PubMed Health⌕ Search

Biomedical subjects

Kisaburo Deguchi

Publications and source records attributed to Kisaburo Deguchi.

At least 19 recordsLinked to original sources
PubMed2007

Structural assignment of disialylated biantennary N-glycan isomers derivatized with 2-aminopyridine using negative-ion multistage tandem mass spectral matching.

To investigate the possibility of structural assignment based on negative-ion multistage tandem mass (MS(n)) spectral matching, four isomers of disialylated biantennary N-glycans (alpha2-6 and/or alpha2-3 linked sialic acid on alpha1-6 and alpha1-3 antennae) derivatized with 2-aminopyridine (PA) were analyzed by employing high-performance liquid chromatography/electrospray ionization linear ion trap time-of-flight mass spectrometry (HPLC/ESI-LIT-TOFMS), which uses helium gas for ion trapping and collision-induced dissociation (CID). It is shown that the MS(2) spectra derived from each precursor ion [M-2H](2-) are reproducible and useful for distinguishing the four isomers. Thus, they can be assigned by negative-ion MS(2) spectral matching based on correlation coefficients. In addition, MS(3) spectra derived from D-type fragment ions clearly differentiate the alpha2-3- or alpha2-6-linked sialic acid on the alpha1-6 antenna due to their characteristic spectral patterns. The C(4)-type fragment ions, which are produced from both the alpha1-6 and alpha1-3 antennae, show the characteristic MS(3) spectra reflecting alpha2-3- or alpha2-6- linkage type or a mixture of both types. Thus, the differentiation and assignment of these disialylated biantennary N-glycan isomers can also be supported with the MS(3) spectra of C(4)- and D-type ions.

Aminopyridines↗
PubMed2006

N-linked neutral oligosaccharides in the stratum corneum of normal and ichthyotic skin.

N-Glycan oligosaccharides are thought to play multiple, important roles in a variety of biological events. However, N-glycan profiles in the stratum corneum of human skin have not yet been studied in detail. To clarify the N-glycan profiles in the stratum corneum of normal and ichthyotic epidermis, N-glycan profiles were studied by high-performance liquid chromatography using normal human epidermal samples and scales from hyperkeratotic skin of ichthyosis patients. Chromatograms of patient scale samples showed unique alterations in three peaks eluted at 15.8, 18.8 and 26.9 min. The N-glycan profiles were significantly altered in ichthyotic hyperkeratotic skin compared with normal non-hyperkeratotic controls. These findings indicate the reduction of N-acetylglucosaminyltransferase II and fucosyltransferase 8 activities. Alteration of N-glycan structures in hyperkeratotic skin suggests the biological role of N-glycans in keratinization.

Chromatography, High Pressure Liquid↗
PubMed2006

Separation of isomeric 2-aminopyridine derivatized N-glycans and N-glycopeptides of human serum immunoglobulin G by using a zwitterionic type of hydrophilic-interaction chromatography.

Isomeric oligosaccharides and isomeric glycopeptides are sometimes difficult to separate on normal-phase (NP) and reversed-phase (RP) columns. A zwitterionic type of hydrophilic-interaction chromatography column with sulfobetaine groups (called ZIC-HILIC column) was first applied to the separation of 2-aminopyridine derivatized (PA) N-glycans and tryptic peptides of human serum immunoglobulin G (IgG). It is shown that the ZIC-HILIC column has high capability for structural recognition of isomeric N-glycans as well as high selectivity for glycopeptides. The former feature (i.e., structural recognition) was proven by sufficient separation of neutral PA N-glycan isomers, which are usually difficult to separate on NP and RP columns. In addition, it is noteworthy that IgG glycopeptides consisting of isomeric N-glycans and the same peptide sequences can be sufficiently separated on a ZIC-HILIC column. The latter feature (i.e., selectivity) was also demonstrated by easily separating two peptide groups with/without N-glycans. Thus, we note that the ZIC-HILIC column is highly promising for a simple analysis of N-glycans and N-glycopeptide samples.

