MALDI linear-field reflectron TOF post-source decay analysis of underivatized oligosaccharides: Determination of glycosidic linkages and anomeric configurations using anion attachment

Six different anionic species (fluoride, chloride, bromide, iodide, nitrate, and acetate) are tested for their abilities to form anionic adducts with neutral oligosaccharides that are detectable by MALDI-TOF mass spectrometry. Fluoride and acetate cannot form anionic adducts with the oligosaccharides in significant yields. However, bromide, iodide, and nitrate anionic adducts consistently appear in higher abundances relative to [M - H](-), just like the highly stable chloride adducts. Post-source decay (PSD) decompositions of Br(-), I(-), and NO(3)(-) adducts of oligosaccharides provide no structural information, i.e., they yield the respective anions as the main product ions. However, determination of linkage types is achieved by analysis of structurally-informative diagnostic peaks offered by negative ion PSD spectra of chloride adducts of oligosaccharides, whereas the relative peak intensities of pairs of diagnostic fragment ions allow differentiation of anomeric configurations of glycosidic bonds. Thus, simultaneous identification of the linkage types and anomeric configurations of glycosidic bonds is achieved. Our data indicate that negative ion PSD fragmentation patterns of chloride adducts of oligosaccharides are mainly determined by the linkage types. Correlation may exist between the linkage positions and fragmentation mechanisms and/or steric requirements for both cross-ring and glycosidic bond fragmentations. PSD of the chloride adducts of saccharides containing a terminal Glcalpha1-2Fru linkage also yields chlorine-containing fragment ions which appear to be specifically diagnostic for a fructose linked at the 2-position on the reducing end. This also allows differentiation from saccharides with a 1-1 linked pyranose on the same position.     

A comparative study of the fragmentation of neutral lactooligosaccharides in negative-ion mode by UV-MALDI-TOF and UV-MALDI ion-trap/TOF mass spectrometry

Structure analyses of underivatized neutral lacto oligosaccharides are systematically performed by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI TOF MS) and UV-MALDI ion-trap time-of-flight mass spectrometry (ion-trap/TOF MS) acquired in negative-ion mode. Interestingly, their fragmentation significantly differ each other. In postsource decay (PSD) in UV-MALDI TOF MS, cross-ring cleavage at the reducing terminal predominates. On the other hand, glycosyl bond cleavage (C-type fragmentation) takes place preferentially in collision induced dissociation (CID) in UV-MALDI ion-trap/TOF MS. The cross-ring cleavage in PSD similar to that in in-source decay occurs via a prompt reaction path characteristic of the UV-MALDI process itself. The product ion spectra of UV-MALDI ion-trap/TOF MS are similar to the electrospray ionization (ESI) ion-trap or quadrupole/TOF CID product ion spectra. During ion-trap/TOF MS experiments, the deprotonated molecular ions survive for several tens of milliseconds after CID event because the high internal energy chlorinated precursor ions are cooled by collisional cooling in the ion trap. The results obtained suggest that the PSD from the chlorinated precursor ion in UV-MALDI TOF MS might proceed as a two-step reaction; in the first, a high internal energy deprotonated molecular ion is generated as a reaction intermediate during the flight in the drift tube, and in the second, the rapid decomposition from the deprotonated molecular ion takes place.     

