Assignment of the Stereochemistry and Anomeric Configuration of Sugars within Oligosaccharides Via Overlapping Disaccharide Ladders Using MSn

A systematic approach is described that can pinpoint the stereo-structures (sugar identity, anomeric configuration, and location) of individual sugar units within linear oligosaccharides. Using a highly modified mass spectrometer, dissociation of linear oligosaccharides in the gas phase was optimized along multiple-stage tandem dissociation pathways (MSⁿ, n = 4 or 5). The instrument was a hybrid triple quadrupole/linear ion trap mass spectrometer capable of high-efficiency bidirectional ion transfer between quadrupole arrays. Different types of collision-induced dissociation (CID), either on-resonance ion trap or beam-type CID could be utilized at any given stage of dissociation, enabling either glycosidic bond cleavages or cross-ring cleavages to be maximized when wanted. The approach first involves optimizing the isolation of disaccharide units as an ordered set of overlapping substructures via glycosidic bond cleavages during early stages of MSⁿ, with explicit intent to minimize cross-ring cleavages. Subsequently, cross-ring cleavages were optimized for individual disaccharides to yield key diagnostic product ions (m/z 221). Finally, fingerprint patterns that establish stereochemistry and anomeric configuration were obtained from the diagnostic ions via CID. Model linear oligosaccharides were derivatized at the reducing end, allowing overlapping ladders of disaccharides to be isolated from MSⁿ. High confidence stereo-structural determination was achieved by matching MSⁿ CID of the diagnostic ions to synthetic standards via a spectral matching algorithm. Using this MSⁿ (n = 4 or 5) approach, the stereo-structures, anomeric configurations, and locations of three individual sugar units within two pentasaccharides were successfully determined.

Collision-induced dissociation of alkali metal cationized and permethylated oligosaccharides: Influence of the collision energy and of the collision gas for the assignment of linkage position

Tandem mass spectrometry has been used to study the collision-induced decomposition of [M+Na]⁺ ions of permethylated oligosaccharides. It is shown that many linkage positions in one compound may be determined by the presence or absence, in a single spectrum, of specific fragment ions that arise from the cleavage of two ring bonds and that the yield of such ions depends strongly on the collision energy and nature of the collision gas. In contrast to the behavior of monolithiated native oligosaccharides, the collision-induced decomposition of the sodiated and permethylated oligosaccharide samples at low energy leads to preferential cleavage of glycosidic linkages. At high collision energies, the fragment ions formed by cleavage of more than one bond are greatly enhanced, especially when helium is replaced by argon as the collision gas. Furthermore, argon is the more efficient collision gas in inducing fragmentation of the precursor ions. As an example of the application of this method, the discrimination between the 1 → 3 and 1 → 6-linked mannose residues in the common core of N-glycans is described.     

Tandem mass spectrometry of alkali cationized polysaccharides in a quadrupole ion trap

Quadrupole ion trap mass spectrometry is used to study the linkage type dependent dissociation pathways of alkali-cationized disaccharides, mostly of the type glucosyl(1 → X)glucose (X = 1, 2, 3, 4, or 6). The reaction mechanisms of a set of disaccharides containing all possible α anomeric linkage types and some β anomers are probed with tandem mass spectrometry, MS ⁿ , and double resonance experiments. Tandem mass spectrometry experiments on an ¹⁸O-labeled disaccharide show that the dissociation paths for Li and Na cationized species are the same. Experiments on three trisaccharides (isomaltotriose, maltotriose, and panose), a tetrasaccharide (isomaltotetraose), and a pentasaccharide (maltopentaose) show that tandem mass spectrometry provides all available linkage information and MS ⁿ can provide selected linkage information. The mode of alkali binding is examined via semiempirical calculations and by measuring alkali-carbohydrate relative cation affinities.     

