Assessment of Dimeric Metal-Glycan Adducts via Isotopic Labeling and Ion Mobility-Mass Spectrometry.

Adduction of multivalent metal ions to glycans has been shown in recent years to produce altered tandem mass spectra with collision-induced dissociation, electron transfer techniques, and photon-based fragmentation approaches. However, these approaches assume the presence of a well-characterized precursor ion population and do not fully account for the possibility of multimeric species for select glycan-metal complexes. With the use of ion mobility separations prior to mass analysis, doubly charged dimers are not necessarily problematic for tandem MS experiments given that monomer and dimer drift times are sufficiently different.

O sensing-associated glycosylation exposes the F-box-combining site of the Skp1 subunit in E3 ubiquitin ligases.

Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (kp1/ullin-1/-box protein) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In (a social amoeba), (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box-binding domain. Prolyl hydroxylation of Skp1 contributes to O-dependent development, but full glycosylation at that position is required for optimal O sensing.

Enhanced Mixture Separations of Metal Adducted Tetrasaccharides Using Frequency Encoded Ion Mobility Separations and Tandem Mass Spectrometry.

Using five isomeric tetrasaccharides in combination with seven multivalent metals, the impact on mobility separations and resulting CID spectra were examined using a hybrid ion mobility atmospheric pressure drift tube system coupled with a linear ion trap. By enhancing the duty cycle of the drift tube system using a linearly chirped frequency, the collision-induced dissociation spectra were encoded in the mobility domain according to the drift times of each glycan isomer precursor. Differential fragmentation patterns correlated with precursor drift times ensured direct assignment of fragments with precursor structure whether as individual standards or in a mixture of isomers.

Differentiation of Rubidiated Methyl-d-Glycoside Stereoisomers by Infrared Multiple-Photon Dissociation Spectroscopy in the O-H and C-H Stretching Regions.

Four isomeric sugar methylglycosides (α- and β-d-gluco- and galactopyranosides) were evaluated as rubidium cation coordination adducts in the gas phase using variable-wavelength multiple-photon dissociation in the range from 2750 to 3750 cm(-1). The adducts dissociated following photon absorption to yield neutral sugars and the rubidium cation, resulting in infrared "action" spectra. Well-resolved hydroxyl stretching bands clearly differentiate stereoisomers that vary solely in their asymmetry at single carbons.

Determining the isomeric heterogeneity of neutral oligosaccharide-alditols of bovine submaxillary mucin using negative ion traveling wave ion mobility mass spectrometry.

Negative ions produced by electrospray ionization were used to evaluate the isomeric heterogeneity of neutral oligosaccharide-alditols isolated from bovine submaxillary mucin (BSM). The oligosaccharide-alditol mixture was preseparated on an off-line high-performance liquid chromatography (HPLC) column, and the structural homogeneity of individual LC fractions was investigated using a Synapt G2 traveling wave ion mobility spectrometer coupled between quadupole and time-of-flight mass spectrometers. Mixtures of isomers separated by both chromatography and ion mobility spectrometry were studied.

Assignment of the stereochemistry and anomeric configuration of sugars within oligosaccharides via overlapping disaccharide ladders using MS(n).

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 (MS(n), 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.

Evaluation of ion mobility-mass spectrometry for determining the isomeric heterogeneity of oligosaccharide-alditols derived from bovine submaxillary mucin.

Rapid separation and independent analysis of isomeric species are needed for the structural characterization of carbohydrates in glycomics research. Ion mobility-mass spectrometry techniques were used to examine a series of isomeric neutral oligosaccharide-alditols derived from bovine submaxillary mucin. Several analytical techniques were employed: (1) off line separation of the oligosaccharide-alditol mixture by HPLC; (2) direct and rapid evaluation of isomeric heterogeneity of oligosaccharides by electrospray ionization-ion mobility-time of flight mass spectrometry; and (3) mobility-selected MS and MS to evaluate isomeric mobility peaks by dual gate ion mobility-tandem mass spectrometry.

Ion mobility mass spectrometry analysis of isomeric disaccharide precursor, product and cluster ions.

Rationale: Carbohydrates are highly variable in structure owing to differences in their anomeric configurations, monomer stereochemistry, inter-residue linkage positions and general branching features. The separation of carbohydrate isomers poses a great challenge for current analytical techniques.

