Resolution of oligosaccharides in glycopeptides using immobilized Endo-M and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry

The resolution of asparagine-linked oligosaccharides in glycopeptides was carried out by combination of the transglycosylation reaction and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS). The resolution of the oligosaccharides is based on the enzymic transglycosylation reaction with Endo-beta-N-acetylglucosaminidase (Endo-M) isolated from Mucor hiemalis. The oligosaccharides were transferred to a fluorescent acceptor (NDA-Asn-GlcNAc) with Endo-M to produce the fluorescent oligosaccharides. In the present research, the enzyme was also immobilized in the well of a microassay plate by the sol-gel technique. The transglycosylation reaction was easily managed due to the immobilization. Furthermore, multiple use was possible by the encapsulated Endo-M. The resulting fluorescent oligosaccharides were separated by UPLC and efficiently detected by ESI-TOF-MS. Several oligosaccharides in ovalbumin were successfully identified by the proposed procedure.     

Simultaneous determination of hydrophilic amino acid enantiomers in mammalian tissues and physiological fluids applying a fully automated micro-two-dimensional high-performance liquid chromatographic concept

A validated two-dimensional HPLC system combining a microbore-monolithic ODS column and a narrowbore-enantioselective column has been established for a sensitive and simultaneous analysis of hydrophilic amino acid enantiomers (His, Asn, Ser, Gln, Arg, Asp, allo-Thr, Glu and Thr) and the non-chiral amino acid, Gly, in biological samples. To accomplish this goal, the amino acids were first tagged with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to the respective fluorescent NBD derivatives which were separated in the first dimension by a micro-reversed-phase column. The automatically collected fractions of the target peaks were then transferred to the second dimension consisting of a Pirkle type enantioselective column generating separation factors higher than 1.13 for all the enantiomeric target analytes. The system was validated using standard amino acids and a rat plasma sample, and analytically satisfactory calibration and precision results were obtained. The present 2D-HPLC system enables the fully automated determination of hydrophilic amino acid enantiomers in mammalian samples. The d-isomers of all the investigated 9 amino acids were found in rat urine but at various enantiomeric ratios.     

Resolution of N-linked oligosaccharides in glycoproteins based upon transglycosylation reaction by CE-TOF-MS

The resolution of asparagine-type oligosaccharides in glycoproteins was carried out by combination of the transglycosylation reaction and CE-TOF-MS. The oligosaccharides enzymatically transferred to a fluorescent acceptor (NDA-Asn-GlcNAc) with Endo-M. The resulting fluorescent-oligosaccharides were separated by CE and detected by TOF-MS. Disialo-Asn was successfully identified by the proposed procedure. Application to oligosaccharides in ovalbumin was also described in this communication.     

On-line hyphenation of supercritical fluid extraction and two-dimensional high performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometer for the analysis of Ganoderma lucidum

A novel on-line system combining supercritical fluid extraction (SFE) and two-dimensional high performance liquid chromatography (2D-HPLC) was developed. A trap column and two three-port valves were employed to couple SFE and 2D-HPLC system, which was composed of a CN column and a monolithic silica column, connected by a 10-port dual-position valve. The analytes extracted by supercritical CO2 were completely transferred to the 2D-HPLC system. After separation in two orthogonal modes, the eluents were delivered to APCI-tandem-MS for identification of the samples. In this way, sample preparation, separation, detection, and identification were integrated into an on-line system permitting analysis of the fruiting bodies of Ganoderma lucidum, and at least 73 components in the extract were resolved with calculated peak capacity of up to 1643.     

