Surface Glycans Regulate Salmonella Infection-Dependent Directional Switch in Macrophage Galvanotaxis Independent of NanH.

Salmonella invades and disrupts gut epithelium integrity, creating an infection-generated electric field that can drive directional migration of macrophages, a process called galvanotaxis. Phagocytosis of bacteria reverses the direction of macrophage galvanotaxis, implicating a bioelectrical mechanism to initiate life-threatening disseminations. The force that drives direction reversal of macrophage galvanotaxis is not understood. One hypothesis is that Salmonella can alter the electrical properties of the macrophages by modifying host cell surface glycan composition, which is supported by the fact that cleavage of surface-exposed sialic acids with a bacterial neuraminidase severely impairs macrophage galvanotaxis, as well as phagocytosis. Here, we utilize N-glycan profiling by nanoLC-chip QTOF mass cytometry to characterize the bacterial neuraminidase-associated compositional shift of the macrophage glycocalyx, which revealed a decrease in sialylated and an increase in fucosylated and high mannose structures. The Salmonella nanH gene, encoding a putative neuraminidase, is required for invasion and internalization in a human colonic epithelial cell infection model. To determine whether NanH is required for the Salmonella infection-dependent direction reversal, we constructed and characterized a nanH deletion mutant and found that NanH is partially required for Salmonella infection in primary murine macrophages. However, compared to wild type Salmonella, infection with the nanH mutant only marginally reduced the cathode-oriented macrophage galvonotaxis, without canceling direction reversal. Together, these findings strongly suggest that while neuraminidase-mediated N-glycan modification impaired both macrophage phagocytosis and galvanotaxis, yet to be defined mechanisms other than NanH may play a more important role in bioelectrical control of macrophage trafficking, which potentially triggers dissemination.

The proteomics of roadside hawk (Rupornis magnirostris), broad-snouted caiman (Caiman latirostris) and loggerhead sea turtle (Caretta caretta) tears.

BACKGROUND: Tears play an important role in ocular surface protection, and help wild animals maintain visual acuity in the face of air and water friction. The proteomics of tears has only been described for mammals. The knowledge of the proteomics of wild animal tears can aid not only in the setting of normal standards for ocular disease studies in these animals, but also to base the search for new molecules to be used in ophthalmology therapeutics. We therefore set out to describe the proteomic profile of roadside hawk (Rupornis magnirostris), broad-snouted caiman (Caiman latirostris) and loggerhead sea turtle (Caretta caretta) tears. Tears were collected from healthy animals, their spectral profiles were obtained with an LTQ Orbitrap XL mass spectrometer, and the dataset was analyzed against reference taxa. RESULTS: For roadside hawk, 446 proteins were identified, the most abundant being albumin, transferrin, globulin and actin. For broad-snouted caiman and loggerhead sea turtle, 1358 and 163 proteins were identified, respectively. Uncharacterized proteins and transferrin were highly abundant in both species. The roadside hawk tear components and their properties were similar to those described for humans, but with a higher albumin concentration. Broad-snouted caiman tears presented a wide diversity of ontological functions, with an abundant presence of enzymatic compounds. In loggerhead sea turtle tears, the predominance of proteins with ion-transport functions was consistent with possible osmolality-maintenance mechanisms. CONCLUSION: These data enhance our understanding of birds and reptiles' tears microcomposition and may be used to base the discovery of new molecules with high biotechnological potential.

Use of real-time, label-free analysis in revealing low-affinity binding to blood group antigens by Helicobacter pylori.

Infectious diseases are often initiated by microbial adherence that is mediated by the binding of attachment molecules, termed adhesins, to cell surface receptors on host cells. We present an experimental system, oblique-incidence reflectivity difference (OI-RD) microscopy, which allows the detection of novel, low-affinity microbial attachment mechanisms that may be essential for infectious processes. OI-RD microscopy was used to analyze direct binding of the oncopathogen, Helicobacter pylori ( H. pylori ) to immobilized glycoconjugates in real time with no need for labeling tags. The results suggest the presence of additional Lewis b blood group antigen (Le(b)) binding adhesins that have not been detected previously. OI-RD microscopy also confirmed the high-affinity binding of H. pylori outer-membrane protein BabA to Le(b). The OI-RD microscopy method is broadly applicable to real-time characterization of intact microbial binding to host receptors and offers new strategies to elucidate the molecular interactions of infectious agents with human host cells.