Sulfate migration in oligosaccharides induced by negative ion mode ion trap collision‐induced dissociation

Migration of sulfate groups between hydroxyl groups was identified after collision‐induced dissociation (CID) of sulfated oligosaccharides in an ion trap mass spectrometer in negative ion mode. Analysis of various sulfated oligosaccharides showed that this was a common phenomenon and was particularly prominent in sulfated oligosaccharides also containing sialic acid. It was also shown that the level of migration was increased when the sulfate was positioned on the flexible areas of the oligosaccharides not involved in the pyranose ring, such as the extra‐cyclic C‐6 carbon of hexoses or N‐acetylhexosamines, or on reduced oligosaccharide. This suggested that migration is dependent on the spatial availability of the sulfate in the ion trap during collision. It is proposed that the migration is initiated when the negatively charged ‐SO₃^– residue attached to the oligosaccharide precursor becomes protonated by a CID‐induced proton transfer. This is supported by the CID fragmentation of precursor ions depleted of acidic protons such as doubly charged [M – 2H]^2– ions or the sodiated [M + Na – 2H]^– ions of oligosaccharides containing one sulfate and one sialic acid in the same molecule. Compared to the CID fragmentation of their monocharged [M – H]^– ions, no migration was observed in CID of proton depleted precursors. Alternative fragmentation parameters to suppress migration of sulfated oligosaccharides also showed that it was not present when sulfated oligosaccharides were fragmented by HCD (High‐Energy C‐trap Dissociation) in an Orbitrap mass spectrometer. Copyright © 2011 John Wiley & Sons, Ltd.     

Differentiation of the Stereochemistry and Anomeric Configuration for 1-3 Linked Disaccharides Via Tandem Mass Spectrometry and <Superscript>18</Superscript>O-labeling

Collision-induced dissociation (CID) of deprotonated hexose-containing disaccharides (m/z 341) with 1–2, 1–4, and 1–6 linkages yields product ions at m/z 221, which have been identified as glycosyl-glycolaldehyde anions. From disaccharides with these linkages, CID of m/z 221 ions produces distinct fragmentation patterns that enable the stereochemistries and anomeric configurations of the non-reducing sugar units to be determined. However, only trace quantities of m/z 221 ions can be generated for 1–3 linkages in Paul or linear ion traps, preventing further CID analysis. Here we demonstrate that high intensities of m/z 221 ions can be built up in the linear ion trap (Q3) from beam-type CID of a series of 1–3 linked disaccharides conducted on a triple quadrupole/linear ion trap mass spectrometer. ¹⁸O-labeling at the carbonyl position of the reducing sugar allowed mass-discrimination of the “sidedness” of dissociation events to either side of the glycosidic linkage. Under relatively low energy beam-type CID and ion trap CID, an m/z 223 product ion containing ¹⁸O predominated. It was a structural isomer that fragmented quite differently than the glycosyl-glycolaldehydes and did not provide structural information about the non-reducing sugar. Under higher collision energy beam-type CID conditions, the formation of m/z 221 ions, which have the glycosyl-glycolaldehyde structures, were favored. Characteristic fragmentation patterns were observed for each m/z 221 ion from higher energy beam-type CID of 1–3 linked disaccharides and the stereochemistry of the non-reducing sugar, together with the anomeric configuration, were successfully identified both with and without ¹⁸O-labeling of the reducing sugar carbonyl group.     

Analysis of underivatized oligosaccharides by liquid chromatography/electrospray ionization tandem mass spectrometry with post‐column addition of formic acid

Underivatized oligosaccharides were analyzed by electrospray ionization (ESI) using a linear ion trap mass spectrometer in the negative ion mode with post‐column addition of an aqueous solution of formic acid. Under these conditions all oligosaccharides showed the presence of the corresponding formate adduct [M + HCOO]^? with high intensity and easy subsequent low‐energy collision‐induced dissociation (CID) fragmentation using successive MSⁿ experiments. A careful examination of the mass spectra obtained from these MSⁿ experiments pointed out some significant differences useful to identify and quantify the single components in mixtures of coeluted disaccharides. This new sensitive and rapid method was successfully applied to the quantification of oligosaccharides in some juices minimizing sample handling. Copyright © 2009 John Wiley & Sons, Ltd.     