Electron detachment dissociation of dermatan sulfate oligosaccharides

The structural characterization of glycosaminoglycans (GAG) oligosaccharides has been a long-standing challenge in the field of mass spectrometry. In this work, we present the application of electron detachment dissociation (EDD) Fourier transform mass spectrometry to the analysis of dermatan sulfate (DS) oligosaccharides up to 10 residues long. The EDD mass spectra of DS oligosaccharides were compared with their infrared multiphoton dissociation (IRMPD) mass spectra. EDD produces more abundant fragmentation than IRMPD with far less loss of SO3 from labile sulfate modifications. EDD cleaves all glycosidic bonds, yielding both conventional glycosidic bond fragmentation as well as satellite peaks resulting from the additional loss of 1 or 2 hydrogen atoms. EDD also yields more cross-ring fragmentation than IRMPD. For EDD, abundant cross-ring fragmentation in the form of A- and X-ions is observed, with 1,5Xn cleavages occurring for all IdoA residues and many of the GalNAc4S residues, except at the reducing and nonreducing ends. In contrast, IRMPD produces only A-type cross-ring fragmentation for long oligosaccharides (dp6-dp10). As all the structurally informative fragment ions observed by IRMPD appear as a subset of the peaks found in the EDD mass spectrum, EDD shows great potential for the characterization of GAG oligosaccharides using a single tandem mass spectrometry experiment.     

Matrix-assisted laser desorption/ionization tandem mass spectrometry and post-source decay fragmentation study of phenylhydrazones of <Emphasis Type="Italic">N</Emphasis>-linked oligosaccharides from ovalbumin

N-linked oligosaccharides were released from hen ovalbumin by PNGase F and derivatized with phenylhydrazine. They were then examined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Phenylhydrazones of N-glycans under MALDI-tandem mass spectrometry (MS/MS) and post-source decay (PSD) conditions produced relatively similar fragmentation patterns; however, more cross-ring cleavages and fragment ions corresponding to low abundance isomeric structures were detected by MS/MS and not in PSD. Most fragment ions corresponded to glycosidic cleavages with preferential loss of residues from the chitobiose core and the 3-antenna. Sialylated phenylhydrazone-N-glycans, characterized here for the first time in ovalbumin by tandem mass spectrometry, underwent losses of sialic acid residues followed the same fragmentation pathways observed with neutral derivatized glycans. The relative abundances of some fragment ions indicated the linkage position of sialic acid and provided information on the number of residues attached to the 6-antenna. Also, new structures of ovalbumin glycans were observed as part of this study and are reported here.     

Linkage and Branch Analysis of High-Mannose Oligosaccharides Using Closed-Ring Labeling of 8-Aminopyrene-1,3,6-Trisulfonate and P-Aminobenzoic Ethyl Ester and Negative Ion Trap Mass Spectrometry

A strategy based on negative ion electrospray ionization tandem mass spectrometry and closed-ring labeling with both 8-aminopyrene-1,3,6-trisulfonate (APTS) and p-aminobenzoic acid ethyl ester (ABEE) was developed for linkage and branch determination of high-mannose oligosaccharides. X-type cross-ring fragment ions obtained from APTS-labeled oligosaccharides by charge remote fragmentation provided information on linkages near the non-reducing terminus. In contrast, A-type cross-ring fragment ions observed from ABEE-labeled oligosaccharides yielded information on linkages near the reducing terminus. This complementary information provided by APTS- and ABEE-labeled oligosaccharides was utilized to delineate the structures of the high-mannose oligosaccharides. As a demonstration of this approach, the linkages and branches of high-mannose oligosaccharides Man(5)GlcNAc(2), Man(6)GlcNAc(2), Man(8)GlcNAc(2), and Man(9)GlcNAc(2) cleaved from the ribonuclease B were assigned from MS(2) spectra of ABEE- and APTS-labeled derivatives.     

Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides

We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared with infrared multiphoton dissociation. Neutral losses and satellite ions such as C-2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA-labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared with 2-AA-labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag.     

Ultraviolet photodissociation of chromophore-labeled oligosaccharides via reductive amination and hydrazide conjugation

The fragmentation patterns of hydrazide-conjugated and reductively aminated oligosaccharides, including lacto-N-fucopentaoses and lacto-N-difucohexaoses, produced on collisionally induced dissociation (CID) and ultraviolet photodissociation (UVPD) in a quadrupole ion trap are presented. The two derivatization methods generate different cross-ring cleavages on UVPD and CID. UVPD of hydrazide-conjugated oligosaccharides yield predominant (2, 4)A-type cross-ring cleavage ions. In contrast, UVPD of aminated oligosaccharides results mainly in (0, 1)A-type ions. Moreover, more extensive dual-cleavage pathways (i.e. internal fragment ions) were observed on UVPD.     

