Combination of SDS-PAGE and intact mass analysis for rapid determination of heterogeneities in monoclonal antibody therapeutics.

mAbs are currently mainstream in biopharmaceuticals, and their market has been growing due to their high target specificity. Characterization of heterogeneities in mAbs is performed to secure their quality and safety by physicochemical analyses. However, they require time-consuming task, which often strain the resources of drug development in pharmaceuticals. Rapid and direct method to determine the heterogeneities should be a powerful tool for pharmaceutical analysis. Considering the advantages of electrophoresis and MS, this study addresses the combination of SDS-PAGE and intact mass analysis, which provides direct, rapid, and orthogonal determination of heterogeneities in mAb therapeutics. mAb therapeutics that migrated in SDS-PAGE were recovered from gel by treatment with SDC-containing buffer. Usage of SDC-containing buffer as extraction solvent and ethanol-based staining solution enhanced the recovery of intact IgG from SDS-PAGE gels. Recovery of mAbs reached more than 86% with 0.2% SD. The heterogeneities, especially N-glycan variants in the recovered mAb therapeutics, were clearly determined by intact mass analysis. We believe that the study is important in pharmaceuticals‧ perspective since orthogonal combination of gel electrophoresis and intact mass analysis should be pivotal role for rapid and precise characterization of mAbs.

Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines : HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.

Expression of the clustered NeuAcα2-3Galß O-glycan determines the cell differentiation state of the cells.

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of >239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAcα2-3Galß O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1-60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1-60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.

Common glycoproteins expressing polylactosamine-type glycans on matched patient primary and metastatic melanoma cells show different glycan profiles.

Recently, we reported comparative analysis of glycoproteins which express cancer-specific N-glycans on various cancer cells and identified 24 glycoproteins having polylactosamine (polyLacNAc)-type N-glycans that are abundantly present in malignant cells [ Mitsui et al., J. Pharm. Biomed. Anal. 2012 , 70 , 718 - 726 ]. In the present study, we applied the technique to comparative studies on common glycoproteins present in the matched patient primary and metastatic melanoma cell lines. Metastatic melanoma cells (WM266-4) contained a large amount of polyLacNAc-type N-glycans in comparison with primary melanoma cells (WM115). To identify the glycoproteins expressing these N-glycans, glycopeptides having polyLacNAc-type N-glycans were captured by a Datura stramonium agglutinin (DSA)-immobilized agarose column. The captured glycopeptides were analyzed by LC/MS after removing N-glycans, and some glycoproteins such as basigin, lysosome-associated membrane protein-1 (LAMP-1), and chondroitin sulfate proteoglycan 4 (CSPG4) were identified in both WM115 and WM266-4 cells. The expression level of polyLacNAc of CSPG4 in WM266-4 cells was significantly higher than that in WM115 cells. In addition, sulfation patterns of chondroitin sulfate (CS) chains in CSPG4 showed dramatic changes between these cell lines. These data show that characteristic glycans attached to common proteins observed in different stages of cancer cells will be useful markers for determining degree of malignancies of tumor cells.

Analysis of O-glycans as 9-fluorenylmethyl derivatives and its application to the studies on glycan array.

A method is proposed for the analysis of O-glycans as 9-fluorenylmethyl (Fmoc) derivatives. After releasing the O-glycans from the protein backbone in the presence of ammonia-based media, the glycosylamines thus formed are conveniently labeled with Fmoc-Cl and analyzed by HPLC and MALDI-TOF MS after easy purification. Fmoc labeled O-glycans showed 3.5 times higher sensitivities than those labeled with 2-aminobenzoic acid in fluorescent detection. Various types of O-glycans having sialic acids, fucose, and/or sulfate residues were successfully labeled with Fmoc and analyzed by HPLC and MALDI-TOF MS. The method was applied to the comprehensive analysis of O-glycans expressed on MKN45 cells (human gastric adenocarcinoma). In addition, Fmoc-derivatized O-glycans were easily converted to free hemiacetal or glycosylamine-form glycans that are available for fabrication of glycan array and neoglycoproteins. To demonstrate the availability of our methods, we fabricate the glycan array with Fmoc labeled glycans derived from mucin samples and cancer cells. The model studies using the glycan array showed clear interactions between immobilized glycans and some lectins.

