Analysis of High-Molecular-Weight Polyrotaxanes by MALDI-TOF-MS Using 3-Aminoquinoline-Based Ionic Liquid Matrix.

Polyrotaxane (PR) is a necklace-like supramolecule composed of cyclic components, such as cyclodextrin (CD), and a threading polymer capped with bulky end groups. PR exhibits peculiar mechanical properties attributed to the intermolecular cross-links with CD. Various CD molecules threaded on a linear PEG chain are often modified with chemical groups to add specific physicochemical properties. In general, the stoichiometry between CD and the PEG chain is a significant parameter that defines the unique physical properties of CD-based polyrotaxane (CD-PR). To date, mass spectrometry (MS) has been applied to investigate the molecular distribution of CD-PR, modifications of CD, and the threaded ratio of CD. However, only molecular weights (MWs) up to several 10s of kDa can be subjected to such analysis, whereas the MW of CD-PR used as industrial materials is much greater. Herein, we applied two ionic liquid matrices composed of 3-aminoquinoline and a high mass detector to analyze PRs using MALDI-TOF-MS. High to very high MW PRs in the range of 90-700 kDa were successfully analyzed using this method. The threaded ratio of CD was estimated from a single MW of CD, PEG, and PR. The ratios obtained were consistent with that obtained using 1H NMR. Furthermore, a single-stranded form of PR in γ-cyclodextrin threaded PR (γCD-PR) was clearly distinguished from a double-stranded form, which is only possible in γCD -PR because of its large host cavity.

Comparison of Japanese and Indian intestinal microbiota shows diet-dependent interaction between bacteria and fungi.

The bacterial species living in the gut mediate many aspects of biological processes such as nutrition and activation of adaptive immunity. In addition, commensal fungi residing in the intestine also influence host health. Although the interaction of bacterium and fungus has been shown, its precise mechanism during colonization of the human intestine remains largely unknown. Here, we show interaction between bacterial and fungal species for utilization of dietary components driving their efficient growth in the intestine. Next generation sequencing of fecal samples from Japanese and Indian adults revealed differential patterns of bacterial and fungal composition. In particular, Indians, who consume more plant polysaccharides than Japanese, harbored increased numbers of Prevotella and Candida. Candida spp. showed strong growth responses to the plant polysaccharide arabinoxylan in vitro. Furthermore, the culture supernatants of Candida spp. grown with arabinoxylan promoted rapid proliferation of Prevotella copri. Arabinose was identified as a potential growth-inducing factor in the Candida culture supernatants. Candida spp. exhibited a growth response to xylose, but not to arabinose, whereas P. copri proliferated in response to both xylose and arabinose. Candida spp., but not P. copri, colonized the intestine of germ-free mice. However, P. copri successfully colonized mouse intestine already harboring Candida. These findings demonstrate a proof of concept that fungal members of gut microbiota can facilitate a colonization of the intestine by their bacterial counterparts, potentially mediated by a dietary metabolite.

Mass spectrometric identification of citrullination sites and immunohistochemical detection of citrullinated glial fibrillary acidic protein in Alzheimer's disease brains.

Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that convert protein arginine to citrulline residues in a calcium ion-dependent manner. Previously, we reported the abnormal accumulation of citrullinated proteins and the increase in the amount of PAD2 in hippocampi from Alzheimer's disease (AD) patients. Moreover, glial fibrillary acidic protein (GFAP), an astrocyte-specific marker protein, and vimentin were identified as citrullinated proteins by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. To clarify the substrate specificity of PADs against GFAP, we prepared recombinant human (rh)PAD1, rhPAD2, rhPAD3, rhPAD4, and rhGFAP. After incubation of rhGFAP with rhPAD1, rhPAD2, rhPAD3, and rhPAD4, citrullinated (cit-)rhGFAP was detected by Western blotting. The citrullination of rhGFAP by rhPAD2 was unique, specific, and time dependent; additionally, rhPAD1 slightly citrullinated rhGFAP. We then generated eight anti-cit-rhGFAP monoclonal antibodies, CTGF-125, -128, -129, -1212, -1213, -1221, -122R, and -1224R, which reacted specifically with cit-rhGFAP. Two of those eight monoclonal antibodies, CTGF-122R and -1224R, reacted with both cit-rhGFAP and rhGFAP in Western blots. By using the CTGF-1221 antibody and a tandem mass spectrometer, we identified the two independent citrullination sites (R270Cit and R416Cit) of cit-rhGFAP. Immunohistochemical analysis with CTGF-1221 antibody revealed cit-GFAP staining in the hippocampus of AD brain, and the cit-GFAP-positive cells appeared to be astrocyte-like cells. These collective results strongly suggest that PAD2 is responsible for the citrullination of GFAP in the progression of AD and that the monoclonal antibody CTGF-1221, reacting with cit-GFAP at R270Cit and R416Cit, is useful for immunohistochemical investigation of AD brains.

A Cytotoxic Antibody Recognizing Lacto-N-fucopentaose I (LNFP I) on Human Induced Pluripotent Stem (hiPS) Cells.

We have generated a mouse monoclonal antibody (R-17F, IgG1 subtype) specific to human induced pluripotent stem (hiPS)/embryonic stem (ES) cells by using a hiPS cell line as an antigen. Triple-color confocal immunostaining images of hiPS cells with R-17F indicated that the R-17F epitope was expressed exclusively and intensively on the cell membranes of hiPS cells and co-localized partially with those of SSEA-4 and SSEA-3. Lines of evidence suggested that the predominant part of the R-17F epitope was a glycolipid. Upon TLC blot of total lipid extracts from hiPS cells with R-17F, one major R-17F-positive band was observed at a slow migration position close to that of anti-blood group H1(O) antigen. MALDI-TOF-MS and MS(n) analyses of the purified antigen indicated that the presumptive structure of the R-17F antigen was Fuc-Hex-HexNAc-Hex-Hex-Cer. Glycan microarray analysis involving 13 different synthetic oligosaccharides indicated that R-17F bound selectively to LNFP I (Fucα1-2Galß1-3GlcNAcß1-3Galß1-4Glc). A critical role of the terminal Fucα1-2 residue was confirmed by the selective disappearance of R-17F binding to the purified antigen upon α1-2 fucosidase digestion. Most interestingly, R-17F, when added to hiPS/ES cell suspensions, exhibited potent dose-dependent cytotoxicity. The cytotoxic effect was augmented markedly upon the addition of the secondary antibody (goat anti-mouse IgG1 antibody). R-17F may be beneficial for safer regenerative medicine by eliminating residual undifferentiated hiPS cells in hiPS-derived regenerative tissues, which are considered to be a strong risk factor for carcinogenesis.

The multiplicity of N-glycan structures of bovine milk 18 kda lactophorin (milk GlyCAM-1).

Lactophorin is a heat-stable phosphoglycoprotein, also known as milk glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1). Bovine 18 kDa lactophorin was purified by heparin affinity chromatography from cow's milk whey. Its N-glycans were obtained by proteomic techniques, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by in-gel digestion with peptide-N(4)-(N-acetyl-beta-glucosaminyl)-asparagine amidase (PNGase F). The released N-glycans were derivatized with 2-aminopryridine (PA) and analyzed by matrix-assisted laser desorption ionization quadruple ion trap time of flight mass spectrometry (MALDI-QIT-TOF MS). Among the MS analyzed peaks, 15 peaks were found to be N-glycan molecules as detected by MS(2) analysis. These glycans consisted of mono-sialylated bi-, tri-, and tetra-antennary complex-type N-glycans carrying Gal-GlcNAc (LacNAc) or GalNAc-GlcNAc (LacdiNAc) with and without core-fucose.

Comparative studies on the structural features of O-glycans between leukemia and epithelial cell lines.

