Oligosaccharide analysis by capillary-scale high-pH anion-exchange chromatography with on-line ion-trap mass spectrometry

A capillary-scale high-pH anion-exchange chromatography (HPAEC) system for the analysis of carbohydrates was developed, in combination with two parallel on-line detection methods of sub-picomolar sensitivity: (1) pulsed amperometric detection (PAD); (2) capillary-scale desalting followed by electrospray ion-trap (IT) mass spectrometry (MS). The capillary chromatographic system combined the superb selectivity of HPAEC that allows routine separation of isomeric oligosaccharides with the information on monosaccharide sequence and linkage positions obtained by MS/MS fragmentation using the IT-MS. The applicability of the system in biomedical research was demonstrated by its use for the analysis of a urine sample of a GM1-gangliosidosis patient. Isomeric glycans in the sample could be resolved by HPAEC and assigned on the basis of the monosaccharide linkage information revealed by on-line IT-MS/MS.     

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. By labeling the reducing carbonyl oxygen of disaccharides with 18O to mass discriminate between monosaccharides, it was established that the m/z 221 ions are comprised solely of an intact nonreducing sugar with a two-carbon aglycon derived from the reducing sugar, regardless of the disaccharide linkage position. This enabled the anomeric configuration and ring form of the ion to be assigned and the location of the ion to the nonreducing side of a glycosidic linkage to be ascertained. Detailed studies of experimental factors necessary for reproducibility in a Paul trap demonstrated that the unique dissociation patterns that discriminate between the isomeric m/z 221 ions could be obtained from month-to-month in conjunction with an internal energy-input calibrant ion that ensures reproducible energy deposition into isolated m/z 221 ions. In addition, MS/MS fragmentation patterns of disaccharide m/z 341 anions in a Paul trap enabled linkage positions to be assigned, as has been previously reported with other types of mass spectrometers.     

莲子心碳苷黄酮与黄酮异构体的色谱质谱研究

采用电喷雾质谱法(ESI-MS),对从莲子心分离得到的碳苷类黄酮化合物进行质谱碎裂规律研究。结果表明,负离子模式下,六碳糖碳苷黄酮主要发生糖环裂解,通过丢失特征性的碎片(90 u、120 u、150 u)与氧苷黄酮区分;单糖取代的六碳醛糖氧苷黄酮直接丢失单糖部分(162 u),六碳醛糖种类无法通过质谱区分,但由于它们在液相上的保留时间不同,可通过液相色谱-质谱(LC-MS)联用方法分离鉴定;二糖取代的氧苷黄酮主要碎片离子通过丢失糖部分(146 u、162 u、308 u)所得,二糖的种类及连接方式可通过质谱图上的碎片离子峰及其相对丰度辨别。莲子心中多种碳苷黄酮和氧苷黄酮质谱的不同裂解规律,不仅有助于莲子心黄酮化合物的快速鉴定,而且可以通过与液相色谱联用实现莲子心中同分异构体的快速区分。     

Identification and the developmental formation of carotenoid pigments in the yellow/orange Bacillus spore-formers

Spore-forming Bacillus species capable of synthesising carotenoid pigments have recently been isolated. To date the detailed characterisation of these carotenoids and their formation has not been described. In the present article biochemical analysis on the carotenoids responsible for the yellow/orange pigmentation present in Bacilli has been carried out and the identity of the carotenoids present was elucidated. Chromatographic, UV/Vis and Mass Spectral (MS) data have revealed the exclusive presence of a C(30) carotenoid biosynthetic pathway in Bacillus species. Apophytoene was detected representing the first genuine carotenoid formed by this pathway. Cultivation in the presence of diphenylamine (DPA), a known inhibitor of pathway desaturation resulted in the accumulation of apophytoene along with other intermediates of desaturation (e.g. apophytofluene and apo-ζ-carotene). The most abundant carotenoids present in the Bacillus species were oxygenated derivatives of apolycopene, which have either undergone glycosylation and/or esterification. The presence of fatty acid moieties (C(9) to C(15)) attached to the sugar residue via an ester linkage was revealed by saponification and MS/MS analysis. In source fragmentation showed the presence of a hexose sugar associated with apolycopene derivatives. The most abundant apocarotenoids determined were glycosyl-apolycopene and glycosyl-4'-methyl-apolycopenoate esters. Analysis of these carotenoids over the developmental formation of spores revealed that 5-glycosyl-4'-methyl-apolycopenoate was related to sporulation. Potential biosynthetic pathways for the formation of these apocarotenoids in vegetative cells and spores have been reconstructed from intermediates and end-products were elucidated.     

Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

We have studied rapid and simple sugar mapping using liquid chromatography/electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column. The oligosaccharide mixture was separated on the basis of the sequence, branching structure, and linkage, and each oligosaccharide was characterized based on its molecular mass. In this study we demonstrated the usefulness of capillary LC/MS (CapLC/MS) and capillary liquid chromatography/tandem mass spectrometry (CapLC/MS/MS) as sensitive means for accomplishing the structural analysis of oligosaccharides in a low-abundance glycoprotein. The carbohydrate heterogeneity and molecular mass information of each oligosaccharide can be readily obtained from CapLC/MS of a small amount of glycoprotein. CapLC/MS/MS provided b-ion series, which is informative with regard to monosaccharide sequence. Exoglycosidase digestion followed by CapLC/MS elucidated a carbohydrate residue linkage. Using this method, we characterized N-linked oligosaccharides in hepatocyte growth factor produced in mouse myeloma NS0 cells as the complex-type bi-, tri-, and tetraantennary terminated with N-glycolylneuraminic acids and alpha-linked galactose residues. Sugar mapping with CapLC/MS and CapLC/MS/MS is useful for monitoring glycosylation patterns and for structural analysis of carbohydrates in a low-abundance glycoprotein and thus will become a powerful tool in biological, pharmaceutical, and clinical studies.     

Presence of terminal N-acetylgalactosamineβ1-4N-acetylglucosamine residues on O-linked oligosaccharides from gastric MUC5AC: involvement in Helicobacter pylori colonization?

Isolation of MUC5AC mucins from the gastric mucosa from two secretor individuals (one from normal mucosa from a patient with gastric cancer and one from a control) showed different abilities to bind and induce the proliferation of the Helicobacter pylori strain J99. Analysis of the released O-linked oligosaccharides by LC-MS from these individuals showed a very heterogeneous mixture of species from the cancer patient containing both neutral and sialylated structures, whereas the normal sample showed dominating neutral blood group H terminating structures as well as neutral structures containing the di-N-acetyllactosamine (lacdiNAc) unit GalNAcβ1-4GlcNAcβ1- on the C-6 branch of the reducing end GalNAc. The linkage configuration of these epitopes were determined using C-4-specific fragmentation for the GalNAcβ1-4GlcNAcβ1- glycosidic linkage, comparison of the MS(3) fragmentation with standards for linkage configuration and N-acetylhexosamine type as well as exoglycosidase treatment. It was also shown that the lacdiNAc epitope is present in both human and porcine gastric mucins, indicating that this is an epitope preserved between species. We hypothesize that the termination on gastric MUC5AC with lacdiNAc is in competition with complex glycosylation such as the Le(b) and H type 1 as well as complex sialylated structures. These are epitopes known to bind the H. pylori BabA and SabA adhesins.     

Androgen receptor activation results in metabolite signatures of an aggressive prostate cancer phenotype: an NMR-based metabonomics study

Metabolomic studies have proven to provide a unique perspective of the cellular dysfunction developing as a result of prostate cancer (PCa) onset and progression, facilitated primarily by mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques. PCa develops as an androgen-dependent disease with the expression of the androgen receptor (AR), where patient treatment typically involves androgen ablation therapy. In response, it is theorized that PCa transforms to an androgen-hypersensitive or androgen-independent state, where treatment options are severely reduced. Under the hypothesis that AR stimulation increases the aggressivity of pre-existing PCa, NMR spectroscopy was utilized in the delineation of the metabonomic response of an androgen-dependent PCa cell line (LnCAP) as a result of AR activation. Metabolite profiles were determined after 12, 24, and 48?h of exposure to methyltrienolone (R1881), an AR agonist. Principal components analysis revealed the relative myo-inositol and phosphocholine levels were severely altered after androgen treatment. Furthermore, univariate analysis revealed multiple metabolic perturbations in response to R1881 exposure, including amino acid, choline, the phosphocholine/glycerophosphocholine ratio, and UDP-coupled sugar metabolism, which are consistent with reported measurements between normal and PCa samples. These results suggest that androgen-sensitive PCa may transform to an aggressive phenotype upon AR activation.     