Ion mobility-mass spectrometry analysis of isomeric carbohydrate precursor ions

The rapid separation of isomeric precursor ions of oligosaccharides prior to their analysis by mass spectrometry to the nth power (MS ⁿ ) was demonstrated using an ambient pressure ion mobility spectrometer (IMS) interfaced with a quadrupole ion trap. Separations were not limited to specific types of isomers; representative isomers differing solely in the stereochemistry of sugars, in their anomeric configurations, and in their overall branching patterns and linkage positions could be resolved in the millisecond time frame. Physical separation of precursor ions permitted independent mass spectra of individual oligosaccharide isomers to be acquired to at least MS³, the number of stages of dissociation limited only practically by the abundance of specific product ions. IMS–MS ⁿ analysis was particularly valuable in the evaluation of isomeric oligosaccharides that yielded identical sets of product ions in tandem mass spectrometry experiments, revealing pairs of isomers that would otherwise not be known to be present in a mixture if evaluated solely by MS dissociation methods alone. A practical example of IMS–MSⁿ analysis of a set of isomers included within a single high-performance liquid chromatography fraction of oligosaccharides released from bovine submaxillary mucin is described.     

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.     

Electron Detachment Dissociation of Underivatized Chloride-Adducted Oligosaccharides

Chloride anion attachment has previously been shown to aid determination of saccharide anomeric configuration and generation of linkage information in negative ion post-source decay MALDI tandem mass spectrometry. Here, we employ electron detachment dissociation (EDD) and collision activated dissociation (CAD) for the structural characterization of underivatized oligosaccharides bearing a chloride ion adduct. Both neutral and sialylated oligosaccharides are examined, including maltoheptaose, an asialo biantennary glycan (NA2), disialylacto-N-tetraose (DSLNT), and two LS tetrasaccharides (LSTa and LSTb). Gas-phase chloride-adducted species are generated by negative ion mode electrospray ionization. EDD and CAD spectra of chloride-adducted oligosaccharides are compared to the corresponding spectra for doubly deprotonated species not containing a chloride anion to assess the role of chloride adduction in the stimulation of alternative fragmentation pathways and altered charge locations allowing detection of additional product ions. In all cases, EDD of singly chloridated and singly deprotonated species resulted in an increase in observed cross-ring cleavages, which are essential to providing saccharide linkage information. Glycosidic cleavages also increased in EDD of chloride-adducted oligosaccharides to reveal complementary structural information compared to traditional (non-chloride-assisted) EDD and CAD. Results indicate that chloride adduction is of interest in alternative anion activation methods such as EDD for oligosaccharide structural characterization.     

Selective ion-molecule reactions of lactams with dimethyl ether ions

The ion-molecule reactions of dimethyl ether ions CH₃OCH₃ ⁺ and (CH₃OCH₃)H⁺, and four- to seven-membered ring lactams with methyl substituents in various positions were characterized by using a quadrupole ion trap mass spectrometer and a triple-quadrupole mass spectrometer. In both instruments, the lactams were protonated by dimethyl ether ions and formed various combinations of [M + 13] ⁺, [M + 15] ⁺, and [M + 45] ⁺ adduct ions, as well as unusual [M + 3] ⁺ and [M + 16] ⁺ adduct ions. An additional [M + 47] ⁺ adduct ion was formed in the conventional chemical ionization source of the triple-quadrupole mass spectrometer. The product ions were isolated and collisionally activated in the quadrupole ion trap to understand formation pathways, structures, and characteristic dissociation pathways. Sequential activation experiments were performed to elucidate fragment ion structures and stepwise dissociation sequences. Protonated lactams dissociate by loss of water, ammonia, or methylamine; ammonia and carbon monoxide; and water and ammonia or methylamine. The [M + 16] ⁺ products, which are identified as protonated lactone structures, are only formed by those lactams that do not have an N-methyl substituent. The ion-molecule reactions of dimethyl ether ions with lactams were compared with those of analogous amides and lactones.     

