20.
J Chem Phys ; 151(14): 141101, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31615217

RESUMO

UV-excited aromatic molecules with N-H/O-H moieties often possess an important nonradiative relaxation pathway, from an optically bright ππ* state to a dark dissociative πσ* state. We apply a new time-selected photofragment translational spectroscopy method to disclose a previously unknown triplet-mediated N-H dissociation of aniline prevented by the multiphoton dissociative ionization in conventional methods. We further determined the branching fractions of aniline dissociated in the πσ*, triplet, and ground states at 248 nm. Additionally, we selectively captured the population changes in the singlet and triplet states with ionization from different laser wavelengths, 355 or 266 nm, in time-resolved photoion yields. The combination of experimental data enables us to uniquely determine the relative ionization cross sections of the singlet and triplet states at an ionization laser wavelength of 266 nm and allows us to extensively measure the rate constants of intersystem crossing and the branching fractions at various excitation wavelengths.

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