Extent of modifications in human proteome samples and their effect on dynamic range of analysis in shotgun proteomics

The complexity of the human proteome, already enormous at the organism level, increases further in the course of the proteome analysis due to in vitro sample evolution. Most of in vitro alterations can also occur in vivo as post-translational modifications. These two types of modifications can only be distinguished a posteriori but not in the process of analysis, thus rendering necessary the analysis of every molecule in the sample. With the new software tool ModifiComb applied to MS/MS data, the extent of modifications was measured in tryptic mixtures representing the full proteome of human cells. The estimated level of 8-12 modified peptides per each unmodified tryptic peptide present at >or=1% level is approaching one modification per amino acid on average. This is a higher modification rate than was previously thought, posing an additional challenge to analytical techniques. The solution to the problem is seen in improving sample preparation routines, introducing dynamic range-adjusted thresholds for database searches, using more specific MS/MS analysis using high mass accuracy and complementary fragmentation techniques, and revealing peptide families with identification of additional proteins only by unfamiliar peptides. Extensive protein separation prior to analysis reduces the requirements on speed and dynamic range of a tandem mass spectrometer and can be a viable alternative to the shotgun approach.     

Quantification of CLR1401, a novel alkylphosphocholine anticancer agent,in rat plasma by hydrophilic interaction liquid chromatography–tandem mass spectrometric detection

A rapid and specific LC–MS/MS based bioanalytical method was developed and validated for the determination of 18-(p-iodophenyl)octadecyl phosphocholine (CLR1401), a novel phosphocholine drug candidate, in rat plasma. The optimal chromatographic behavior of CLR1401 was achieved on a Kromasil silica column (50 mm × 3 mm, 5 μm) under hydrophilic interaction chromatography. The total LC analysis time per injection was 2.8 min with a flow rate of 1.5 mL/min under gradient elution. Liquid–liquid extraction in a 96-well format using ethyl acetate was developed and applied for method validation and sample analysis. The method validation was conducted over the curve range of 2.00–1000 ng/mL using 0.0500 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤ 5.9% relative standard deviation (RSD) and −10.8 to −1.4% relative error (RE). The method was successfully applied to determine the toxicokinetics of CLR1401 in rats from three dose groups of 0.4, 4.0, and 10.0 mg/kg/day via intravenous administration.     

Mass spectrometric assignment of Smith degradation glycopeptides derived from ribonuclease B

We established a method to determine the glycosyl linkage structure by a combination of Smith degradation and liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-Q-TOF-MS) and tandem MS (MS/MS). To assign the sugar linkage of N-glycoprotein, we employed a typical ribonuclease B containing oligosaccharides (Man5-9GlcNAc2). Tryptic digestion of ribonuclease B provided a mixture of high-mannose glycopeptides consisting of the four amino acids, Asn34-Leu-Thr-Lys37 (NLTK, T6). The mixture of glycopeptides was separated by high-performance liquid chromatography (HPLC) in a reversed phase column and was characterized by ESI-Q-TOF-MS and MS/MS. Comparison of the data with and without Smith degradation allowed us to make reasonable assignments to support such linkage patterns as (1-->2), (1-->3), (1-->6) and their multiples. These assignments were limited to six mannoses or lower due to the unstable nature of the higher derivatives. This method should be applicable to determine the linkage pattern of an unknown glycoprotein in about a 6-microgram amount.     

Early stage fetal neocortex exhibits a complex ganglioside profile as revealed by high resolution tandem mass spectrometry

