Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry

High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.     

RIKEN tandem mass spectral database (ReSpect) for phytochemicals: A plant-specific MS/MS-based data resource and database

The fragment pattern analysis of tandem mass spectrometry (MS/MS) has long been used for the structural characterization of metabolites. The construction of a plant-specific MS/MS data resource and database will enable complex phytochemical structures to be narrowed down to candidate structures. Therefore, a web-based database of MS/MS data pertaining to phytochemicals was developed and named ReSpect (RIKEN tandem mass spectral database). Of the 3595 metabolites in ReSpect, 76% were derived from 163 literature reports, whereas the rest was obtained from authentic standards. As a main web application of ReSpect, a fragment search was established based on only the m/z values of query data and records. The confidence levels of the annotations were managed using the MS/MS fragmentation association rule, which is an algorithm for discovering common fragmentations in MS/MS data. Using this data resource and database, a case study was conducted for the annotation of untargeted MS/MS data that were selected after quantitative trait locus analysis of the accessions (Gifu and Miyakojima) of a model legume Lotus japonicus. In the case study, unknown metabolites were successfully narrowed down to putative structures in the website.     

Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion

Cetuximab is a novel therapeutic monoclonal antibody with two N-glycosylation sites: a conserved site in the CH2 domain and a second site within the framework 3 of the variable portion of the heavy chain. The detailed structures of these oligosaccharides were successfully characterized using orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight mass spectrometry (oMALDI Qq-TOF MS) and tandem mass spectrometry (MS/MS) in combination with exoglycosidase digestion. The N-linked oligosaccharides were released by treatment with N-glycanase F, reductively aminated with anthranilic acid, and fractionated by normal phase high-performance liquid chromatography (NP-HPLC). The fluorescent-labeled oligosaccharide pool and fractions were analyzed by oMALDI Qq-TOF MS and MS/MS in negative ion mode. Each fraction was further digested with an array of exoglycosidase mixtures, and subsequent MALDI TOF MS analysis of the resulting products yielded information about structural features of the oligosaccharide. The combined data revealed the presence of 21 distinct oligosaccharide structures in cetuximab. These oligosaccharides differ mainly in degree of sialylation with N-glycolyl neuraminic acid and extent of galactosylation (zero-, mono-, di-, and alpha(1-3)-galactosidase). The individual oligosaccharides were further assigned to the specific sites in the Fab and Fc regions of the antibody. This study represents a unique approach in that MS/MS data were used to identify and confirm the oligosaccharide structures of a protein.     

Characterization of N-linked oligosaccharides in chorion peroxidase of Aedes aegypti mosquito

A peroxidase is present in the chorion of Aedes aegypti eggs and catalyzes chorion protein cross-linking during chorion hardening, which is critical for egg survival in the environment. The unique chorion peroxidase (CPO) is a glycoprotein. This study deals with the N-glycosylation site, structures, and profile of CPO-associated oligosaccharides using mass spectrometric techniques and enzymatic digestion. CPO was isolated from chorion by solubilization and several chromatographic methods. Mono-saccharide composition was analyzed by HPLC with fluorescent detection. Our data revealed that carbohydrate (D-mannose, N-acetyl D-glucosamine, D-arabinose, N-acetyl D-galactosamine, and L-fucose) accounted for 2.24% of the CPO molecular weight. A single N-glycosylation site (Asn328-Cys- Thr) was identified by tryptic peptide mapping and de novo sequencing of native and PNGase A-deglycosylated CPO using matrix-assisted laser/desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Asn328 was proven to be a major fully glycosylated site. Potential tryptic glycopeptides and profile were first assessed by MALDI/TOF/MS and then by precursor ion scanning during LC/MS/MS. The structures of N-linked oligosaccharides were elucidated from the MS/MS spectra of glycopeptides and exoglycosidase sequencing of PNGase A-released oligosaccharides. These CPO-associated oligosaccharides had dominant Man3GlcNAc2 and Man3 (Fuc) GlcNAc2 and high mannose-type structures (Man(4-8)GlcNAc2). The truncated structures, Man2GlcNAc2 and Man2 (Fuc) GlcNAc2, were also identified. Comparison of CPO activity and Stokes radius between native and deglycosylated CPO suggests that the N-linked oligosaccharides influence the enzyme activity by stabilizing its folded state.     

Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure

The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-beta-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-beta-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.     

Ultrastructural, histochemical, and freeze-fracture evaluation of multilamellated structures in human pulmonary alveolar proteinosis

Ultrastructural, histochemical, and freeze-fracture studies of material recovered by bronchoalveolar lavage from patients with pulmonary alveolar proteinosis revealed four types (A, B, C, and D) of multilamellated structures (MS). Type A, the major component, consisted of concentric, trilaminar structures which were composed of two electron-dense layers and a central lucent layer (5.7-7.5 nm in overall width) alternating with wider (25-30 nm) electron-lucent intervening layers. Type B MS were formed by concentric lamellae with a 5-5.3-nm periodicity. Type C MS were composed of wavy, electron-dense lamellae with a 4-4.5-nm periodicity. Type D MS were conglomerated masses of intricately arranged double or triple electron-dense layers (7.5-13.5 nm wide) alternating with wider (30-40-nm) electron-lucent layers. The electron-dense lamellae of type A, type C, and type D MS were stained with ruthenium red, the Thiéry method, and concanavalin A, indicating the presence of carbohydrate components. Freeze-fracture studies revealed smooth inner and outer surfaces in type A MS, with the fracture planes passing through the central parts of the trilaminar structures; the intervening layers contained 10-nm particles, which probably are proteins. Type B MS had smooth surfaces, and type C MS had slightly particulate surfaces; while type D MS showed tubular or polygonal structures, 350 nm wide, with rows of particles 7-8 nm in diameter. It is concluded that type A and type D MS contain proteins and carbohydrates, probably in the form of glycoproteins, as well as phospholipids, and are related to tubular myelin. Type B and type C MS are considered to contain mainly phospholipids; type C MS are also considered to contain carbohydrates and to be related to lamellar bodies of type II alveolar epithelial cells.     

Using MS-FINDER for identifying 19 natural products in the CASMI 2016 contest

In its fourth year, the CASMI 2016 contest was organized to evaluate current chemical structure identification strategies for 19 natural products using high-resolution LC–MS and LC–MS/MS challenge datasets using automated methods with or without the combination of other tools. These natural products originate from plants, fungi, marine sponges, algae, or micro-algae. Every compound annotation workflow must start with determination of elemental compositions. Of these 19 challenges, one was excluded by the organizers after submission. For the remaining 18 challenges, three software programs were used. MS-FINDER version 1.62 was able to correctly identify 89% of the molecular formulas using an internal database that comprised of 13 metabolomics repositories with 45,181 formulas. SIRIUS correctly identified 61% compositions using PubChem formulas and Seven Golden Rules correctly identified 83% by using the Dictionary of Natural Products as a targeted database. Next, we performed structural dereplication for which we used the consensus formula from the three software programs. We submitted two solution sets for these challenges. In the first solution set, avaniya001, we only used the internal MS-FINDER functions for predicting and ranking structures, correctly identifying 53% of the structures as top-hit, 72% within the top-3 structures, and 78% within the top-10 hits. For our second set, avaniya002, we used both MS-FINDER predictions as well as MS/MS queries against the commercial NIST 14, METLIN, and the public MassBank of North America libraries. Here we correctly identified 78% of the structures as top-hit and 83% within the top-3 hits. Three challenge spectra remained unidentified in either of our submissions within the top-10 hits.     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Analysis of folate binding protein N-linked glycans by mass spectrometry

The folate binding protein (FBP), also known as the folate receptor (FR), is a glycoprotein which binds the vitamin folic acid and its analogues. FBP contains multiple N-glycosilation sites, is selectively expressed in tissues and body fluids, and mediates targeted therapies in cancer and inflammatory diseases. Much remains to be understood about the structure, composition, and the tissue specificities of N-glycans bound to FBP. Here, we performed structural characterization of N-linked glycans originating from bovine and human milk FBPs. The N-linked glycans were enzymatically released from FBPs, purified, and permethylated. Native and permethylated glycans were further analyzed by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their structural characterization. The assignment of putative glycan structures from MS and MS/MS data was achieved using Functional Glycomics glycan database and SimGlycan software, respectively. It was found that FBP from human milk contains putative structures that have composition consistent with high-mannose (Hex(5-6)HexNAc(2)) as well as hybrid and complex N-linked glycans (NeuAc(0-1)Fuc(0-3)Hex(3-6)HexNAc(3-5)). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex(4-9)HexNAc(2)) as well as hybrid and complex N-linked glycans (Hex(3-6)HexNAc(3-6)), but these glycans mostly do not contain fucose and sialic acid. Glycomic characterization of FBP provides valuable insight into the structure of this pharmacologically important glycoprotein and may have utility in tissue-selective drug targeting and as a biomarker.     