Novel fluorescent asparaginyl-N-acetyl-d-glucosamines (Asn-GlcNAc) for the resolution of oligosaccharides in glycopeptides, based on enzyme transglycosylation reaction

Several fluorescent asparaginyl-N-acetyl-d-glucosamines (Asn-GlcNAcs), i.e., DBD-Asn-GlcNAc, NBD-Asn-GlcNAc, NDA-Asn-GlcNAc, PS-Asn-GlcNAc, FITC-Asn-GlcNAc, DMEQ-Asn-GlcNAc and DBD-PZ-Boc-Asn-GlcNAc, were synthesized as the acceptors for the resolution of oligosaccharides in glycopeptides. The resolution of oligosaccharides is based on the transglycosylation reaction with end-β-N-acetylglucosaminidase (Endo-M), isolated from Mucor hiemalis. The synthesis of each fluorescent acceptor was carried out in a one-pot reaction of Asn-GlcNAc and the corresponding fluorescent tagging reagent. The transglycosylation reaction using Endo-M proceeds at RT in neutral phosphate buffer (pH 6.0) and reached maxima at around 30 min. When Fmoc-Asn-GlcNAc (acceptor) was enzymatically reacted with Disialo-Asn (donor) in the presence of Endo-M, the ratio of Disialo-Asn-Fmoc (transglycosylation product) was almost comparable with the decreasing ratio of Fmoc-Asn-GlcNAc. Therefore, the transglycosylation activity of Endo-M from Disialo-Asn (donor) and fluorescent-Asn-GlcNAc (acceptor) was calculated from the decreasing ratio of fluorescent-Asn-GlcNAc. The order was NDA-Asn-GlcNAc > DBD-Asn-GlcNAc Fmoc-Asn-GlcNAc > NBD-Asn-GlcNAc DMEQ-Asn-GlcNAc > DNS-Asn-GlcNAc > PS-Asn-GlcNAc > FITC-Asn-GlcNAc. On the other hand, the activity with a fluorescent acceptor (DBD-PZ-Boc-Asn-GlcNAc), labeled to a carboxylic acid group in the Asn residue, was the strongest among the synthesized acceptors.     

Development of novel active acceptors possessing a positively charged structure for the transglycosylation reaction with Endo-M and their application to oligosaccharide analysis

With Boc-Asn-GlcNAc as a basic structure, four permanently positively charged kinds of new acceptors (GP-Boc-Asn-GlcNAc, GT-Boc-Asn-GlcNAc, HMP-Boc-Asn-GlcNAc, MPDPZ-Boc-Asn-GlcNAc) and five kinds of similar structure acceptors (2-PA-Boc-Asn-GlcNAc, 3-PA-Boc-Asn-GlcNAc, 4-PA-Boc-Asn-GlcNAc, HP-Boc-Asn-GlcNAc, PDPZ-Boc-Asn-GlcNAc) were synthesized as acceptors for the resolution of oligosaccharides in glycopeptides. The synthesized acceptors enzymatically reacted with Disialo-Asn (donor) in the presence of Endo-M. The reaction yields of each transglycosylation product were not obvious, because we do not have all the authentic Disialo-Asn-Boc-acceptors. Therefore, we used the peak area of the transglycosylation product detected by mass spectrometry and evaluated the utility of each acceptor. Among the Boc-Asn-GlcNAc acceptors, the positively charged MPDPZ derivative peak area was the highest, MPDPZ-Boc-Asn-GlcNAc with a positively charged structure showed about a 2.2 times greater sensitivity of the transglycosylation product compared to the conventional fluorescence acceptor DBD-PZ-Boc-Asn-GlcNAc. As a result, the MPDPZ-Boc-Asn-GlcNAc acceptor was suitable for the transglycosylation reaction with Endo-M. The development of a qualitative determination method for the N-linked oligosaccharides in glycoproteins was attempted by combination of the transglycosylation reaction and semi-micro high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC/ESI-QTOF-MS/MS). The asparaginyl-oligosaccharides in glycoproteins, liberated by treatment with Pronase E, were separated, purified and labeled with positively charged MPDPZ. The resulting derivatives were separated by a semi-micro HPLC system. The eluted N-linked oligosaccharide derivatives were then introduced into a QTOF-MS instrument and sensitively detected in the ESI(+) mode. Various fragment ions based on the carbohydrate units appeared in the MS/MS spectra. Among the peaks, m/z 782.37 corresponding to MPDPZ-Boc-Asn-GlcNAc is the most important one for identifying the asparaginyl-oligosaccharides. Disialo-Asn-Boc-MPDPZ was easily identified by the selected-ion chromatogram at m/z 782.37 by MS/MS detection. Therefore, the identification of N-linked oligosaccharides in glycoproteins seems to be possible by the proposed semi-micro HPLC separations followed by the QTOF-MS/MS detection. Furthermore, several oligosaccharides in ovalbumin and ribonuclease B were successfully identified by the proposed procedure.     