Electron detachment dissociation of neutral and sialylated oligosaccharides

Electron detachment dissociation (EDD) has recently been shown by Amster and coworkers to constitute a valuable analytical approach for structural characterization of glycosaminoglycans. Here, we extend the application of EDD to neutral and sialylated oligosaccharides. Both branched and linear structures are examined, to determine whether branching has an effect on EDD fragmentation behavior. EDD spectra are compared to collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) spectra of the doubly and singly deprotonated species. Our results demonstrate that EDD of both neutral and sialylated oligosaccharides provides structural information that is complementary to that obtained from both CAD and IRMPD. In all cases, EDD resulted in additional cross-ring cleavages. In most cases, cross-ring fragmentation obtained by EDD is more extensive than that obtained from IRMPD or CAD. Our results also indicate that branching does not affect EDD fragmentation, contrary to what has been observed for electron capture dissociation (ECD).     

Fragmentation of oligosaccharide ions with 157 nm vacuum ultraviolet light

The 157 nm photofragmentation of native and derivatized oligosaccharides was studied in a linear ion trap and in a home-built matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometer, and the results were compared with collision-induced dissociation (CID) experiments. Photodissociation produces product ions corresponding to high-energy fragmentation pathways; for cation-derivatized oligosaccharides, it yields strong cross-ring fragment ions and provides better sequence coverage than low- and high-energy CID experiments. On the other hand, for native oligosaccharides, CID yielded somewhat better sequence coverage than photodissociation. The ion trap enables CID hybrid MS3 experiments on the high-energy fragment ions obtained from photodissociation.     

Systematic identification of N-acetylheparosan oligosaccharides by tandem mass spectrometric fragmentation

Recently, a useful procedure for the preparation of both even- and odd-numbered series of N-acetylheparosan (NAH) oligosaccharides was established. The present report describes findings when these NAH oligosaccharides were subjected to comparative mass spectrometry (MS)/MS fragmentation analysis by matrix-assisted laser desorption/ionization (MALDI)-LIFT-time-of-flight (TOF)/TOF-MS/MS, and electrospray ionization (ESI) collision-induced dissociation (CID) MS/MS. The resultant fragment ions were systematically assigned to elucidate fragmentation characteristics. In the MALDI-LIFT-MS/MS experiments, all the NAH oligosaccharides underwent unique glycosidic cleavages that included B-Y ion cleavages (nomenclature system of Domon and Costello, Glycoconjugate J. 1988; 5: 397) at the C-1 side, and C-Z ion cleavages at the C-4 side, with respect to glucuronic acid (GlcA). In addition, (0,2)A and/or (0,2)X cross-ring cleavages were observed for relatively small oligosaccharides. The former observation clearly reflects the occurrence of a GlcA-N-acetylglucosamine (GlcNAc) alternating structure of NAH, while the latter feature implies the occurrence of the -beta-1-4-glucuronide linkage. Extensive glycosidic cleavages were also observed in the ESI-CID-MS/MS fragmentation, though cleavage specificity was less evident than in the case of MALDI-LIFT-TOF/TOF-MS/MS. The information obtained in this study should be valuable for understanding both biosynthetic and degradation processes of NAH and its derivatives including heparin and heparan sulfate, as well as artificially modified NAH oligosaccharides.     