Determination of linkage position and anomeric configuration in Hex-Fuc disaccharides using electrospray ionization tandem mass spectrometry

Fixed-energy sequential tandem mass spectrometry (MS(n)) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of six isomers of Gal-Fucalpha-OBenzyl disaccharides. Under collision-induced dissociation (CID), sodiated molecular species generated in the positive-ion electrospray ionization mode yield simple and predictable mass spectra. Information on interglycosidic linkages and configurations can be deduced from the relative intensities of the selected diagnostic fragments arising from the glycosidic bond cleavages and corroborated by the fragments arising from cross-ring cleavages. As the CID patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by matching the spectra with a library. The rules governing the fragmentation behavior of this class of oligosaccharides were tested for a representative isomeric disaccharide, Glcbeta1,3Fucalpha-OAllyl. The findings establish a basis for using MS(n) with a quadrupole ion trap instrument to elucidate structures of hexose-fucose subunits from more complicated oligosaccharides. Energy-resolved mass spectra were also acquired by CID tandem triple-quadrupole mass spectrometry. The breakdown behavior of the molecular ions revealed patterns which could differentiate stereoisomers of Gal-Fuc disaccharides over a range of collision energy from 20 to 50 eV.     

Analysis of the linkage positions and isomers of monosaccharide units in oligosaccharides by negativ secondary ion mass spectrometry in combination with the stereoselective reaction with substituted boronic acid

The negative secondary ion mass spectrometry, in combination with the stereoselective derivatizations with substituted boronic acid RB(OH)₂, was used in the analysis of fourteen oligosaccharides. The mass spectra of the derivatives provide information on their linkage positions and isomerism of the individual monosacaccharide units. The results indicated that among the derivatives of the oligosaccharides analyzed, those with 1–4 and 1–6 linkages all presented the ion peaks at m/z 287, sometimes one more peak at m/z 449. Furthermore, a relationship was found between the linkage positions and the intensity orders of the derivative ions. Finally, the derivatives of the disaccharides with a galactose presented an intense ion peak at m/z 347, and those of oligosaccharides with 1–6 linkage to a galactose at terminal presented the ion at m/z 317. In the case of oligosaccharides with a fructose residue, characteristic ion of m/z 155 was produced. The conditions of stereoselective derivatizations and mass spectrometry were studied, in order to obtain a better reproducibility of the mass spectra.     

Mass-analysed ion kinetic energy and collisionally induced dissociation mass spectra of clusters of acetylated xylo-oligosaecharides with protonated ammonia and methylannine

Per-O-acetylated methyl glycosides of D -xylan-type di- and trisaccharides were studied by mass-analysed ion kinetic energy (MIKE) and collisionally induced dissociation (CID) mass Spectrometry using protonated ammonia and methylamine, respectively, as reaction gases in chemical ionization (CI). The oligosaccharides form abundant cluster ions, [M + NH₄]⁺ or [M + CH₃NH₃]⁺, and the main fragmentation of these ions in the MIKE and CID spectra is the cleavage of interglycosidic linkages. Thus, CI (NH₃) or CI (CH₃NH₂) spectra in combination with the MIKE or CID spectra allow the molecular masses, the masses of monosaccharide units and the branching point in oligosaccharides to be established. In the case of disaccharides, it is possible to distinguish the (1 → 2) linkage from the other types of linkages.     

Incremented alkyl derivatives enhance collision induced glycosidic bond cleavage in mass spectrometry of disaccharides

Electrospray ionization and collision induced dissociation on a triple quadrupole mass spectrometer were used to determine the effect of spatial crowding of incremented alkyl groups of two anomeric pairs of peralkylated (methyl to pentyl) disaccharides (maltose/cellobiose and isomaltose/gentiobiose). Protonated molecules were generated which underwent extensive fragmentation under low energy conditions. For both the 1 --> 4 and 1 --> 6 alpha and beta isomers, at comparable collision energies the methyl derivative exhibited the least fragmentation followed by ethyl, propyl, butyl, and pentyl. Collision energy is converted to rotational-vibrational modes in competition with bond cleavage, as represented by the slope of product/parent ion (D/P) ratio versus offset energy. Variable rotational freedom at the glycosidic linkage with incremented alkyl groups is hypothesized to be responsible for this effect. Discrimination of anomeric configuration was also assessed for these stereoiosmeric disaccharides. A systematic study showed that an increasing discrimination was attained for the 1 --> 4 isomeric pair as the size of the derivative increased from methyl to pentyl. No anomeric discrimination was attained for the 1 --> 6 isomeric pair. Parent and product ion scans confirmed the consistency of fragmentation pathways among derivatives. Chem-X and MM3 molecular modeling programs were used to obtain minimum energy structures and freedom of motion volumes for the permethylated disaccharides. The modeling results correlated with the fragmentation ratios obtained in the mass spectrometer giving strong indication that the collision induced spectra are dependent on the freedom of rotational motion around the glycosidic bond.     