Isomer and glycomer complexities of core GlcNAcs in Caenorhabditis elegans

Analysis of protein glycosylation within the nematode Caenorhabditis elegans has revealed an abundant and unreported set of core chitobiose modifications (CCMs) to N-linked glycans. With hydrazine release, an array of glycomers and isobars were detected with hexose extensions on the 3- and 3,6-positions of the penultimate and reducing terminus, respectively. A full complement of structures includes a range of glycomers possessing a Galbeta(1-4)Fuc disaccharide at the 3- and 6-positions of the protein-linked GlcNAc. Importantly, enzymatic (PNGase F/A) release failed to liberate many of these extended structures from reduced and alkylated peptides and, as a consequence, such profiles were markedly deficient in a representation of the worm glycome. Moreover, the 3-linked Galbeta(1-4)Fuc moiety was notably resistant to a range of commercial galactosidases. For identification, the fragments were spectrum-matched with synthetic products and library standards using sequential mass spectrometry (MS(n)). A disaccharide observed at the 3-position of penultimate GlcNAc, indicating a Hex-Fuc branch on some structures, was not further characterized because of low ion abundance in MS(n). Additionally, a Hex-Hex-Fuc trisaccharide on the 6-position of proximal GlcNAc was also distinguished on select glycomers. Similar branch extensions on 6-linked core fucosyl residues have recently been reported among other invertebrates. Natural methylation and numerous isobars complement the glycome, which totals well over 100 individual structures. Complex glycans were detected at lower abundance, indicating glucosaminyltransferase-I (GnT-I) and GnT-II activity. A range of phosphorylcholine (PC)-substituted complex glycans were also confirmed following a signature two-stage loss of PC during MS(n) analysis, although the precursor ion was not observed in the mass profiles. In a similar manner, numerous other minor glycans may be present but unobserved in hydrazine-release profiles dominated by fucosylated structures. All CCM structures, including multiple isomers, were determined without chromatography by gas-phase disassembly (MS(n)) in Paul and linear ion trap (IT) instruments.     

Dermatoxin and phylloxin from the waxy monkey frog, Phyllomedusa sauvagei: cloning of precursor cDNAs and structural characterization from lyophilized skin secretion

Amphibian skin is a morphologically, biochemically and physiologically complex organ that performs the wide range of functions necessary for amphibian survival. Here we describe the primary structures of representatives of two novel classes of amphibian skin antimicrobials, dermatoxin and phylloxin, from the skin secretion of Phyllomedusa sauvagei, deduced from their respective precursor encoding cDNAs cloned from a lyophilized skin secretion library. A degenerate primer, designed to a highly conserved domain in the 5'-untranslated region of analogous peptide precursor cDNAs from Phyllomedusa bicolor, was employed in a 3'-RACE reaction. Peptides with molecular masses coincident with precursor-deduced mature toxin peptides were identified in LC/MS fractions of skin secretion and primary structures were confirmed by MS/MS fragmentation. This integrated experimental approach can thus rapidly expedite the primary structural characterization of amphibian skin peptides in a manner that circumvents specimen sacrifice whilst preserving robustness of scientific data.     