Structural characterization of pyridylaminated oligosaccharides derived from neutral glycosphingolipids by high-sensitivity capillary electrophoresis-mass spectrometry.

High-sensitivity capillary electrophoresis-electrospray ionization quadrupole ion trap time-of-flight mass spectrometry (CE-ESI-QIT-TOF MS) was developed to structurally characterize four kinds of pyridylaminated (PA) oligosaccharides, i.e., lactose (Lac)-PA, globotriose (Gb3)-PA, globotetraose (Gb4)-PA, and IV(3) αGalNAc-Gb4 (Forssman antigen)-PA, derived from neutral glycosphingolipids. The CE-MS system included the head-column field-amplified sample stacking (HC-FASS) method for effective sample injection into a capillary column in CE, a sheathless interface between CE and a mass spectrometer, and MS and tandem MS (MS(2)) measurements with narrow mass range repeated high-speed switching. The total sensitivity of the developed CE-MS system was about 20,000 times higher than that of the conventional CE-MS system consisting of pressure injection, a sheath-flow interface, and a wide mass range measurement. The MS and MS(2) spectra of the four PA-oligosaccharides at a concentration of 25 amol/µL in mixtures (each 250 amol/10 µL in a tube) clearly showed protonated molecular ions ([M + H](+)) and the fragment ions responsible for the sequential elimination of saccharides. The developed CE-MS system is a powerful method for the structural characterization of glycosphingolipids extracted from very small amounts of biological materials and could be extended to the structural characterization of oligosaccharides derived from glycoproteins.

A novel condition for capillary electrophoretic analysis of reductively aminated saccharides without removal of excess reagents.

We have identified novel CE conditions for the separation of 7-amino-4-methylcoumarin-labeled monosaccharides and oligosaccharides from glycoproteins. Using a neutrally coated capillary and alkaline borate buffer containing hydroxypropylcellulose and ACN, saccharide derivatives form anionic borate complexes, which move from the cathode to the anode in an electric field and are detected near the anodic end. Excess labeling reagents and other fluorescent products remain at the cathodic end. Fluorimetric detection using an LED as a light source enables determination of monosaccharide derivatives with good linearity between at least 0.4 and 400 µM, may correspond to 140 amol to 140 fmol. The lower LOD (S/N = 5) is only 80 nM in the sample solution (ca. 28 amol). The results were comparable to reported values using fluorometric detection LC. The method was also applied to the analysis of oligosaccharides that were enzymatically released from glycoproteins. Fine resolution enables profiling of glycans in glycoproteins. The applicability of the method was examined by applying it to other derivatives labeled with nonacidic tags such as ethyl p-aminobenzoate- and 2-aminoacridone-labeled saccharides.

Structural characterization of neutral glycosphingolipids using high-performance liquid chromatography-electrospray ionization mass spectrometry with a repeated high-speed polarity and MSn switching system.

Four types of neutral glycosphingolipids (LacCer, Gb3Cer, Gb4Cer, and IV3αGalNAc-Gb4Cer; 10 pmol each) were analyzed using high-performance liquid chromatography (HPLC)-electrospray ionization quadrupole ion trap time-of-flight (ESI-QIT-TOF) mass spectrometry (MS) with a repeated high-speed polarity and MSn switching system. This system can provide six types of mass spectra, including positive and negative ion MS, MS2, and MS3 spectra, within 1 s per cycle. Using HPLC with a normal-phase column, information on the molecular weights of major molecular species of four neutral glycosphingolipids was obtained by detecting [M+Na]+ in the positive ion mode mass spectra and [M−H]− in the negative ion mode mass spectra. Sequences of glycosphingolipid oligosaccharide were obtained in the negative ion MS2 spectra. In addition, information on the ceramide structures was clearly obtained in the negative ion MS3 mass spectra. GlcCer molecular species were analyzed by HPLC-ESI-QIT-TOF MS with a reversed-phase column using 1 pmole of GlcCer. The structures of the seven molecular species of GlcCer, namely, d18:1-C16:0, d18:1-C18:0, d18:1-C20:0, d18:1-C22:0, d18:1-C23:0, d18:1-C24:1, and d18:1-C24:0, were characterized using positive ion MS and negative ion MS, MS2, and MS3. The established HPLC-ESI-QIT-TOF MS with MSn switching and a normal phase column has been successfully applied to the structural characterization of LacCer and Gb4Cer in a crude mixture prepared from human erythrocytes.