Recently, we developed an automated apparatus for rapid releasing of O-glycans from mucin-type glycoproteins and proteoglycans ( Anal. Biochem. 2007 , 362 , 245 - 251 ; 2007 , 371 , 52 - 61 ). In the present paper, we released O-glycans from some leukemia and epithelial cells using the apparatus, and compared the profiles of O-glycans among these cells after fluorescent labeling of the released glycans with 2-aminobenzoic acid. The fluorescent labeled glycans were analyzed using a combination of HPLC and off-line MALDI-(QIT)TOF mass spectrometry We found that leukemia cells generally showed simple glycan profiles and commonly contained sialyl-T (NeuAcalpha2-3Galbeta1-3GalNAc) and disialyl-T (NeuAcalpha2-3Galbeta1-3(NeuAcalpha2-6)GalNAc) antigens as major O-glycans. In contrast, epithelial cancer cell lines usually showed extremely complex profiles. We found that polylactosamine-type O-glycans were abundantly present in MKN45 cells. Especially, we found characteristic glycans, of which Galbeta1-3 residue of core1 structure is modified with biantennary polylactosamine units. In contrast, this cell line did not contain polylactosamine-type N-glycans ( J. Proteome Res. 2006 , 5 , 88 - 97 ). These results suggest that the different biosynthetic pathways for N- and O-glycans are proposed. The method presented here will accelerate the speed for comprehensive analysis of O-glycans in biological samples and will be a powerful tool for clinical/biochemical analysis in cancer biology.

Ionic liquid matrixes optimized for MALDI-MS of sulfated/sialylated/neutral oligosaccharides and glycopeptides.

1,1,3,3-tetramethylguanidium (TMG) salt of alpha-cyano-4-hydroxycinnamic acid (CHCA) (G(2)CHCA) was reported by Tatiana et al. as a useful ionic liquid matrix (ILM) for sulfated oligosaccharides to suppress the loss of sulfate groups. However, the report mainly referred to positive ion spectra only and amounts of 10 pmol or more of the analyte were used. Herein, we demonstrated highly sensitive detection of sulfated/sialylated/neutral oligosaccharides and preferential ionization of glycopeptides by optimizing a newly synthesized ILM: TMG salt of p-coumaric acid (G(3)CA) and the existing G(2)CHCA in both positive and negative ion extraction modes. Sulfated oligosaccharides were detected with high sensitivity (e.g., 1 fmol) in both ion extraction modes, and the dissociation of sulfate groups was suppressed especially using G(3)CA. Sialylated and neutral oligosaccharides were also detected with high sensitivity (e.g., 1 fmol) with positive ion extraction while the dissociation of sialic acids was suppressed especially using G(3)CA. Additionally, glycopeptide ions were detected preferentially using the ILMs among the digest of a glycoprotein, ribonuclease B, in both ion extraction modes but particularly in the negative ion mode. As a result, the use of optimized ILMs provides an effective method for carbohydrate analysis due to the highly sensitive soft-ionization achieved in both ion extraction modes as well as the homogeneity of analyte-matrix mixtures.

Direct on-membrane glycoproteomic approach using MALDI-TOF mass spectrometry coupled with microdispensing of multiple enzymes.

We report a novel approach for direct on-membrane glycoproteomics by digestion of membrane-blotted glycoproteins with multiple enzymes using piezoelectric chemical inkjet printing technology and on-membrane direct MALDI-TOF mass spectrometry. With this approach, both N-linked glycan analyses and peptide mass fingerprinting of several standard glycoproteins were successfully performed using PNGase F and trypsin microscale digestions of the blotted spots on membrane from an SDS-PAGE gel. In addition, we performed a similar analysis for 2-DE separated serum glycoproteins as a demonstration of how the system could be used in human plasma glycoproteomics.

Strategy for simulation of CID spectra of N-linked oligosaccharides toward glycomics.