On-target derivatization of keratan sulfate oligosaccharides with pyrenebutyric acid hydrazide for MALDI-TOF/TOF-MS

In the present work, a rapid and novel method of on-target plate derivatization of keratan sulfate (KS) oligosaccharides for subsequent analysis by matrix-assisted laser desorption and ionization (MALDI) mass spectrometry is described. MALDI-(time-of-flight)-TOF spectra of labeled KS oligosaccharides revealed that significantly improved ionization can be accomplished through derivatization with pyrenebutyric acid hydrazide (PBH), and the most abundant peak in each spectrum corresponds to the singly charged molecular ion [M - H]- or [M + (n - 1)Na - nH]-, where n = the number of sulfates (n = 1, 2, 3...). The high-energy collision-induced dissociation (heCID) spectra of labeled KS oligosaccharides displayed fragments of compounds similar to those observed with laser-induced dissociation (LID) analysis, suggesting that both heCID and LID fragmentations can be used to analyze KS oligosaccharides. Moreover, fragmentation analysis of all labeled KS oligosaccharides was performed by MALDI-TOF/TOF-MS. With LID mode, sodium adducts showed fragmentation of glycosidic linkages with mainly Y/B/C ions, as well as various cross-ring cleavages providing exact information for the positions of sulfate groups along the KS oligosaccharide chains. This one-step on-target derivatization method makes MALDI-TOF/TOF-MS identification of KS fast, simple and highly throughput for trace amounts of biological samples.     

Electron Transfer Dissociation of Milk Oligosaccharides

For structural identification of glycans, the classic collision-induced dissociation (CID) spectra are dominated by product ions that derived from glycosidic cleavages, which provide only sequence information. The peaks from cross-ring fragmentation are often absent or have very low abundances in such spectra. Electron transfer dissociation (ETD) is being applied to structural identification of carbohydrates for the first time, and results in some new and detailed information for glycan structural studies. A series of linear milk sugars was analyzed by a variety of fragmentation techniques such as MS/MS by CID and ETD, and MS(3) by sequential CID/CID, CID/ETD, and ETD/CID. In CID spectra, the detected peaks were mainly generated via glycosidic cleavages. By comparison, ETD generated various types of abundant cross-ring cleavage ions. These complementary cross-ring cleavages clarified the different linkage types and branching patterns of the representative milk sugar samples. The utilization of different MS(3) techniques made it possible to verify initial assignments and to detect the presence of multiple components in isobaric peaks. Fragment ion structures and pathways could be proposed to facilitate the interpretation of carbohydrate ETD spectra, and the main mechanisms were investigated. ETD should contribute substantially to confident structural analysis of a wide variety of oligosaccharides.     

Structural characterization of 2-aminobenzamide-derivatized oligosaccharides using a matrix-assisted laser desorption/ionization two-stage time-of-flight tandem mass spectrometer

Oligosaccharides were derivatized by reductive amination using 2-aminobenzamide (2-AB) and analyzed by matrix-assisted laser desorption/ionization two-stage time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS) in the positive ion mode. The major signals were obtained under these conditions from the [M+Na]+ ions for all 2-AB-derivatized oligosaccharides. A systematic study was conducted on a series of 2-AB-derivatized oligosaccharides to allow rationalization of the fragmentation processes. The MALDI-TOF/TOF-MS/MS spectra of the [M+Na]+ ions of 2-AB-derivatized oligosaccharides were dominated by glycosidic cleavages. These fragments originating both from the reducing and the non-reducing ends of the oligosaccharide yield information on sequence and branching. Moreover, the MALDI-TOF/TOF-MS/MS spectra were also characterized by abundant cross-ring fragments which are very informative on the linkages of the monosaccharide residues constituting these oligosaccharides. MALDI-TOF/TOF-MS/MS analysis of 2-AB-derivatized oligosaccharides, by providing structural information at the low-picomole level, appears to be a powerful tool for carbohydrate structural analysis.     

Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MS(n) (part 1: methodology)

Underivatized neutral oligosaccharides from human milk were analyzed by nano-electrospray ionization (ESI) using a quadrupole ion trap mass spectrometer (QIT-MS) in the negative-ion mode. Under these conditions neutral oligosaccharides are observed as deprotonated molecules [M-H]- with high intensity. CID-experiments of these species with the charge localized at the reducing end lead to C-type fragment ions forming a "new" reducing end. Fragmentations are accompanied by cross-ring cleavages that yield information about linkages of internal monosaccharides. Several isomeric compounds with distinct structural features, such as different glycosidic linkages, fucosylation and branching sites were investigated. The rules governing the fragmentation behavior of this class of oligosaccharides were elucidated and tested for a representative number of certain isomeric glycoforms using the MS/MS and MS(n) capabilities of the QIT. On the basis of the specific fragmentation behavior of deprotonated molecules, the position of fucoses and the linkage type (Gal beta-->3 GlcNAc or Gal beta1-->4 GlcNAc) could be determined and linear and branched could be differentiated. Rules could be established which can be applied in further investigations of these types of oligosaccharides even from heterogenous mixtures.     