Isolation and recovery of acidic oligosaccharides from polyacrylamide gels by semi-dry electrotransfer.

Acidic oligosaccharides derived from glycosaminoglycan heparin were separated by polyacrylamide gradient gel electrophoresis (PAGE). The gel could be visualized using Alcian Blue dye to give a pattern of highly resolved, well defined bands. The particular banding pattern obtained was the result of a heparinase catalyzed depolymerization which afforded oligosaccharide products that differed in size by one disaccharide unit. The separated oligosaccharides could be recovered prior to staining by electroelution onto a positively charged nylon membrane by a semi-dry transfer procedure. Subsequent elution and quantitative recovery of individual oligosaccharides from the membrane was achieved. By using multiple membrane layers a second separation dimension was obtained, resulting in increased oligosaccharide purity proportional to transfer depth. Preparative gradient polyacrylamide gel electrophoresis followed by semi-dry electro-transfer and recovery represents a novel method for the preparation of homogeneous acidic oligosaccharides.     

Three-dimensional mapping of N-linked oligosaccharides using anion-exchange, hydrophobic and hydrophilic interaction modes of high-performance liquid chromatography

To determine the structure of N-linked oligosaccharides, a three-dimensional (3-D) sugar mapping technique for pyridylaminated neutral and sialyl oligosaccharides is proposed. The pyridylaminated oligosaccharide mixture is first separated by HPLC on a diethylaminoethyl anion-exchange column and the elution data are placed on the Z-axis. Neutral and mono-, di-, tri- and tetrasialyloligosaccharides are then individually separated on both a hydrophobic octadecylsilylsilica column and a hydrophilic amide-silica column under the same conditions as described previously for neutral oligosaccharides. The validity of the 3-D mapping technique was tested using sialyl pyridylaminated oligosaccharides from human serum glycoproteins.     

On-line high-performance liquid chromatography/mass spectrometric characterization of native oligosaccharides from glycoproteins

An on-line high-performance liquid chromatography/mass spectrometry (HPLC/MS) method is described for the rapid characterization of any type of oligosaccharide released from glycoproteins. The procedure can be applied without further manipulation to fractions collected from a high-performance anion-exchange chromatography-pulse amperometric detection (HPAEC-PAD) system commonly used for glycosylation mapping of glycoproteins, or to a pool of oligosaccharides directly released from glycoproteins. The system consists of a porous graphitized high-performance chromatography column (Hypercarb) coupled to a quadrupole time-of-flight (TOF) mass spectrometer. Oligosaccharides are eluted from the column with a gradient of ammonium acetate/acetonitrile and directly identified following in-source fragmentation. Some applications of the method are presented, as well as information about the spectra and fragmentation behavior observed for N- and O-linked oligosaccharides released from some recombinant glycoproteins. Low femtomole limits of detection are achieved using proper miniaturization.     

Multi-dimensional mapping of pyridylamine-labeled N-linked oligosaccharides by capillary electrophoresis

A simple, sensitive and reproducible multi-dimensional capillary electrophoresis (CE) oligosaccharide mapping method is reported. The structures of 20 identified N-linked oligosaccharides have been assigned mapping positions from which co-migrating unknown oligosaccharides can be characterized. The separation protocols developed have been demonstrated to separate both charged and neutral oligosaccharides. One dimension involves electroendosmotic flow-assisted CE in a sodium acetate buffer, pH 4.0. A second dimension involves separation based on borate complexation electrophoresis in a polyethylene glycol-containing buffer. A third dimension developed specifically for neutral oligosaccharides, using a sodium phosphate buffer, pH 2.5, has been shown to resolve neutral species not able to be separated by the other two dimensions. Thus, a three-dimensional map was generated to facilitate structural characterization of these oligosaccharides.     

Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid: application to the enzymological structural analysis of oligosaccharides

Mono- and oligosaccharides, each containing a reducing end group, were labeled with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid and the resulting derivatives were separated with high resolution by polyacrylamide gel electrophoresis using a method developed recently (P. Jackson, Biochem. J. 1990, 270, 705-713) but with an alternative electrophoretic buffer system. The fluorescent derivatives of glucose and all its straight chain, alpha 1-4 linked, oligomers from maltose to maltoheptaose were well resolved. Various isomers such as maltose and lactose could be separated, as were maltose and cellobiose and some epimers, for instance glucose and galactose. The method was applied to the analysis of the partial sequential degradation of a complex oligosaccharide with neuraminidase and beta-galactosidase. Gels showing fluorescent saccharide band patterns were recorded in picomolar quantities either photographically or using an imaging system based on a cooled charge-coupled device.     

Structural characterization of oligosaccharides in recombinant soluble human interferon receptor 2 using fluorophore-assisted carbohydrate electrophoresis

The N-linked oligosaccharide profiles (banding patterns in gels) and structures of recombinant soluble human interferon receptor 2 (r-shIFNAR2) were determined using fluorophore-assisted carbohydrate electrophoresis (FACE, Glyko, Novato, CA). The method involves releasing N-linked oligosaccharide moieties from a glycoprotein by digestion with peptide-N glycanase (PNGase F), labeling the released oligosaccharides with the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separating the labeled oligosaccharides by gel electrophoresis. The isolated oligosaccharides in the bands from the profiling gels can then be sequenced using exoglycosidases to reveal the oligosaccharide structures. The oligosaccharide profile of r-shIFNAR2 consists of at least nine oligosaccharide bands. The relative amount of oligosaccharide in each band can vary, depending on the culture conditions of the source cells. FACE structural analysis shows that r-shIFNAR2 contains only core-fucosylated N-linked oligosaccharides, most of which are fully sialylated (approximately 92%). The major types and relative amounts of the oligosaccharides from a representative sample are: disialylated, galactosylated, biantennary (15%); trisialylated, galactosylated, triantennary (19%), tetrasialylated, galactosylated, tetraantennary (30%), and N-acetyllactosamine-containing higher-order oligosaccharides including tri-, tetra-, and pentaantennary (28%). The remaining oligosaccharides are not fully sialylated and/or not fully galactosylated di-, tri-, and tetraantennary structures (approximately 5%) and unidentified structures (approximately 3%). A method for determining the types and structures of the N-acetyllactosamine containing oligosaccharides is also reported in this study.     

Preparative isolation of novel antioxidant flavonoids of alfalfa by stop-and-go counter-current chromatography and following on-line liquid chromatography desalination

In this work, we have established a new stop-and-go two-dimensional chromatography coupling of counter-current chromatography and liquid chromatography (2D CCC × LC) for the preparative separation of two novel antioxidant flavonoids from the extract of alfalfa (Medicago sativa L.). The CCC column has been used as the first dimension to purify the target flavonoids using a solvent system of isopropanol and 20% sodium chloride aqueous solution (1:1, v/v) with the stop-and-go flow technique, and the LC column packed with macroporous resin has been employed as the second dimension for on-line absorption, desalination and desorption of the targeting effluents purified from the first CCC dimension. As a result, two novel flavonoids, 6,8-dihydroxy-flavone-7-O-β-D-glucuronide (15.3 mg) and 6-methoxy-8-hydroxy-flavone-7-O-β-D-glucuronide (13.7 mg), have been isolated from 126.8 mg of crude sample pre-enriched by macroporous resin column. Their structures have been identified by electrospray ionization mass spectrometry (ESI-MS), electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) and one- and two-dimensional nuclear magnetic resonance spectra (1D and 2D NMR). Further antioxidant assays showed that the first component possess a strong antioxidant activity. All the results demonstrated that the stop-and-go 2D CCC × LC method is very efficient for the separation of flavonoids of alfalfa and it can also be applied to isolate other comprehensive multi-component natural products.     