Neutral and acidic oligosaccharides in Holstein-Friesian colostrum during the first 3 days of lactation measured by high performance liquid chromatography on a microfluidic chip and time-of-flight mass spectrometry.

Oligosaccharides (OS) from bovine milk are a class of bioactive molecules that are receiving increasing commercial attention for their potential health benefits. In the present work we measured, comprehensively and systematically, free milk OS in the colostrum of 7 Holstein-Friesian cows during the first 3 d of lactation in 12-h intervals by HPLC-chip/time-of-flight mass spectrometry to determine the biological variation of free milk OS in early lactation. The high sensitivity and resolution of the analytical technique made it possible to monitor all OS species, thus providing a comprehensive and quantitative analysis of OS variations during colostrum production. This study confirmed that although sialyllactose is the major OS in bovine colostrum, several neutral OS species are present in significant abundance even at the third day of lactation. Furthermore, variation in terms of OS species and relative abundances of OS between cows suggest individual animal variation. These variations are likely due to genetic factors because environmental factors such as nutrition, lactation number, and accommodation were the same for all cows. This investigation revealed that colostrum milk from Holstein-Friesian cows is a rich source of neutral and acidic OS for the food and pharmaceutical industries.

Variations in bovine milk oligosaccharides during early and middle lactation stages analyzed by high-performance liquid chromatography-chip/mass spectrometry.

Milk oligosaccharides (OS) are not only a source of nutrition for newborns, but also provide numerous important biological functions including the prevention of pathogen binding to the intestinal epithelium and serving as nutritive sources for beneficial bacteria. High-performance mass spectrometry and separation methods were used to evaluate changes of bovine milk oligosaccharides (bMO) in different lactation stages. Previously, 40 bMO were identified in bovine milk with many consisting of short oligomeric chains that were less complex than human milk oligosaccharides (hMO). The bMO are also significantly more anionic than hMO, with nearly 70% in measured abundances containing either N-acetylneuraminic acid or N-glycolylneuraminic acid, and no fucosylated OS. In this study, we examined factors that could affect the abundances of bMO including stage of lactation and breed. The total concentrations dropped rapidly in the first several days of lactation. Moreover, the anionic oligosaccharides (including N-glycolylneuraminic acid) decreased more rapidly compared with the neutral oligosaccharides.

The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome.

Following birth, the breast-fed infant gastrointestinal tract is rapidly colonized by a microbial consortium often dominated by bifidobacteria. Accordingly, the complete genome sequence of Bifidobacterium longum subsp. infantis ATCC15697 reflects a competitive nutrient-utilization strategy targeting milk-borne molecules which lack a nutritive value to the neonate. Several chromosomal loci reflect potential adaptation to the infant host including a 43 kbp cluster encoding catabolic genes, extracellular solute binding proteins and permeases predicted to be active on milk oligosaccharides. An examination of in vivo metabolism has detected the hallmarks of milk oligosaccharide utilization via the central fermentative pathway using metabolomic and proteomic approaches. Finally, conservation of gene clusters in multiple isolates corroborates the genomic mechanism underlying milk utilization for this infant-associated phylotype.

Bovine milk glycome.

Bovine milk oligosaccharides have several potentially important biological activities including the prevention of pathogen binding to the intestinal epithelial and as nutrients for beneficial bacteria. It has been suggested that milk oligosaccharides are an important source of complex carbohydrates as supplements for the food and the pharmaceutical industries. However, only a small number of structures of bovine milk oligosaccharides (bMO) are known. There have been no systematic studies on bMO. High-performance mass spectrometry and separation methods are used to evaluate bMO, and nearly 40 oligosaccharides are present in bovine milk. Bovine milk oligosaccharides are composed of shorter oligomeric chains than are those in human milk. They are significantly more anionic with nearly 70%, measured abundances, being sialylated. Additionally, bMO are built not only on the lactose core (as are nearly all human milk oligosaccharides), but also on lactose amines. Sialic acid residues include both N-acetyl and N-glycolylneuraminic acid, although the former is significantly more abundant.