Characterization of the Dissociation Behavior of Gas-Phase Protonated and Methylated Lactones

The dissociation behavior of gas-phase protonated and methylated four-, five-, six-, and seven-membered ring lactones, some with methyl substituents in various positions, has been characterized by using a quadrupole ion trap mass spectrometer and a triple quadrupole mass spectrometer. The energy dependence of collisionally activated dissociation pathways was determined by energy-resolved mass spectrometry, and the dissociation behavior of the various protonated lactones was compared to that observed for protonated cyclic ketones and ethers of analogous ring size. The protonated cyclic ethers and ketones predominantly dissociated via dehydration, whereas the protonated lactones dissociated via losses of an alkene, ketene, and water. The dissociation behavior of the gas-phase methylated lactones formed from ion/molecule reactions with dimethyl ether ions was compared to the collisionally activated dissociation behavior of isomeric protonated methyl-substituted lactones. The methylation experiments indicated that the gas-phase addition of a methyl group may dramatically alter the favored dissociation pathways when compared to the simple protonated ions.     

Linkage Determination of Linear Oligosaccharides by MSn (n > 2) Collision-Induced Dissociation of Z1 Ions in the Negative Ion Mode

Obtaining unambiguous linkage information between sugars in oligosaccharides is an important step in their detailed structural analysis. An approach is described that provides greater confidence in linkage determination for linear oligosaccharides based on multiple-stage tandem mass spectrometry (MSⁿ, n >2) and collision-induced dissociation (CID) of Z₁ ions in the negative ion mode. Under low energy CID conditions, disaccharides ¹⁸O-labeled on the reducing carbonyl group gave rise to Z₁ product ions (m/z 163) derived from the reducing sugar, which could be mass-discriminated from other possible structural isomers having m/z 161. MS³ CID of these m/z 163 ions showed distinct fragmentation fingerprints corresponding to the linkage types and largely unaffected by sugar unit identities or their anomeric configurations. This unique property allowed standard CID spectra of Z₁ ions to be generated from a small set of disaccharide samples that were representative of many other possible isomeric structures. With the use of MSⁿ CID (n = 3 – 5), model linear oligosaccharides were dissociated into overlapping disaccharide structures, which were subsequently fragmented to form their corresponding Z₁ ions. CID data of these Z₁ ions were collected and compared with the standard database of Z₁ ion CID using spectra similarity scores for linkage determination. As the proof-of-principle tests demonstrated, we achieved correct determination of individual linkage types along with their locations within two trisaccharides and a pentasaccharide.

The stereochemical dependence of unimolecular dissociation of monosaccharide-glycolaldehyde anions in the gas phase: a basis for assignment of the stereochemistry and anomeric configuration of monosaccharides in oligosaccharides by mass spectrometry via a key discriminatory product ion of disaccharide fragmentation, m/z 221