Structural Characterization of Neutral Oligosaccharides by Laser-Enhanced In-Source Decay of MALDI-FTICR MS

MALDI in-source decay (ISD) technique described to date has proven to be a convenient and rapid method for sequencing purified peptides and proteins. However, the general ISD still can not produce adequate fragments for the detailed structural elucidation of oligosaccharides. In this study, an efficient and practical method termed the laser-enhanced ISD (LEISD) technique of MALDI-FTICR MS allows highly reliable and abundant fragmentation of the neutral oligosaccharides, which was attributed to the ultrahigh irradiation laser of mJ level. The yield of ISD fragmentation was evaluated under different laser powers for 7 neutral oligosaccharides using DHB as matrix. Better quality ISD spectra including fragment ions in low-mass region were obtained at higher laser power. Results from the LEISD of oligosaccharides demonstrated that a significantly better signal-to-noise ratio (S/N) and more structural information could be obtained in comparison to the conventional CID. It was also suggested that the valuable A ions derived from cross-ring cleavage of the linear oligosaccharides allowed the distinction among α(1 → 4)-, α(1 → 6)-, β(1 → 4)-, and β(1 → 3)-linked isobaric structures according to fragment types and intensities. In addition, ideal fragmentation ions observed by LEISD method facilitated the determination of the sequences and branched points of complex oligosaccharides from human milk.     

Matrix-assisted laser desorption/ionization in-source decay combined with tandem time-of-flight mass spectrometry of permethylated oligosaccharides: targeted characterization of specific parts of the glycan structure

Matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) has been introduced in recent years as a valuable tool for the structural characterization of permethylated oligosaccharides. In this report, we describe the combination of MALDI in-source decay (ISD) with the subsequent TOF/TOF-MS analyses of specific fragments, allowing the detailed characterization of the selected part of the oligosaccharide molecule. Part of the second-generation fragment ions were different from those observed in conventional MALDI-TOF/TOF-MS experiments. Other fragments, which had already been observed in conventional MALDI-TOF/TOF-MS and again showed up in second-generation fragment analysis, could be assigned to specific parts of the molecule. Our approach disclosed different structural features of the oligosaccharides: due to permethylation, the glycosidic linkage fragments allowed the distinction between terminal, monosubstituted and disubstituted monosaccharides and indicated the oligosaccharide sequence. Moreover, substitution positions were deduced based on characteristic cross-ring fragmentation by high-energy collision-induced fragmentation. In conclusion, combination of MALDI-ISD with TOF/TOF-MS allows the detailed characterization of specific moieties of permethylated oligosaccharides and is, therefore, a powerful technique for structural glycomics.     

Matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometric study of 2,3-dihydro-1,4-benzoxazepines

Fragmentations of the protonated adduct ions [M+H](+) of seven 1,4-benzoxazepine derivatives were studied using 'post-source decay' matrix-assisted laser desorption/ionization (PSD MALDI) and electrospray nozzle-skimmer collisionally induced dissociation (ESI-CID) mass spectrometric methods. It was found that both methods generated mainly product ions arising from the cross-ring cleavages of the benzoxazepine ring. Similar product ions were generated under MALDI and ESI conditions; however, it was observed that the loss of the alkylene unit from the N-substituted benzoxazepine, and the loss of a H(2)X molecule (where X = O or S), are more preferred under ESI conditions. Based on the experimental results a mechanism is also proposed for the fragmentation of the oxazepines studied.     