Methods: The isomeric heterogeneity of disaccharide ions and monosaccharide-glycolaldehyde product ions was evaluated using electrospray traveling wave ion mobility mass spectrometry (Synapt G2 high-definition mass spectrometer) in both positive and negative ion modes.

Linkage determination of linear oligosaccharides by MS(n) (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 (MS(n), n >2) and collision-induced dissociation (CID) of Z1 ions in the negative ion mode. Under low energy CID conditions, disaccharides (18)O-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.

Ion Mobility-Mass Correlation Trend Line Separation of Glycoprotein Digests without Deglycosylation.

A high-throughput ion mobility mass spectrometer (IMMS) was used to rapidly separate and analyze peptides and glycopeptides derived from glycoproteins. Two glycoproteins, human α-1-acid glycoprotein and antithrombin III were digested with trypsin and subjected to electrospray traveling wave IMMS analysis. No deglycosylation steps were performed; samples were complex mixtures of peptides and glycopeptides.

Carbohydrate structure characterization by tandem ion mobility mass spectrometry (IMMS)2.

A high resolution ion mobility spectrometer was interfaced to a Synapt G2 high definition mass spectrometer (HDMS) to produce IMMS-IMMS analysis. The hybrid instrument contained an electrospray ionization source, two ion gates, an ambient pressure linear ion mobility drift tube, a quadrupole mass filter, a traveling wave ion mobility spectrometer (TWIMS), and a time-of-flight mass spectrometer. The dual gate drift tube ion mobility spectrometer (DTIMS) could be used to acquire traditional IMS spectra but also could selectively transfer specific mobility selected precursor ions to the Synapt G2 HDMS for mass filtration (quadrupole).

Resolving structural isomers of monosaccharide methyl glycosides using drift tube and traveling wave ion mobility mass spectrometry.

Monosaccharide structural isomers including sixteen methyl-D-glycopyranosides and four methyl-N-acetylhexosamines were subjected to ion mobility measurements by electrospray ion mobility mass spectrometry. Two ion mobility-MS systems were employed: atmospheric pressure drift tube ion mobility time-of-flight mass spectrometry and a Synapt G2 HDMS system which incorporates a low pressure traveling wave ion mobility separator. All the compounds were investigated as [M + Na](+) ions in the positive mode.

Discrimination between coupling networks of glucopyranosides varying at a single stereocenter using two-dimensional vibrational correlation spectroscopy.

A combination of two-dimensional infrared (2DIR) correlation spectroscopy, linear absorption spectroscopy, and density functional theory quantum calculations was used to identify characteristic spectral features of two anomers of acetylated 2-azido-2-deoxy-D-glucopyranose. While the linear absorption spectra for the α and β anomers were distinctive, a substantial difference between them was observed only in the spectral region below 1200 cm(-1). The infrared correlation spectra of the two anomers differed significantly, even in regions where their linear absorption spectra were similar.

Differentiation of the stereochemistry and anomeric configuration for 1-3 linked disaccharides via tandem mass spectrometry and 18O-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.

Differentiation of closely related isomers: application of data mining techniques in conjunction with variable wavelength infrared multiple photon dissociation mass spectrometry for identification of glucose-containing disaccharide ions.

Data mining algorithms have been used to analyze the infrared multiple photon dissociation (IRMPD) patterns of gas-phase lithiated disaccharide isomers irradiated with either a line-tunable CO(2) laser or a free electron laser (FEL). The IR fragmentation patterns over the wavelength range of 9.2-10.

Direct evidence for the ring opening of monosaccharide anions in the gas phase: photodissociation of aldohexoses and aldohexoses derived from disaccharides using variable-wavelength infrared irradiation in the carbonyl stretch region.

All eight D-aldohexoses and aldohexoses derived from the non-reducing end of disaccharides were investigated by variable-wavelength infrared multiple-photon dissociation (IRMPD) as anions in the negative-ion mode. Spectroscopic evidence supports the existence of a relatively abundant open-chain configuration of the anions in the gas phase, based on the observation of a significant carbonyl absorption band near 1710 cm(-1). The abundance of the open-chain configuration of the aldohexose anions was approximately 1000-fold or greater than that of the neutral sugars in aqueous solution.

Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O₂ signaling in dictyostelium.