Glycomics analysis of serum: a potential new biomarker for ovarian cancer?

We recently reported the use of matrix-assisted laser desorption ionization (MALDI) Fourier transformation mass spectrometry (FTMS) techniques to identify unique glycan markers in ovarian cancer cell lines which may be biomarkers for diagnosis of ovarian cancer. Glycan markers and CA125 levels are compared in a series of ovarian cancer patients and normal control subjects. Oligosaccharides (OS) were cleaved from the serum glycoproteins and isolated using solid phase extraction. MALDI-FTMS was then used to identify unique mass spectrometry (MS) peaks. Sensitivity, specificity, and the area under the receiver operating characteristic (ROC) curve were calculated to measure the test performance of glycan markers. Sixteen unique OS MS signals were identified in ovarian cancer patient sera. Their additive mass/charge intensities were used to determine their presence or absence. The ovarian cancer patients varied in their disease status, with initial cancer stages ranging from IC to IV. Forty-four of 48 patients had detectable OS signals, with CA125 values between 2 and 17,044. Four patients had undetectable signals and their CA125 ranged between 7 and 10. Twenty-three of 24 control subjects had no detectable glycan markers, with CA125 levels between 10 and 64. Sensitivity and specificity values were determined to be 91.6% and 95.8%, respectively. The area under the ROC curve for all 72 samples was 0.954 (95% CI: 0.896, 1.0) using the glycomics assay, which was superior to CA125 in discriminating between cases and controls. This preliminary study suggests that glycomics profiling may be useful for the detection of ovarian cancer.

Bilateral, difunctional nanosphere aggregates and their assembly mediated by polymer chains.

We have developed an efficient method for producing difunctional, bilateral nanospheres. A monolayer of nanoparticles was prepared followed by deposition of a thin layer of metal. By varying the base particle and metal deposited, bilateral nanoparticles were formed. The different regions of the nanoparticles were selectively functionalized with polymer linkers containing specific terminal groups, thereby creating bilateral, difunctional nanoparticles. Subsequent covalent cross-linking of different nanoparticles enabled the formation of stable architectures with programmed hierarchy and controlled chemical composition.

Protein composition of the intranuclear inclusions of FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by premutation expansions (55-200 CGG repeats) in the fragile X mental retardation 1 (FMR1) gene. The pathologic hallmark of FXTAS is the ubiquitin-positive intranuclear inclusion found in neurons and astrocytes in broad distribution throughout the brain. The pathogenesis of FXTAS is likely to involve an RNA toxic gain-of-function mechanism, and the FMR1 mRNA has recently been identified within the inclusions. However, little is known about the proteins that mediate the abnormal cellular response to the expanded CGG repeat allele. As one approach to identify the protein mediators, we have endeavoured to define the protein complement of the inclusion itself. Fluorescence-activated flow-based methods have been developed for the efficient purification of inclusions from the post-mortem brain tissue of FXTAS patients. Mass spectrometric analysis of the entire protein complement of the isolated inclusions, combined with immunohistochemical analysis of both isolated nuclei and tissue sections, has been used to identify inclusion-associated proteins. More than 20 inclusion-associated proteins have been identified on the basis of combined immunohistochemical and mass spectrometric analysis, including a number of neurofilaments and lamin A/C. There is no dominant protein species in the inclusions, and ubiquitinated proteins represent only a minor component; thus, inclusion formation is not likely to reflect a breakdown in proteasomal degradation of nuclear proteins. The list of proteins includes at least two RNA binding proteins, heterogeneous nuclear ribonucleoprotein A2 and muscle blind-like protein 1, which are possible mediators of the RNA gain-of-function in FXTAS.

Identification and structural elucidation of lectin-binding oligosaccharides by bioaffinity matrix-assisted laser desorption/ionization Fourier transform mass spectrometry.