Aminopyridines↗
PubMed2006

Simple separation of isomeric sialylated N-glycopeptides by a zwitterionic type of hydrophilic interaction chromatography.

Asparagine-linked oligosaccharides (N-glycans) usually show structural heterogeneity, especially in proteins with sialylated N-glycans and, therefore, their structural analysis is still very difficult. A zwitterionic type of hydrophilic interaction chromatography column with sulfobetaine functional groups (called a ZIC-HILIC column) was applied to the separation of tryptic peptides of alpha-1-acid glycoprotein. It was demonstrated that the ZIC-HILIC separation column has a selectivity for sialylated N-glycopeptides and a high capability for separation based on the structural recognition of sialylated N-glycan isomers as well as for the previously reported neutral N-glycans and N-glycopeptides. The retention characteristics of neutral and sialylated N-glycans derivatized with 2-aminopyridine (PA N-glycans) demonstrate that the retentions of the N-glycans are based primarily on hydrophilic interaction with the water-rich liquid layer generated on the surface of the ZIC-HILIC column. In addition, the electrostatic repulsion interaction shielded with counter ions effectively tunes the separation and recognition of sialylated N-glycan isomers.

Aminopyridines↗
PubMed2006

Structural assignment of isomeric 2-aminopyridine-derivatized monosialylated biantennary N-linked oligosaccharides using negative-ion multistage tandem mass spectral matching.

To investigate the possibility of structural assignment based on negative-ion tandem multistage (MSn) mass spectral matching, four isomers of 2-aminopyridine (PA)-derivatized monosialylated oligosaccharides (i.e., complex-type N-glycans with an alpha2-3- or alpha2-6-linked sialic acid on alpha1-6 or alpha1-3 antennae) were analyzed using high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry (HPLC/ESI-IT-TOFMS). The negative ion [M-2H]2- is observed predominantly in the MS1 spectra without the loss of a sialic acid. The MS2 spectra derived from it are sufficiently reproducible that MS2 spectral matching based on correlation coefficients can be applied to the assignment of these isomers. The isomers containing a sialic acid on alpha1-6 or alpha1-3 antennae can be distinguished by MS2 spectral matching, but the alpha2-3 and alpha2-6 linkage types of sialic acid cannot be distinguished by their MS2 spectra. However, MS3 spectra derived from fragment ions containing a sialic acid (i.e., C4- and D-type ions) clearly differentiate the alpha2-3 and alpha2-6 linkage types of sialic acid in their MS3 spectral patterns. This difference might be rationalized in terms of a proton transfer from the reducing-end mannose to the negatively charged sialic acid. These two moieties are very close in the structural conformations of the precursor C4-type fragment ions of alpha2-6 linkage type, as predicted by molecular mechanics calculations. Thus, negative-ion MSn (n = 2, 3) spectral matching was demonstrated to be useful for the structural assignment of these four monosialylated PA N-glycan isomers.

Aminopyridines↗
PubMed2006

Complementary structural information of positive- and negative-ion MSn spectra of glycopeptides with neutral and sialylated N-glycans.