To develop a novel glycomics tool that can enable anyone to identify oligosaccharides very easily and quickly, we have recently constructed a library of observed multistage tandem mass (MS(n)) spectra for oligosaccharides. However, this approach requires the preparation of a large variety of structurally defined oligosaccharides. Therefore, simulation of the tandem mass spectrum for any given structure would be another powerful approach with which to improve the above method. By performing collision-induced dissociation (CID) experiments of sets of oligosaccharides complementarily labeled with (13)C(6)-D-galactose, we identified characteristic fragment patterns for each branch type of N-linked oligosaccharides. On the basis of these characteristic fragment patterns, we could simulate CID spectra for three isomeric oligosaccharides. In addition, we successfully demonstrated the identification of an oligosaccharide by matching its CID spectrum against the library of simulated tandem mass spectra. This strategy will be a useful tool for glycomics, as well as for approaches based on the library of observed MS(n) spectra.

Cell-cell interaction-dependent regulation of N-acetylglucosaminyltransferase III and the bisected N-glycans in GE11 epithelial cells. Involvement of E-cadherin-mediated cell adhesion.

Changes in oligosaccharide structures are associated with numerous physiological and pathological events. In this study, the effects of cell-cell interactions on N-linked oligosaccharides (N-glycans) were investigated in GE11 epithelial cells. N-glycans were purified from whole cell lysates by hydrazinolysis and then detected by high performance liquid chromatography and mass spectrometry. Interestingly, the population of the bisecting GlcNAc-containing N-glycans, the formation of which is catalyzed by N-acetylglucosaminyltransferase III (GnT-III), was substantially increased in cells cultured under dense conditions compared with those cultured under sparse conditions. The expression levels and activities of GnT-III but not other glycosyltransferases, such as GnT-V and alpha1,6-fucosyltransferase, were also consistently increased in these cells. However, this was not observed in mouse embryonic fibroblasts or MDA-MB231 cells, in which E-cadherin is deficient. In contrast, perturbation of E-cadherin-mediated adhesion by treatment with EDTA or a neutralizing anti-E-cadherin antibody abolished the up-regulation of expression of GnT-III. Furthermore, we observed the significant increase in GnT-III activity under dense growth conditions after restoration of the expression of E-cadherin in MDA-MB231 cells. Our data together indicate that a E-cadherin-dependent pathway plays a critical role in regulation of GnT-III expression. Given the importance of GnT-III and the dynamic regulation of cell-cell interaction during tissue development and homeostasis, the changes in GnT-III expression presumably contribute to intracellular signaling transduction during such processes.

A strategy for identification of oligosaccharide structures using observational multistage mass spectral library.

Glycosylation is the most widespread posttranslational modification in eukaryotes; however, the role of oligosaccharides attached to proteins has been little studied because of the lack of a sensitive and easy analytical method for oligosaccharide structures. Recently, tandem mass spectrometric techniques have been revealing that oligosaccharides might have characteristic signal intensity profiles. We describe here a strategy for the rapid and accurate identification of the oligosaccharide structures on glycoproteins using only mass spectrometry. It is based on a comparison of the signal intensity profiles of multistage tandem mass (MSn) spectra between the analyte and a library of observational mass spectra acquired from structurally defined oligosaccharides prepared using glycosyltransferases. To smartly identify the oligosaccharides released from biological materials, a computer suggests which ion among the fragment ions in the MS/MS spectrum should yield the most informative MS3 spectrum to distinguish similar oligosaccharides. Using this strategy, we were able to identify the structure of N-linked oligosaccharides in immunoglobulin G as an example.

The carbohydrate sequence markup language (CabosML): an XML description of carbohydrate structures.

UNLABELLED: Bioinformatics resources for glycomics are very poor as compared with those for genomics and proteomics. The complexity of carbohydrate sequences makes it difficult to define a common language to represent them, and the development of bioinformatics tools for glycomics has not progressed. In this study, we developed a carbohydrate sequence markup language (CabosML), an XML description of carbohydrate structures. AVAILABILITY: The language definition (XML Schema) and an experimental database of carbohydrate structures using an XML database management system are available at http://www.phoenix.hydra.mki.co.jp/CabosDemo.html CONTACT: kikuchi@hydra.mki.co.jp.