Cortical granule lectin (CGL) is released by the egg of the South African toad Xenopus laevis upon fertilization. The lectin binds to oligosaccharides in the extracellular matrix of the egg to form a physical block to prevent additional sperm penetration or polyspermy. To identify the oligosaccharides that bind to CGL, the lectin was immobilized on the surface of a matrix-assisted laser desorption/ionization probe. This bioaffinity probe was used to determine oligosaccharides that bind preferentially to CGL. Structural analyses based on collision-induced dissociation was used to determine that oligosaccharides with the sulfate esters at the nonreducing ends preferentially bind to the lectin.

The gas-phase chemistry of cyclodextrin inclusion complexes.

Complexes of cyclodextrins with amino acid analytes are produced in the gas phase and are ideal for the study of molecular recognition. In this Account, we discuss the evidence for the presence of gas-phase inclusion complexes and the nature of the interaction. The use of cyclodextrins as chiral selectors in gas-phase guest-exchange reactions is illustrated, and the nature of the enantioselectivity is discussed. The development of the enantioselective reaction into an analytical method for determining enantiomeric excess is also described.

Targeted use of exoglycosidase digestion for the structural elucidation of neutral O-linked oligosaccharides.

Exoglycosidase digestion in combination with the catalog-library approach (CLA) is used with matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) to obtain the complete structure of oligosaccharides. The CLA is a collision-induced dissociation (CID)-based method used to determine the structure of O-linked neutral oligosaccharides. It provides both linkage and stereochemical information. Exoglycosidases are used to confirm independently the validity of the CLA. In some cases, the CLA provides structural information on all but a single residue. Exoglycosidase is used to refine these structures. In this way, exoglycosidase use is targeted employing only a small number of enzymes. Exoglycosidase arrays, which have been used with N-linked oligosaccharides, is avoided despite the larger variations in structures of O-linked species.

Enantiomeric analysis of pharmaceutical compounds by ion/molecule reactions.

Protonated complexes involving cyclodextrin hosts and guest compounds that are pharmacologically important are produced in the gas phase and reacted with a gaseous amine. The guest is exchanged to produce a new protonated complex with the amine. The reaction is enantioselective and is used to develop a method for determining enantiomeric excess using only mass spectrometry. The pharmaceutical compounds include DOPA, amphetamine, ephedrine, and penicillamine. The presence of more than one reacting species is observed with DOPA and penicillamine. Molecular dynamics calculations are used to understand the nature of the interactions and the possible source of the variations in the reactivities.

Electrophoresis separation in open microchannels. A method for coupling electrophoresis with MALDI-MS.

The separation of biological mixtures in open micro-channels using electrophoresis with rapid and simple coupling to mass spectrometry is introduced. Rapid open-access channel electrophoresis employs microchannels that are manufactured on microchips. Separation is performed in the open channels, and the chips are transferred to a matrix-assisted laser desorption/ionization (MALDI) source after the solvent is evaporated. The matrix (2,5-dihydroxybenzoic acid) is placed in the solution with the run buffer before the separation of the analyte components. After separation, the solvent is evaporated and the microchip is ready for MALDI-MS analysis. The microchip is placed directly into a specially designed ion source of an external source Fourier transform mass spectrometry instrument. Separation of simple mixtures containing oligosaccharides and peptides is shown.

Chiral recognition in gas-phase cyclodextrin: amino acid complexes--is the three point interaction still valid in the gas phase?

The validity of the "three-point interaction" model is examined in the guest exchange reaction involving complexes of cyclodextrins and amino acids. The amino acid guest is exchanged in the gas phase in the presence of a gaseous alkyl amine. The net reaction is proton transfer between the protonated amino acid and the alkyl amine. The amino acid is lost as a neutral species. This reaction is sensitive to the chirality of the amino acid. Several amino acids are examined as well as the respective methyl esters to determine the role of the three interacting groups (ammonium, carboxylic acid, and side chain) in enantioselectivity. We find that the three-point interaction model is indeed valid in the gas phase. Enantioselectivity is optimal when two points of attraction and one repulsion is present in the gas-phase complex. The results are supported by molecular modeling calculations. A mechanism for the exchange is proposed.