Positive- and negative-ion MSn spectra of chicken egg yolk glycopeptides binding a neutral and a sialylated N-glycan were acquired by using electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) and collision-induced dissociation (CID) with helium as collision gas. Several characteristic differences were observed between the positive- and negative-ion CID MSn (n = 2, 3) spectra. In the positive-ion MS2 spectra, the peptide moiety was presumably stable, but the neutral N-glycan moiety caused several B-type fragmentations and the sialylated N-glycan almost lost sialic acid(s). In contrast, in the negative-ion MS2 spectra, the peptide moiety caused several side-chain and N-glycan residue (e.g., N-acetylglucosamine (GlcNAc) residue) fragmentations in addition to backbone cleavages, but the N-glycan moieties were relatively stable. The positive-ion MS3 spectra derived from the protonated peptide ion containing a GlcNAc residue (203.1 Da) provided enough information to determine the peptide amino-acid sequence including the glycosylation site, while the negative-ion MS3 spectra derived from the deprotonated peptide containing a 0,2X1-type cross-ring cleavage (83.1 Da) complicated the peptide sequence analysis due to side-chain and 0,2X1 residue related fragmentations. However, for the structural information of the N-glycan moiety of the glycopeptides, the negative-ion CID MS3 spectra derived from the deprotonated 2,4A6-type cross-ring cleavage ion (neutral N-glycan) or the doubly deprotonated B6-type fragment ion (sialylated N-glycan) are more informative than are those of the corresponding positive-ion CID MS3 spectra. Thus, the positive-ion mode of CID is useful for the analyses of peptide amino-acid sequences including the glycosylation site. The negative-ion mode of CID is especially useful for sialylated N-glycan structural analysis. Therefore, in the structural analysis of N-glycopeptides, their roles are complementary.

Animals↗
PubMed2006

Direct structural assignment of neutral and sialylated N-glycans of glycopeptides using collision-induced dissociation MSn spectral matching.

Mass spectrometric analyses of various N-glycans binding to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on collision-induced dissociation (CID) MS(n) spectra acquired by electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) in the positive- and negative-ion modes has been proposed as a direct method of assigning N-glycans without releasing them from N-glycopeptides. In the positive-ion mode of this approach, the MS(2) spectrum of N-glycopeptide was acquired so that a glycoside-bond cleavage occurs in the chitobiose residue (i.e., GlcNAcbeta1-4GlcNAc, GlcNAc: N-acetylglucosamine) attached to asparagine (N), and two charges on the [M+H+Na](2+) precursor ion are shared with both of the resulting fragments. These fragments are sodiated B(n)-type fragment ions of oligosaccharide (N-glycan) and a protonated peptide ion retaining one GlcNAc residue on the asparagine (N) residue. The structure of N-glycan was assigned by comparing MS(3) spectra derived from both the sodiated B(n)-type fragment ions of N-glycopeptide and the PA (2-aminopyridine) N-glycan standard (i.e., MS(n) spectral matching). In a similar manner, the structural assignment of sialylated N-glycan was performed by employing the negative-ion CID MS(n) spectra of deprotonated B(n)-type fragment ions of N-glycopeptide and the PA N-glycan standard. The efficacy of this approach was tested with chicken egg yolk glycopeptides with a neutral and a sialylated N-glycan, and human serum IgG glycopeptides with neutral N-glycan isomers. These results suggest that the approach based on MS(n) spectral matching is useful for the direct and simple structural assignment of neutral and sialylated N-glycans of glycopeptides.

Algorithms↗
PubMed2006

Structural analysis of sialyl N-glycan using pyridylamination and chromatography followed by multistage tandem mass spectrometry.

Multiple-dimensional mapping (n-DM) methods consisting of pyridylamination and high-performance liquid chromatography (HPLC) are widely used for oligosaccharide analysis. These methods are quantitative, sensitive, and suitable for separating isomers. Oligosaccharide structures are suggested by elution positions on two or three kinds of columns and then confirmed by enzymatic and chemical treatments or mass spectrometry (MS). Multiple-stage tandem MS (MS(n)) analyses have been used to determine oligosaccharide structures by spectrum matching, comparing standard oligosaccharides, and offering detailed MS(n) fragment analysis. However, oligosaccharides usually exist as a mixture including isomers. The 3-DM method provides quantitative information and well-isolated sialyl- or neutral-oligosaccharides; on the other hand, the MS(n) method saves time by eliminating complicated enzyme degradation steps. The combination of both methods, which support each other, represents a reasonable and efficient strategy. This chapter describes 3-DM on HPLC after negative-ion MS(n) spectral matching for sialyl N-glycans.