电喷雾质谱在寡糖序列分析中的应用

选取具有不同结构特征的N-糖链、硫酸软骨素寡糖、人乳寡糖以及海洋来源的壳寡糖、褐藻胶寡糖、卡拉胶寡糖和硫酸岩藻寡糖等,对电喷雾质谱在寡糖的主链序列、分支位点、硫酸基取代位置确定、单糖组成和聚合度分析等方面的应用技术及碎片离子的断裂规律进行了总结.根据相邻同类碎片离子之间的质荷比差值可初步判断寡糖的单糖组成类型;通过与色谱分离技术联用或衍生化方法可提高寡糖的分辨率和离子化效率,并测得寡糖的分子量及聚合度;借助串联质谱及对寡糖还原端的特异性标记,可获得寡糖的还原端残基和部分序列信息;根据寡糖产生的特征碎片离子及其丰度大小可判断残基的特定位置和类型.另外,寡糖的分支通常作为一个整体发生糖苷键断裂或产生D离子,据此可判断分支点的位置;根据硫酸寡糖产生的特异性跨环断裂碎片,可以确定硫酸基的连接位置.这些规律和方法的总结为未知寡糖的结构和序列的分析提供了启发和指导.     

Structural analysis of oligosaccharides by atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap mass spectrometry.

An ion source incorporating a fibre optic interface has been constructed for atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap mass spectrometry. The configuration has been applied to the study of linear and complex oligosaccharides. Multi-stage tandem mass spectrometry (MSn, n = 2-4) experiments carried out in the ion trap enable extended fragmentation pathways to be investigated that yield structural information. Collisional activation of sodiated oligosaccharides, as demonstrated on the model compound maltoheptaose, produces primarily B and Y fragments resulting from cleavage of glycosidic bonds; fragments from cross-ring cleavages are also observed following further stages of tandem mass spectrometry, providing additional linkage information. The analyses of mixtures of complex oligosaccharides are demonstrated for N-linked glycans from chicken egg glycoproteins and a ribonuclease glycan mixture. Mass spectrometric and tandem mass spectrometric data for sugars with molecular weights up to 4000 Da is shown for mixtures of linear dextrans and N-linked glycans. The use of MSn (n = 3, 4) on these complex molecules enabled structural information to be elucidated that confirms data observed in the MS/MS spectra.     

Structural characterization by both positive and negative electrospray ion trap mass spectrometry of oligogalacturonates purified by high-performance anion-exchange chromatography

The off-line coupling of high-performance anion-exchange chromatography to electrospray ion trap mass spectrometry (ESI-IT-MS) is described. Two sets of isocratic conditions were optimised for the semi-preparative purification of oligogalacturonates of degree of polymerisation from 4 to 6 by monitoring eluates with either pulsed amperometric detection or evaporative light scattering detection in the presence of an online Dionex Carbohydrate Membrane Desalter (CMD). In these conditions, purified oligogalacturonate solutions were suitable, without further desalting steps, for infusion ESI-IT-MS experiments. This paper provides some interesting features of positive and negative ESI-IT-multiple MS (MSn) of these acidic oligosaccharides. The spectra acquired in both ion modes show characteristic fragments resulting from glycosidic bond and cross-ring cleavages. Under negative ionization conditions, the fragmentation of the singly-charged [M-H]- ions, as well as the Ci-, and Zi-, fragment ions through sequential MSn experiments, was always dominated by product ions from C- and Z-type glycosidic cleavages. All spectra also displayed 0.2 A-type cross-ring cleavage ions which carry linkage information. Collision-induced dissociation (CID) spectra of sodium-cationized species obtained under positive ionization conditions were more complex. Successive MSn experiments also led to the 0.2 A-type cross-ring cleavage ions observed together with B- and Y-type ions. The presence of the 0.2 A ion series was related to Mr 60 (C2H4O2) losses. Combined with the absence of the Mr 30 (CH2O) and the Mr 90 (C3H6O3) ions, these ions were indicative of 1-4 type glycosidic linkage.