Mass spectrometry of hexose-containing disaccharides often yields product ions of m/z 221 in the negative ion mode. Using a Paul trap, isolation and collision-induced dissociation of the m/z 221 anions yielded mass spectra that easily differentiated their stereochemistry and anomeric configuration, for all 16 stereochemical variants. The ions were shown to be glycopyranosyl-glycolaldehydes through chemical synthesis of their standards. The stereochemistry dramatically affected fragmentation which was dependent on four relative stereochemical arrangements: (1) the relationship between the hydroxyl group at position 2 and the anomeric configuration, (2) a cis relationship of the anomeric position and positions 2 and 3 (1,2,3-cis), (3) a 1,2 trans-2,3 cis relationship, and (4) the relationship between the hydroxyl group at position 4 and the anomeric configuration. After labeling the reducing carbonyl oxygen of a series of disaccharides with 18O to mass-discriminate between their monosaccharide components, it was demonstrated that m/z 221 anions are comprised of an intact nonreducing sugar glycosidically linked to a 2-carbon aglycon derived from the reducing sugar, irrespective of the linkage position between monosaccharides. This enabled the location of the intact sugar to be assigned to the nonreducing side of a glycosidic linkage. Detailed studies of experimental factors necessary for reproducibility demonstrated that the unique mass spectrum for each m/z 221 anion could be obtained from month-to-month through the use of an internal energy-input calibrant ion that ensured reproducible energy deposition into the ions. The counterparts to these ions for the 2-acetamido-2-deoxyhexoses were m/z 262 anions, and the anomeric configuration and stereochemistry of these anions could also be reproducibly discriminated for N-acetylglucosamine and N-acetylgalactosamine. The fragmentation patterns of m/z 221 anions provide a firm reproducible basis for assignment of sugar stereochemistries in the gas phase.     

Characterizing protein glycosylation sites through higher‐energy C‐trap dissociation

Assigning glycosylation sites of glycoproteins and their microheterogeneity is still a very challenging analytical task despite the rapid advancements in mass spectrometry. It is shown here that glycopeptide ions can be fragmented efficiently using the higher‐energy C‐trap dissociation (HCD) feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap). An attractive aspect of this dissociation option is the generation of distinct Y1 ions (peptide+GlcNAc), thus allowing unequivocal assignment of N‐glycosylation sites of glycoproteins. The combination of the very informative collision‐induced dissociation spectra acquired in the linear ion trap with the distinct features of HCD offers very useful information aiding in the characterization of the glycosylation sites of glycoproteins. The HCD activation energy needed to obtain optimum Y1 ions was studied in terms of glycan structure and charge state, and size and structure of the peptide backbone. The latter appeared to be primarily dictating the needed HCD energy. The distinct Y1 ion formation in HCD facilitated an easy assignment of such an ion and its subsequent isolation and dissociation through multiple‐stage tandem mass spectrometry. The resulting MS³ spectrum of the Y1 ion facilitates database searching and de novo sequencing thus prompting the subsequent identification of the peptide backbone and associated glycosylation sites. Moreover, fragment ions formed by HCD are detected in the Orbitrap, thus overcoming the 1/3 cut‐off limitation that is commonly associated with ion trap mass spectrometers. As a result, in addition to the Y1 ion, the common glycan oxonium ions are also detected. The high mass accuracy offered by the LTQ Orbitrap mass spectrometer is also an attractive feature that allows a confident assignment of protein glycosylation sites and the microheterogeneity of such sites. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparison of Triple Quadrupole and Double Focusing Mass Spectrometers to Investigate Collision-Induced Dissociation and Metastable Ions of Glycoconjugates

Glycoconjugates, such as chromophore-labeled disaccharides and permethylated glycosphingolipids (GSL) were used for comparison of triple quadrupole and double focusing mass spectrometers in analysis of product ions. A profound effect of collision energy was observed in the product ion spectra of ceramide ions (fragment ions of permethylated GSL): more product ions were observed from a double focusing mass spectrometer. Besides collision energy, the structure of the analyte had a significant effect on the formation of product ions. Despite the fact that masses of protonated molecular ions (MH⁺) of permethylated GSL are significantly larger than their ceramide fragments, the low-energy and high-energy product ion spectra of MH⁺ are, in general, similar. In a double focusing mass spectrometer of reversed geometry, more metastable ions were observed in the first field free region (FFR) than in the second FFR. The metastable ions observed in the second FFR were similar to those observed in low-energy collision-induced dissociation (CID). Although a double focusing mass spectrometer is superior to triple quadrupole instrument for detection of product ions, the poor resolution in either the selection of precursor ion or in the product ion spectra can be a serious problem in analysis of a mixture with similar masses.