On-line liquid chromatography-electrospray ionisation mass spectrometry for kappa-carrageenan oligosaccharides with a porous graphitic carbon column

Enzymatically digested oligosaccharides of kappa-carrageenans were separated on a porous graphitic carbon (PGC) column and characterised on-line by electrospray ionisation mass spectrometry (ESI-MS). Two different developing ions were applied. Among them ammonium hydrogencarbonate showed more eluting power as it should on normal anion-exchange stationary phases. The oligosaccharides were detected by ESI-MS as fully deprotonated oligosaccharides.     

Isotope tag method for quantitative analysis of carbohydrates by liquid chromatography-mass spectrometry

We have previously demonstrated that liquid chromatography/mass spectrometry equipped with a graphitized carbon column (GCC-LC/MS) is useful for the structural analysis of carbohydrates in a glycoprotein. Here, we studied the monosaccharide composition analysis and quantitative oligosaccharide profiling by GCC-LC/MS. Monosaccharides were labeled with 2-aminopyridine and then separated and monitored by GCC-LC/MS in the selective ion mode. The use of tetradeuterium-labeled pyridylamino (d4-PA) monosaccharides as internal standards, which were prepared by the tagging of standard monosaccharides with hexadeuterium-labeled 2-aminopyridine (d6-AP), afforded a good linearity and reproducibility in ESIMS analysis. This method was successfully applied to the monosaccharide composition analysis of model glycoproteins, fetuin, and erythropoietin. For quantitative oligosaccharide profiling, oligosaccharides released from an analyte and a standard glycoprotein were tagged with d0- and d6-AP, respectively, and an equal amount of d0- and d4-PA oligosaccharides were coinjected into GCC-LC/MS. In this procedure, the oligosaccharides that existed in either analyte or a standard glycoprotein appeared as single ions, and the oligosaccharides that existed in both analyte and a standard glycoprotein were detected as paired ions. The relative amount of analyte oligosaccharides could be determined on the basis of the analyte/internal standard ion-pair intensity ratio. The quantitative oligosaccharide profiling enabled us to make a quantitative and qualitative comparison of glycosylation between the analyte and standard glycoproteins. The isotope tag method can be applicable for quality control and comparability assessment of glycoprotein products as well as the analysis of glycan alteration in some diseases.     

Microscale analysis of mucin-type O-glycans by a coordinated fluorophore-assisted carbohydrate electrophoresis and mass spectrometry approach

The total glycan moiety was released in a single step from native glycoproteins by a nonreductive beta-elimination procedure. The generated oligosaccharides were further derivatized either with the hydrophobic fluorophore 2-aminoacridone (AMAC) or the charged 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) fluorophore, and the resulting fluorescent derivatives were separated according to their hydrodynamic size or charge with high-resolution gel electrophoresis. Both N- and O-glycans released by this beta-elimination procedure might be analyzed simultaneously. AMAC derivatization allows a rapid separation of neutral and charged oligosaccharides without prior fractionation. Derivatized oligosaccharide species were then eluted from the gel slices and analyzed by mass spectrometry. This methodology allowed the rapid structural characterization of each glycan in term of monosaccharide composition and sequence. Using this technique we were able to screen several heterogeneous O-glycan mixtures isolated at the picomolar range from reference glycoproteins or mucins.     

Analysis of 8-aminonaphthalene-1,3,6-trisulfonate-derivatized oligosaccharides by capillary electrophoresis-electrospray ionization quadrupole ion trap mass spectrometry.