Animals↗
PubMed2005

Capillary high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry using a novel nanoflow gradient generator.

A type of high-performance liquid chromatography (HPLC) based on a novel nanoflow gradient generator (Asymptotic-Trace-10-Port-Valve (AT10PV) nanoGR generator) was developed and coupled with an electrospray ion trap time-of-flight mass spectrometer (ESI-IT-TOF MS). Stability of the nanoflow GR HPLC system was tested at flow rates of 20 and 50 nL/min by using a nanoflow meter. Average flow rates in a 2-h run were 51.2 nL/min with RSD 0.7% and 21.0 nL/min with RSD 1.8%. Repeatability of analysis of the nanoHPLC/ESI-IT-TOF MS system was also tested by injecting 1.0 microL of trypsin digested bovine serum albumin (BSA) (100 fmol) into a monolithic silica-ODS column (30 microm i.d., 150 mm in length) through a packed silica-ODS trapping column (particle size 5 microm, 150 microm i.d., 10 mm in length). At a flow rate of 50 nL/min, the result demonstrated a reasonably good repeatability of peak retention times (RSD: 0.32-1.1%) and base-ion peak areas (RSD: 4.4-6.6%).

Chromatography, High Pressure Liquid↗
PubMed2005

High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis.

Despite recent advances in our understanding of the significance of the protein glycosylation, the throughput of protein glycosylation analysis is still too low to be applied to the exhaustive glycoproteomic analysis. Aiming to elucidate the N-glycosylation of murine epidermis and dermis glycoproteins, here we used a novel approach for focused proteomics. A gross N-glycan profiling (glycomics) of epidermis and dermis was first elucidated both qualitatively and quantitatively upon N-glycan derivatization with novel, stable isotope-coded derivatization reagents followed by MALDI-TOF(/TOF) analysis. This analysis revealed distinct features of the N-glycosylation profile of epidermis and dermis for the first time. A high abundance of high mannose type oligosaccharides was found to be characteristic of murine epidermis glycoproteins. Based on this observation, we performed high mannose type glycoform-focused proteomics by direct tryptic digestion of protein mixtures and affinity enrichment. We identified 15 glycoproteins with 19 N-glycosylation sites that carry high mannose type glycans by off-line LC-MALDI-TOF/TOF mass spectrometry. Moreover the relative quantity of microheterogeneity of different glycoforms present at each N-glycan binding site was determined. Glycoproteins identified were often contained in lysosomes (e.g. cathepsin L and gamma-glutamyl hydrolase), lamellar granules (e.g. glucosylceramidase and cathepsin D), and desmosomes (e.g. desmocollin 1, desmocollin 3, and desmoglein). Lamellar granules are organelles found in the terminally differentiating cells of keratinizing epithelia, and desmosomes are intercellular junctions in vertebrate epithelial cells, thus indicating that N-glycosylation of tissue-specific glycoproteins may contribute to increase the relative proportion of high mannose glycans. The striking roles of lysosomal enzymes in epidermis during lipid remodeling and desquamation may also reflect the observed high abundance of high mannose glycans.

Amino Acid Sequence↗
PubMed2005

Structural analysis of an N-glycan with "beta1-4 bisecting branch" from human serum IgG by negative-ion MSn spectral matching and exoglycosidase digestion.

A novel N-linked oligosaccharide (N-glycan) with "beta1-4 bisecting branch (galactose beta1-4 bisecting N-acetylglucosamine)" was found in human serum IgG. Its structure was efficiently analyzed by using beta-galactosidase digestion, a MSn spectral library database, and negative-ion MS2 spectral matching. For confirmation, the novel N-glycan was synthesized by using an expected standard N-glycan (acceptor), UDP-galactose (donor), and beta1-4 galactosyltransferase. This work also demonstrates that the MSn spectral library database, in particular, negative-ion MS2 spectral matching, can efficiently reduce the number of specific, sequential exoglycosidase digestions required and is useful for rapid structural analysis of unknown glycans not in the database.