Factor analysis of brain structure reorganization in patient with multiple sclerosis (based on pet data)

The goal of present study was to investigate the functional reorganization of brain structures in patients with multiple sclerosis (MS). The patterns of distribution of relative estimations of local cerebral metabolic rate of glucose (ICMRglu) in regions of interest (ROIs), corresponding to anatomo-functional brain areas are obtained in groups of healthy volunteers (n=31 subjects) and patients with relapsing-remitting and progressive types of MS (n=59 and 39 accordingly). The analysis of factor structure of the obtained patterns allowed to make a conclusion about the existence of a common features with the factor structure of the distribution of another functional parameter--a regional cerebral blood flow (rCBF). This indicates that both factor solutions mainly reflect the functional organization of a brain. The differences revealed in factor structures of ICMRglu distribution in groups of patients with various types of MS and healthy volunteers allowed to assume that even at early stages of the disease despite the close anatomic and functional connectivity that normally exists between basal ganglia, MS patients have a functional dissociation of these structures. The bipolarity of revealed factors probably reflects the different directionality of the processes: relative decrease of functional activity in the areas which are directly responsible for performance of broken functions, caused by the deafferentation of the specified areas and its compensatory relative increase in functionally connected zones.     

超高效液相色谱与串联四级杆飞行时间质谱仪联用技术结合质谱裂解规律快速分析Alternaria panax中二酮哌嗪类化学成分

【目的】利用超高效液相色谱与串联四级杆飞行时间质谱仪联用技术(ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry,UPLC-Q-TOF-MS/MS)结合质谱裂解规律分析,靶向分离Alternaria panax发酵液粗提物中次生代谢产物。【方法】用马铃薯葡萄糖(potato dextrose broth,PDB)培养基液体发酵A.panax 14 d,将滤液用乙酸乙酯萃取后减压浓缩得粗提物;基于UPLC-Q-TOF-MS/MS方法(高分辨质谱、分子式与碎片峰等)分析粗提物化学成分及质谱裂解规律;采用半制备高效液相色谱(high-performance liquid chromatography,HPLC)方法进一步分离纯化;结合核磁共振波谱(nuclear magnetic resonance,NMR)和质谱(mass spectrometry,MS)等谱学技术以及与文献数据对照确定化合物结构。【结果】利用UPLC-Q-TOF-MS/MS技术分析出...     

Ion-pair high-speed countercurrent chromatography in fractionation of a high-molecular weight variation of acyl-oligosaccharide linked betacyanins from purple bracts of Bougainvillea glabra

The natural pigment composition of purple bracts of Bougainvillea glabra (Nyctaginaceae) consists of a highly complex mixture of betacyanins solely differing by the substitution with a variety of acyl-oligoglycoside units. This study was focused on a two-dimensional chromatography approach, a combination of preparative high-speed countercurrent chromatography (HSCCC) and analytical C18-HPLC with ESI–DAD–MS/MS detection which finally enabled a more detailed view into the pigment profile and elucidated the existence of an overwhelming amount of varying betacyanin structures occurring in Bougainvillea bracts. The detected molecular weights of the pigments reached so far unknown high values and ranged up to maximum values of 1653 and 1683 Da for the largest molecules due to oligosaccharide linkage and multiple acyl substitutions. The preparative IP–HSCCC separation yielded 15 complex fractions containing betacyanins of enhanced polarity as well as structures with highly increased lipophilicity. Betacyanin structures extended by large oligosaccharide chains with bigger number of glycoside units and also carrying a reduced number of hydroxycinnamic acid substitutions were characteristic for polar pigments occurring mainly in the early eluting CCC fractions. IP–HSCCC was proven to be extremely effective for fractionating this complex crude betalain pigment extract into more defined ‘polarity-windows’. Structural analysis by analytical LC–ESI–MS/MS in the positive ionization mode detected a total sum of 146 different betacyanin pigments in the CCC fractions of reduced complexity.     

A New Lariciresinol-Type Butyrolactone Lignan Derived from Hydroxymatairesinol and Its Identification in Spruce Wood

When the natural lignan hydroxymatairesinol ( 1 ) was treated with an alkaline aqueous solution, it partially rearranged to isomeric forms of a lariciresinol-type butyrolactone lignan. The two major diastereomers formed (2 and 3) were isolated by column and medium-pressure chromatography, and their structures were elucidated by MS and NMR techniques. These previously unknown butyrolactone lignans were identified as naturally occurring in spruce knotwood by GC, GC-MS, and HPLC-ESI MS/MS analyses. The formation of isohydroxymatairesinol (2) and epi-isohydroxymatairesinol (3) from hydroxymatalresinol ( 1 ), and their detection in rat urine after administration of 1, is discussed.     