In this study we report on the first mass spectrometric (MS) investigation of gangliosides and preliminary assessment of the expression and structure in normal fetal neocortex in early developmental stages: 14th (Neo14) and 16th (Neo16) gestational weeks. Ganglioside analysis was carried out using a hybrid quadrupole time-of-flight (QTOF) MS with direct sample infusion by nanoelectrospray ionization (nanoESI) in the negative ion mode. Under optimized conditions a large number of glycoforms i.e. 75 in Neo14 and 71 in Neo16 mixtures were identified. The ganglioside species were found characterized by a high diversity of the ceramide constitution, an elevated sialylation degree (up to pentasialylated gangliosides-GP1) and sugar cores modified by fucosylation (Fuc) and acetylation (O-Ac). Direct comparison between Neo14 and Neo16 revealed a prominent expression of monosialylated structures in the Neo16 as well as the presence of a larger number of polysialylated species in Neo14 which constitutes a clear marker of rapid development-dependant changes in the sialylation. Also the MS screening results highlighted that presumably O-acetylation process occurs faster than fucosylation. CID MS/MS under variable collision energy applied for the first time for structural analysis of a fucosylated pentasialylated species induced an efficient fragmentation with generation of ions supporting Fuc-GP1d isomer in early stage fetal brain neocortex.     

Quadrupole ion-trap mass spectrometry to locate fatty acids on lipid A from Gram-negative bacteria

The structure of lipid A released by mild acid hydrolysis from lipopolysaccharide from two strains of Shigella flexneri with different degrees of acylation was characterized using electrospray ionization (ESI) and ion-trap mass spectrometry. The lipid A was analyzed underivatized with ESI in negative-ion mode. With multiple stages of fragmentation (MS(n)), both the degree of acylation and the positions of the fatty acids on the disaccharide backbone could be determined. It was possible to determine the degree of acylation by the MS(n) technique, where in each MS stage the parent ion was an ion where one fatty acid had been eliminated. One way to determine the location of the fatty acids was by identifying cross-ring fragments of the reducing sugar from parent ions containing different numbers of fatty acids. Another was by identifying a possible charge-driven release of fatty acids situated close to a phosphate group. The fatty acids were otherwise eliminated by a charge-remote fragmentation mechanism. The combined data show the usefulness of ion-trap mass spectrometers for this type of analysis.     

Metabolite profiling of saponins in <Emphasis Type="Italic">Balanites</Emphasis><Emphasis Type="Italic">aegyptiaca</Emphasis> plant tissues using LC (RI)-ESI/MS and MALDI-TOF/MS

Saponins are a major family of secondary metabolites which consist of a sugar moiety glycosidically linked to a hydrophobic aglycone (sapogenin). In recent years the interest in saponins has increased significantly because of their diverse properties as natural detergents and foaming agents, their cardiac, immunostimulating, and anti-cancer activity, as well as other health promoting functions. This study deals with metabolitic analysis of saponins from methanolic extracts of fruit mesocarp (ME), seed kernel (KE) and root (RE) of Balanites aegyptiaca (L.) Del. (desert date) plant grown in Israel using LC (RI)-ESI/MS and MALDI-TOF/MS. The structural assignment was carried out by fragmentation experiments of LC (RI)-ESI/MS and literature data. The study has revealed that, all together, twenty-four furostanol saponins were found in ME, KE and RE. Of these, four saponins are found only in ME, five only in KE and six only in RE. Diosgenin was found to be the sole aglycone in all the saponins. The smallest saponin (MW 740 Da) was found with two sugar units (glucose) and the largest saponin (MW 1678 Da) was found with eight sugar units (5 glucose, 2 rhamnose and 1 xylose) attached to diosgenin. The results suggest that MALDI-TOF/MS with positive ion mode is particularly effective for determining the metabolites of saponins in B. aegyptiaca plant tissues. MALDI-TOF/MS not only verified the results of the LC (RI)-ESI/MS, but also identified additional saponins that are now systematically organized in a database of B. aegyptiaca saponins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Monitoring on-line desalted lignocellulosic hydrolysates by microdialysis sampling micro-high performance anion exchange chromatography with integrated pulsed electrochemical detection/mass spectrometry