Dextran was partially hydrolyzed with 0.1 mol/l HCl and the hydrolysate was derivatized with 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) by reductive amination. The derivatized-oligosaccharide mixture was separated by capillary electrophoresis (CE) in a buffer of 1% HAc-NH4OH, pH 3.4, and the separated components were detected on-line by electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT-MS) in the negative ion mode. A mass accuracy lower than 0.01% could be achieved and as low as 1.6 pmol of detxran octaose could be detected. ANTS-derivatized dextran oligosaccharide with a degree of polymerization (DP) lower than 6 produced both [M-H]- and [M-2H]2- ions, whereas those with a DP of 6 or higher than 6 produced only [M-2H]2- ion. As 1< or =DP< or =6, the percentage of [M-2H]2- ion in the total ions of [M-H]- and [M-2H]2- was found to be a linear function of the logarithmic DP. Molecular mass determination with ESI-QIT-MS strengthens the power of CE analysis of oligosaccharides.     

Improved determination of milk oligosaccharides using a single derivatization with anthranilic acid and separation by reversed-phase high-performance liquid chromatography

An improved analytical scheme for human milk neutral oligosaccharides determination was developed, in which, the oligosaccharides were pooled in two fractions (pools 1 and 2) after gel filtration, and then were quantitatively derivatized with a single fluorescent reagent, 2-anthranilic acid. Separation was by reversed-phase HPLC on an ODS-100Z column with a mobile phase of 50 mM ammonium acetate pH 4.0 and 150 mM citrate buffer pH 4.5 and monitored by a fluorescence detector at 360 nm excitation and 425 nm emission wavelengths. The method improved on the separation of neutral tetra- and hexa-saccharide isomers, namely, lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) as well as of lacto-N-difucohexaose I (LNDFH I) and lacto-N-difucohexaose II (LNDFH II). The separation of trisacccharide isomers, 3-fucosyllactose (3-FL) and 2′-fucosyllactose (2′-FL) was also successful. Limits of detection and quantification were in the range of 1–10 ng/l and 2–30 ng/l, respectively. The methods’ accuracy was good with its precision at <20% RSD and <1% RSD, respectively, for oligosaccharide concentration and retention time. The recoveries were in the range of 80–100%. This method was successfully applied to the separation and determination of representative neutral oligosaccharide contents in Samoa women milk.     

Determination of neutral oligosaccharide fractions from human milk by gel permeation chromatography.

A gel permeation chromatographic method for quantifying neutral oligosaccharide fractions from human milk has been developed. Oligosaccharides from monofucosyllactoses to trifucosyllacto-N-hexaoses were separated according to size on a Fractogel TSK HW 40 (S) column. Refractive index detection of monofucosyllactoses to difucosyllacto-N-tetraoses yielded a constant mass response factor of ca. 1 relative to glucose. After the addition of glucose as an internal standard, oligosaccharides were isolated from human milk by ethanol precipitation or two ultrafiltration procedures. The oligosaccharide concentrations found by the ultrafiltration procedures were significantly lower (significance level 0.05) than those determined by the ethanol precipitation procedure.     

Miniaturized on-line proteolysis-capillary liquid chromatography-mass spectrometry for peptide mapping of lactate dehydrogenase

In this study, methodology was developed for on-line and miniaturized enzymatic digestion with liquid chromatographic (LC) separation and mass spectrometric (MS) detection. A packed capillary LC-MS system was combined with on-line trypsin cleavage of a model protein, lactate dehydrogenase, to provide an efficient system for peptide mapping. The protein was injected onto an enzymatic capillary reactor and the resulting peptides were efficiently trapped on a capillary trapping column. Different trapping columns were evaluated to achieve a high binding capacity for the peptides generated in the enzyme reactor. The peptides were further eluted from the pre-column and separated on an analytical capillary column by a buffer more suitable for the following an electrospray ionisation (ESI) MS process. An important aspect of the on-line approach was the desalting of peptides performed in the trapping column to avoid detrimental signal suppression in the ESI process. The developed on-line system was finally compared to a classical digestion in solution, with reference to peptide sequence coverage and sensitivity. It was shown that the on-line system gave more than 100% higher peptide sequence coverage than traditional digestion methods.