New insights in Rapana venosa hemocyanin N-glycosylation resulting from on-line mass spectrometric analyses

The N-glycosylation of structural unit 1 of Rapana venosa hemocyanin was studied. Enzymatically liberated N-glycans were analyzed by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis (CE)-MS following 8-aminopyrene-1,3,6-trisulfonate labeling and labeling with 3-aminopyrazole, a new dedicated sugar reagent. Structural information was obtained by exoglycosidase sequencing, on-line MS/MS, permethylation, and amidation. A mixture of high-mannose and complex glycans with so far unknown and unusual acidic terminal structures was revealed. As the hemocyanin protein sequence is currently unknown, de novo sequencing of the glycopeptides had to be carried out. The N-glycans were therefore enzymatically removed with simultaneous partial (50%) (18)O-labeling of glycosylated asparagine residues prior to proteolysis. Following nano-liquid chromatography-MALDI-TOF-MS, the originally glycosylated peptides could be revealed and their sequences determined by MS/MS. The site occupancies were subsequently elucidated by precursor ion scanning of the intact glycopeptides using a Q-Trap mass spectrometer.     

N-glycosylation of ovomucin from hen egg white

Ovomucin is a bioactive egg white glycoprotein responsible for the gel properties of fresh egg white and is believed to be involved in egg white thinning, a natural process that occurs during storage. Ovomucin is composed of two subunits: a carbohydrate-rich β-ovomucin with molecular weight of 400-610?KDa and a carbohydrate-poor α-ovomucin with molecular mass of 254?KDa. In addition to limited information on O-linked glycans of ovomucin, there is no study on either the N-glycan structures or the N-glycosylation sites. The purpose of the present study was to characterize the N-glycosylation of ovomucin from fresh eggs using nano LC ESI-MS, MS/MS and MALDI MS. Our results showed the presence of N-linked glycans on both glycoproteins. We found 18 potential N-glycosylation sites in α-ovomucin. 15 sites were glycosylated, one site was found in both glycosylated and non-glycosylated forms and two potential glycosylation sites were found unoccupied. The N-glycans of α-ovomucin found on the glycosylation sites are complex-type structures with bisecting N-acetylglucosamine. MALDI MS of the N-glycans released from α-ovomucin by PNGase F revealed that the most abundant glycan structure is a bisected type of composition GlcNAc(6)Man(3). Two N-glycosylated sites were found in β-ovomucin.     

Structural studies on the lipopolysaccharide core of Proteus OX strains used in Weil-Felix test: a mass spectrometric approach

The core region of the lipopolysaccharides of Proteus group OX bacteria, which are used as antigens in Weil-Felix test for serodiagnosis of rickettsiosis, were studied by chemical degradations in combination with ESI FTMS, including infrared multi-photon dissociation (IRMPD) MS/MS and capillary skimmer dissociation. Structural variants of the inner core region were found to be the same as in Proteus non-OX strains that have been studied earlier. The outer core region has essentially the same structure in Proteus vulgaris OX19 (serogroup O1) and OX2 (serogroup O2) and a different structure in Proteus mirabilis OXK (serogroup O3). A fragmentation due to the rupture of the linkage between GlcN or GalN and GalA was observed in IRMPD-MS/MS of core oligosaccharides and found to be useful for screening of Proteus strains to assign structures of the relatively conserved inner core region and to select for further studies strains with distinct structures of a more variable outer core region.     

High-sensitivity analysis of glycosphingolipids by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight imaging mass spectrometry on transfer membranes

Glycosphingolipids are ubiquitous constituents of cells. Yet there is still room for improvement in the techniques for analyzing glycosphingolipids. Here we report our highly sensitive and convenient analytical technology with imaging mass spectrometry for detailed structural analysis of glycosphingolipids. We were able to determine detailed ceramide structures; i.e., both the sphingosine base and fatty acid, by MS/MS/MS analysis on a PVDF membrane with 10 pmol of GM1, with which only faint bands were visible by primuline staining. The limit of detection was approximately 1 pmol of GM1, which is lower than the value in the conventional reports (10 pmol).