The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα) type prolyl 4-hydroxylase (P4H1) and an α-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein) class of E3 ubiquitin ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O(2) regulation of development, suggesting the existence of an ancient O(2)-sensing mechanism related to modification of the transcription factor HIFα by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O(2) signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1.

Assignment of the stereochemistry and anomeric configuration of structurally informative product ions derived from disaccharides: infrared photodissociation of glycosyl-glycolaldehydes in the negative ion mode.

Using mass spectrometry in the negative ion mode, m/z 221 ions are frequently observed as product ion substructures derived from reducing disaccharides having 2, 4, or 6 linkages. The ions have been shown to be glycosyl-glycolaldehydes. All 16 stereochemical variants of their pyranosides were prepared and evaluated by infrared photodissociation, in addition to HexNAc-glycolaldehyde variants (m/z 262) of 2-acetamido-2-deoxy-d-glucose and 2-acetamido-2-deoxy-d-galactose.

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 MS(3), the number of stages of dissociation limited only practically by the abundance of specific product ions.

High-resolution pyrimidine- and ribose-specific 4D HCCH-COSY spectra of RNA using the filter diagonalization method.

The NMR spectra of nucleic acids suffer from severe peak overlap, which complicates resonance assignments. 4D NMR experiments can overcome much of the degeneracy in 2D and 3D spectra; however, the linear increase in acquisition time with each new dimension makes it impractical to acquire high-resolution 4D spectra using standard Fourier transform (FT) techniques. The filter diagonalization method (FDM) is a numerically efficient algorithm that fits the entire multi-dimensional time-domain data to a set of multi-dimensional oscillators.

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.

Rapid resolution of carbohydrate isomers by electrospray ionization ambient pressure ion mobility spectrometry-time-of-flight mass spectrometry (ESI-APIMS-TOFMS).

Carbohydrates are an extremely complex group of isomeric molecules that have been difficult to analyze in the gas phase by mass spectrometry because (1) precursor ions and product ions to successive stages of MS(n) are frequently mixtures of isomers, and (2) detailed information about the anomeric configuration and location of specific stereochemical variants of monosaccharides within larger molecules has not been possible to obtain in a general way. Herein, it is demonstrated that gas-phase analyses by direct combination of electrospray ionization, ambient pressure ion mobility spectrometry, and time-of-flight mass spectrometry (ESI-APIMS-TOFMS) provides sufficient resolution to separate different anomeric methyl glycosides and to separate different stereoisomeric methyl glycosides having the same anomeric configuration. Reducing sugars were typically resolved into more than one peak, which might represent separation of cyclic species having different anomeric configurations and/or ring forms.

Differentiation of the anomeric configuration and ring form of glucosyl-glycolaldehyde anions in the gas phase by mass spectrometry: isomeric discrimination between m/z 221 anions derived from disaccharides and chemical synthesis of m/z 221 standards.

Mass spectrometry of disaccharides in the negative-ion mode frequently generates product anions of m/z 221. With glucose-containing disaccharides, dissociation of isolated m/z 221 product ions in a Paul trap yielded mass spectra that easily differentiated between both anomeric configurations and ring forms of the ions. These ions were shown to be glucosyl-glycolaldehydes through chemical synthesis of their standards.

High-resolution four-dimensional carbon-correlated 1H-1H ROESY experiments employing isotags and the filter diagonalization method for effective assignment of glycosidic linkages in oligosaccharides.

Four-dimensional nuclear magnetic resonance spectroscopy of oligosaccharides that correlates 1H-1H ROESY cross peaks to two additional 13C frequency dimensions is reported. The 13C frequencies were introduced by derivatization of all free hydroxyl groups with doubly 13C-labeled acetyl isotags. Pulse sequences were optimized for processing with the filter diagonalization method.

Differentiation of isomers by wavelength-tunable infrared multiple-photon dissociation-mass spectrometry: application to glucose-containing disaccharides.

Variation in the wavelength of irradiation in infrared multiple-photon dissociation (IR-MPD) of lithium-tagged glucose-containing disaccharide ions (1-2-, 1-3-, 1-4-, and 1-6-linked isomers of both anomeric configurations) resulted in marked differences in their mass spectral fragmentation patterns. Two-dimensional plots of the fragment yield versus infrared wavelength for each mass spectral product ion were unique for each isomer and can be considered a spectral fingerprint. Individual product ions or diagnostic ratios of key product ions can be optimized at specific IR wavelengths.