Animals↗
PubMed2005

Versatile glycoblotting nanoparticles for high-throughput protein glycomics.

We have developed an effective and practical trap-and-release method based on chemoselective ligation of carbohydrates with reactive aminooxyl groups attached to the surface of nanoparticles (referred to as glycoblotting nanoparticles). These glycoblotting nanoparticles were synthesized by UV irradiation of diacetylene-functionalized lipids that contain the aminooxyl group. The glycoblotting nanoparticles captured carbohydrates in aqueous solution under mild conditions and were collected by simple centrifugation. The trapped carbohydrates were effectively released from the nanoparticles under acidic conditions to give pure oligosaccharides. This glycoblotting process reduced the time required for the purification process of carbohydrates to less than 6 h, compared to the several days needed for conventional chromatographic techniques. The oligosaccharides (N-glycan) were released from ovalbumin (glycoprotein) by PNGase F after tryptic digestion. MALDI-TOF mass spectra before purification did not show any significant signals corresponding to N-glycans because these signals were hidden by the large signals of the abundant peptides. However, after purification with the glycoblotting nanoparticles, only signals corresponding to oligosaccharides appeared. We also demonstrated a clear analysis of the oligosaccharides contained in the mice dermis by means of glycoblotting.

Animals↗
PubMed2005

Simultaneous analysis of 2-aminopyridine-derivatized neutral and sialylated oligosaccharides from human serum in the negative-ion mode by sonic spray ionization ion trap mass spectrometry.

Neutral and acidic (sialylated) 2-aminopyridine-derivatized (PA) oligosaccharides were analyzed by using reversed-phase high-performance liquid chromatography/ion trap mass spectrometry (RP-HPLC/IT MS) with a sonic spray ionization (SSI) source. Under the RP-HPLC separation using a buffer of 1 mM ammonium acetate (pH4.3) at a flow rate of 0.2 mL/min, both PA-oligosaccharides in the negative-ion mode showed a comparable degree of ionization efficiency, differing from that of the positive-ion mode, which exhibits a wide gap between their ionization efficiencies. In addition, the ion intensities of both PA-oligosaccharides were higher in the negative-ion mode than in the positive-ion mode. These results strongly suggest that the negative-ion mode of SSI-MS is suitable for simultaneous analysis of neutral and acidic (sialylated) oligosaccharides in RP-HPLC/MS. In the present study, RP-HPLC/SSI-IT MS in the negative-ion mode was used in the analysis of PA-oligosaccharides from human serum and its usefulness was investigated. As a result, 32 neutral and sialylated PA-oligosaccharides from human serum were identified with differentiating isomeric oligosaccharides and relatively quantified by a single HPLC/MS run. This method is useful for simple and rapid analysis of the overall distribution of neutral and sialylated oligosaccharides in a complex sample such as serum.

Aminopyridines↗
PubMed2005

Structural assignment of isomeric 2-aminopyridine-derivatized oligosaccharides using negative-ion MSn spectral matching.

To investigate the possibility of structural assignment based on negative-ion MS2 spectral matching, three isomeric pairs of 2-aminopyridine (PA)-derivatized non-fucosylated, fucosylated, and sialylated oligosaccharides (complex type N-glycans) were analyzed using high-performance liquid chromatography/ion trap mass spectrometry (HPLC/ITMS) with a sonic-spray ionization (SSI) source. In the SSI negative-ion mode the deprotonated molecule [M-2H]2- becomes prominent. Negative-ion MS2 spectra derived from such ions contain many fragment types (B and Y, C and Z, A, and D) and therefore are more informative than the positive-ion MS2 spectra derived from [M+H+Na]2+ ions, which usually consist mainly of B and Y fragment ions. In particular the internal ions (D- and E-type ions) provided useful information about the alpha1-6 branching patterns and the bisecting GlcNAc residue. Spectral matching based on the correlation coefficients between negative-ion MS2 spectra was performed in a manner similar to the positive-ion MS2 spectral matching previously reported. It was demonstrated that negative-ion MS2 spectral matching is as useful and applicable to the structural assignment of relatively large non-fucosylated, fucosylated, and sialylated PA-oligosaccharide isomers as its positive-ion counterpart.