An on-line system based on microdialysis sampling (MD), micro-high performance anion exchange chromatography (micro-HPAEC), integrated pulsed electrochemical detection (IPED), and electrospray ionization mass spectrometry (MS) for the monitoring of on-line desalted enzymatic hydrolysates is presented. Continuous monitoring of the enzymatic degradation of dissolving pulp from Eucalyptus grandis as well as degradation of sugar cane bagasse in a 5-mL reaction vessel was achieved up to 24 h without any additional sample handling steps. Combining MD with micro-HPAEC-IPED/MS and on-line desalting of hydrolysates enabled injection (5 microL) of at least 23 samples in a study of the sequential action of hydrolytic enzymes in an unmodified environment where the enzymes and substrate were not depleted due to the perm-selectivity of the MD membrane (30 kDa cut-off). Xylanase, phenolic acid esterase and a combination of endoglucanase (EG II) with cellobiohydrolase (CBH I) resulted in the production of DP 1 after the addition of esterase, DP 2 and DP 3 after the addition of EG II and CBH I, from the dissolving pulp substrate. Similar sequential enzyme addition to sugar cane bagasse resulted in DP 1 production after the addition of esterase and DP 1, DP 2 and DP 3 production after the addition of the EG II and CBH I mixture. Combining MS on-line with micro-HPAEC-IPED proved to be a versatile and necessary tool for such a study compared to conventional methods. The mass selectivity of MS revealed complementary information, including the co-elution of saccharides as well as the presence of more than one type of DP 2 in the case of dissolving pulp and several types of DP 2 and DP 3 for sugar cane bagasse. This study demonstrates the limitation of the use of retention time alone for confirmation of the identity of saccharides especially when dealing with complex enzymatic hydrolysates. In situ sampling and sample clean-up combined with on-line desalting of the chromatographic effluent, provides a generic approach to achieve real time monitoring of enzymatic hydrolysates when they are detected by a combination of IPED and MS.     

The CTP:phosphocholine cytidylyltransferase encoded by the licC gene of Streptococcus pneumoniae: cloning, expression, purification, and characterization.

Streptococcus pneumoniae is a member of a small group of bacteria that display phosphocholine on the cell surface, covalently attached to the sugar groups of teichoic acid and lipoteichoic acid. The putative pathway for this phosphocholine decoration is, in its first two enzymes, functionally similar to the CDP-choline pathway used for phosphatidylcholine biosynthesis in eukaryotes. We show that the licC gene encodes a functional CTP:phosphocholine cytidylyltransferase (CCT). The enzyme has been expressed and purified to homogeneity. Assay conditions were optimized, particularly with respect to linearity with time, pH, Mg(2+), and ammonium sulfate concentration. The pure enzyme has K(M) values of 890+/-240 microM for CTP, and 390+/-170 microM for phosphocholine. The k(cat) is 17.5+/-4.0 s(-1). S. pneumoniae CTP:phosphocholine cytidylyltransferase (SpCCT) is specific for CTP or dCTP as the nucleotide substrate. SpCCT is strongly inhibited by Ca(2+). The IC(50) values for recombinant and native SpCCT are 0.32+/-0.04 and 0.27+/-0.03 mM respectively. The enzyme is also inhibited by all other tested divalent cations, including Mg(2+) at high concentrations. The cloning and expression of this enzyme sets the stage for design of inhibitors as possible antipneumococcal drugs.     

PTM MarkerFinder,a software tool to detect and validate spectra from peptides carrying post‐translational modifications

Mass spectrometry (MS) analysis of peptides carrying post‐translational modifications is challenging due to the instability of some modifications during MS analysis. However, glycopeptides as well as acetylated, methylated and other modified peptides release specific fragment ions during CID (collision‐induced dissociation) and HCD (higher energy collisional dissociation) fragmentation. These fragment ions can be used to validate the presence of the PTM on the peptide. Here, we present PTM MarkerFinder, a software tool that takes advantage of such marker ions. PTM MarkerFinder screens the MS/MS spectra in the output of a database search (i.e., Mascot) for marker ions specific for selected PTMs. Moreover, it reports and annotates the HCD and the corresponding electron transfer dissociation (ETD) spectrum (when present), and summarizes information on the type, number, and ratios of marker ions found in the data set. In the present work, a sample containing enriched N‐acetylhexosamine (HexNAc) glycopeptides from yeast has been analyzed by liquid chromatography‐mass spectrometry on an LTQ Orbitrap Velos using both HCD and ETD fragmentation techniques. The identification result (Mascot .dat file) was submitted as input to PTM MarkerFinder and screened for HexNAc oxonium ions. The software output has been used for high‐throughput validation of the identification results.     

Structural analysis of sulfated N-linked oligosaccharides in erythropoietin.