MALDI-FTMS characterization of oligosaccharides labeled with 9-aminofluorene.

9-Aminofluorene (9AmFL) was investigated as an oligosaccharide label. The label was amenable to high UV detectability but did not interfere with mass spectrometric analysis. The 9AmFL label has high molar absorptivity (epsilon = 1.4 x 10(4) L cm(-1) mol(-1) at lambda = 267 nm), is chemically stable, and adds easily in reductive amination to the aldehyde terminus of oligosaccharides. Various linear and branched oligosaccharides were labeled with 9AmFL and the products were purified by chromatography on porous graphitized carbon (PGC). The derivatization reaction gave excellent yields (>95%). Up to 100-fold increase in UV sensitivity at lambda = 206 nm, compared to the corresponding alditol, was observed. Mass spectra were recorded for the labeled compounds. In the presence of sodium dopant, series of Y- and B-fragments were observed. Protonation of the labeled compounds prior to mass spectrometric analysis resulted in simplified spectra (Y-fragments only) and allowed for complete sequence analysis. The retention of the positive charge at the label in the protonated species was consistent with the basicity of the amine. The smallest amount of labeled sugar to be detected by photo-diode array (PDA) was 5 pmol (lambda = 267 nm).

Profiling with structural elucidation of the neutral and anionic O-linked oligosaccharides in the egg jelly coat of Xenopus laevis by Fourier transform mass spectrometry.

A strategic method with high speed and sensitivity is outlined for the analysis of mucin-type oligosaccharide from the jelly coat of Xenopus laevis. The method relies primarily on mass spectrometric techniques, in this case matrix-assisted laser desorption/ionization Fourier-transform mass spectrometry (MALDI-FTMS) and collision-induced dissociation (CID). Separation with isolation of the oligosaccharides was streamlined to couple well with mass spectrometry allowing the rapid determination of all detectable components from both neutral and anionic species. Partial structures of anionic components, composed primarily of sulfate esters, were obtained with CID. For neutral species, a method that allowed the complete structural determination using mass spectrometry was used. The method builds on the structure of small number of known compounds to determine unknown structures from the same biological source. In this example, a small number of oligosaccharides, elucidated previously by NMR, were used to develop a set of substructural motifs that were characterized by CID. The presence of the motifs in the CID spectra were then used to determine the structures of unknown compounds that were in abundances too small for NMR analysis.

A mass spectrometry method for the determination of enantiomeric excess in mixtures of D,L-amino acids.

A mass spectrometry method based on ion/molecule reactions for determining enantiomeric excess in mixtures of amino acids is illustrated. Protonated complexes of amino acids with beta-cyclodextrin are produced in the gas phase using electrospray ionization. Under a background pressure of N-propylamine, the amino acid is replaced by the alkylamine. The rates of the guest exchange reaction vary with the chirality of the amino acid. A calibration curve employing varying mixtures of D- and L-amino acids is produced and the fractions of the D isomer in the test mixtures are determined. The quality of the curve is compared to the chiral selectivity of the reaction. In general, the best calibration curves are obtained in systems with the greatest selectivity.

Selection of anionic dopant for quantifying desialylation reactions with MALDI-FTMS.

Alkylsulfonates are examined as anion dopants for the matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) of neutral oligosaccharides. The anion dopants allow neutral oligosaccharides to be examined in the same mixture as acidic oligosaccharides. The alkylsulfonate dopants interact strongly with the oligosaccharide to produce a quasimolecular ion composed of the oligosaccharides and the deprotonated alkylsulfonates. Compounds as small as disaccharides to large branched oligosaccharides can all be examined. The new series of alkylsulfonates works as well as HSO4- in terms of signal-to-noise ratio, sensitivity, and ease of preparation in the negative ion mode. The alkylsulfonates are used to construct a calibration curve for obtaining the relative amounts of varying neutral and acidic oligosaccharide mixtures. With the anion dopants, the MALDI-FTMS signals are shown to have a linear relationship with the molar ratios of the neutral and sialylated oligosaccharides. The method is also used to monitor the product of a desialylation reaction by acid hydrolysis.