Aminopyridines↗
PubMed2005

Characteristic pH and electrolyte concentration dependences of 2-aminopyridine-derivatized oligosaccharides (N-glycans) in sonic-spray ionization mass spectrometry.

The intensities of ion signals from neutral oligosaccharides (N-glycans) derivatized with 2-aminopyridine (PA) were analyzed by ion trap mass spectrometry with a sonic-spray ionization (SSI) source, in both positive- and negative-ion modes, while varying the pH and concentration of ammonium acetate buffer solution. Two characteristic results are reported and discussed. The first characteristic is the pH dependence of the ion intensities; on increasing the solution pH from 4.3 to 8.6, positive ion intensities increase and negative ion intensities decrease. The second characteristic concerns the dependence of ion intensities on electrolyte concentration; on increasing the electrolyte concentration, the SSI efficiency for the PA N-glycans first increases and then decreases. Assuming that the SSI mechanism essentially conforms to the statistical charging model and the charge residue model, a new model that focuses a great deal of attention on the counter (electrolyte) ion distribution surrounding the solvated analyte (PA N-glycan) is proposed, in particular to rationalize the characteristic pH dependence.

Aminopyridines↗
PubMed2004

Assignment and quantification of 2-aminopyridine derivatized oligosaccharide isomers coeluted on reversed-phase HPLC/MS by MSn spectral library.

2-Aminopyridine (PA)-derivatized oligosaccharides from IgG were analyzed by using reversed-phase HPLC/mass spectrometry (RP-HPLC/MS) and a MS(n) spectral library, in particular, focusing on two pairs of isomers incompletely separated or coeluted in chromatograms. We previously reported that MS(n) spectral matching considering both major fragment ions (m/z) and intensities is useful and applicable to the structural assignment of PA-oligosaccharide isomers. In this study, MS(n) spectral matching based on the MS(n) spectral library was applied to the assignment of these PA-oligosaccharide isomers in IgG. Its usefulness was investigated by comparing it to the conventional two-dimensional mapping method based on retention time indexes. Specifically, we focus on the assignment and quantification of the isomers, which are coeluted in chromatograms. From this, we propose a new method using MS(n) spectral matching and the working curve on which are plotted the relative intensities of selected fragment ions in their MS(2) spectra versus various mixtures of the isomers. This new method demonstrated that the obtained quantities coincide very well with those estimated after separating by a combination of lectin and reversed-phase columns. This means that separation by RP-HPLC/MS is greatly simplified because complete separation of the isomers is no longer required. Application of this new method was tested by using the two other pairs of fucosylated and nonfucosylated PA-oligosaccharides from IgG. The results showed that this method works for them as well.

Aminopyridines↗
PubMed2004

Nanoflow gradient generator for capillary high-performance liquid chromatography-nanoelectrospray mass spectrometry.

A novel nanoflow gradient generator using a 10-port switching valve with two injection loops installed, which is referred to here as the "Asymptotic-Trace-10-Port-Valve" (AT10PV) nanoGR generator, has been applied to capillary high-performance liquid chromatography (HPLC)-microelectrospray (microESI) or nanoelectrospray (nanoESI) time-of-flight mass spectrometry (TOF MS). In this study, performance of this capillary HPLC-micro/nanoESI-MS system was tested at a flow rate of 200 nl/min by using three typical peptides (angiotensins I, II, and III: 50 fmol each). The result demonstrated that this system provides reasonably good repeatability of peak retention times (R. S.D. of less than 0.5%). Sequential runs of a series of sample injections were performed in the same manner as conventional analysis at microflow rates.

Chromatography, High Pressure Liquid↗