We previously demonstrated that high-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) is useful for the structural analysis of carbohydrates in glycoproteins. Using LC/MS with GCC, sulfated N-linked oligosaccharides were found in erythropoietin (EPO) expressed in baby hamster kidney cells. Sulfation occurs in a part of the N-linked oligosaccharides in the EPO. Sulfated monosaccharide residue in the sulfated N-linked oligosaccharide was determined by exoglycosidase digestion followed by sugar mapping by LC/MS. The linkage position and branch-location of the sulfate group in the tetraantennary oligosaccharide were analyzed by (1)H-nuclear magnetic resonance. It was suggested that sulfation occurs on the C-6 position of GlcNAc located in the GlcNAcbeta1-4Manalpha1-3 branch.     

High-throughput immunoglobulin G N-glycan characterization using rapid resolution reverse-phase chromatography tandem mass spectrometry

We present an optimized high-throughput method for the characterization of 2-aminobenzamide (2-AB)-labeled N-glycans from recombinant immunoglobulin G (rIgG). This method includes an optimized sample preparation protocol involving microwave-assisted deglycosylation in conjunction with an automated sample cleanup strategy and a rapid resolution reverse-phase high-performance liquid chromatography (RRRP-HPLC) separation of labeled N-glycans. The RRRP-HPLC method permits generation of a comprehensive glycan profile using fluorescence detection in 45 min. In addition, the profiling method is directly compatible with electrospray ionization mass spectrometry (ESI-MS), allowing immediate and sensitive characterization of the glycan moiety by intact MS and tandem MS (MS/MS) fragmentation. We conservatively estimate an efficiency gain of fourfold with respect to the throughput capabilities of this optimized method as compared with traditional protocols (overnight deglycosylation, sample cleanup by graphitized carbon or cellulose cartridge, high-pH anion exchange chromatography, fraction collection, and processing for matrix-assisted laser desorption/ionization time-of-flight [MALDI-TOF] MS analysis) for a single sample. Even greater gains are achieved when processing of multiple samples is considered.     

Comparison of 2-D LC and 3-D LC with post- and pre-tryptic-digestion SEC fractionation for proteome analysis of normal human liver tissue

The current "shotgun" proteomic analysis, strong cation exchange-RPLC-MS/MS system, is a widely used method for proteome research. Currently, it is not suitable for complicated protein sample analysis, like mammal tissues or cells. To increase the protein identification confidence and number, an additional separation dimension for sample fractionation is necessary to be coupled prior to current multi-dimensional protein identification technology (MudPIT). In this work, SEC was elaborately selected and applied for sample prefractionation in consideration of its non-bias against sample and variety of choice of mobile phases. The analysis of the global lysate of normal human liver tissue sample provided by the China Human Liver Proteome Project, were performed to compare the proteome coverage, sequence coverage (peptide per protein identification) and protein identification efficiency in MudPIT, 3-D LC-MS/MS identification strategy with preproteolytic and postproteolytic fractionation. It was demonstrated that 3-D LC-MS/MS utilizing protein level fractionation was the most effective method. A MASCOT search using the MS/MS results acquired by QSTAR(XL) identified 1622 proteins from 3-D LC-MS/MS identification approaches. A primary analysis on molecular weight, pI and grand average hydrophobicity value distribution of the identified proteins in different approaches was made to further evaluate the 3-D LC-MS/MS analysis strategy.     

Determination of sequence and linkage of tissue oligosaccharides in caprine β-mannosidosis by fast atom bombardment,collisionally activated dissociation tandem mass spectrometry

Fast atom bombardment, collisionally activated dissociation tandem mass spectrometry (FAB-CAD-MS/MS), combined withp-aminobenzoic acid ethyl ester (ABEE) derivatization, were used to confirm the sequence and linkage pattern of subnanomolar amounts of the previously characterized three major thyroid gland oligosaccharides accumulated in caprine -mannosidosis. Positive ion FAB-CAD-MS/MS of both the [M + H]⁺ and [M + Na]⁺ ions from the ABEE derivatized oligosaccharides produced product ions derived from cleavage of the glycosidic bonds which allowed the sequences to be determined. Several fragments resulting from cleavages across the sugar ring permitted the assignment, in some cases, of the linkage positions between the sugar residues. The natriated molecule yielded several fragments of this type which were not observed when the protonated molecule was selected as the precursor ion. Use of these techniques gave the complete sequence and linkage characterization of the disaccharide and complete sequence and partial linkage information for the two higher oligosaccharides.