Analysis of nonhuman N-glycans as the minor constituents in recombinant monoclonal antibody pharmaceuticals.

Minor N-linked glycans containing N-glycolylneuraminic acid residues and/or α-Gal epitopes (i.e., galactose-α1,3-galactose residues) have been reported to be present in recombinant monoclonal antibody (mAb) therapeutics. These contaminations are due to their production processes using nonhuman mammalian cell lines in culture media containing animal-derived materials. In case of the treatment of tumors, we inevitably use such mAbs by careful risk-benefit considerations to prolong patients' lives. However, expanding their clinical applications such as for rheumatism, asthma, and analgesia demands more careful evaluation of the product characteristics. The present work for detailed evaluations of N-glycans demonstrates the methods using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and a combination of high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry. The CE-LIF method provides excellent separation of both major and minor N-glycans from six commercial mAb pharmaceuticals within 30 min and clearly indicates that a possible trigger of immunogenicity in humans due to the presence of nonhuman N-glycans is present. We strongly believe that the proposed method will be a powerful tool for the analysis of N-glycans of recombinant mAb products in various development stages, such as clone selection, process control, and routine release testing to ensure safety and efficacy of the products.

Specific detection of N-glycolylneuraminic acid and Galα1-3Gal epitopes of therapeutic antibodies by partial-filling capillary electrophoresis.

The oligosaccharide structure is very important in biopharmaceuticals because of its effects on protein function, including efficacy and half-life. N-glycolylneuraminic acid (Neu5Gc) and Galα1-3Gal (α-Gal) residues are known to show immunogenicity in humans. It is now understood that murine cell lines, such as NS0 or SP2, which are typically used for biopharmaceutical manufacture, produce proteins containing Neu5Gc and α-Gal residues. The expression of these specific residues is affected by the cell line and culture conditions. Therefore, monitoring and controlling the levels of these epitopes are important for the quality control of biopharmaceuticals. To detect the two epitopes on a therapeutic antibody produced by NS0 cells, we applied partial-filling capillary electrophoresis using anti-Neu5Gc antibody and α-galactosidase. In the anti-Neu5Gc antibody filling method, one minor glycan peak with Neu5Gc residues at the nonreducing end disappeared specifically from the electropherogram. In the α-galactosidase filling method, some minor peaks with α1,3-linked Gal residues disappeared. However, in a therapeutic antibody from Chinese hamster ovary cells, no peaks disappeared with the two methods. These results show this method can be used to specifically detect and quantify the two epitopes on biopharmaceuticals with high sensitivity.

One-pot characterization of cancer cells by the analysis of mucin-type glycans and glycosaminoglycans.

We developed an automated apparatus for rapid releasing of O-glycans from mucin-type glycoproteins [Anal. Biochem. 371 (2007) 52-61; Anal. Chem. 82 (2010) 7436-7443] and applied the device to analyze them in some cancer cell lines [J. Proteome Res. 8 (2009) 521-537]. We also found that the device is useful to release glycosaminoglycans from proteoglycans [Anal. Biochem. 362 (2007) 245-251]. Based on these studies, we developed a method for one-pot analysis of mucin-type glycans and glycosaminoglycans after releasing them from total protein pool obtained from some cancer cell lines. Mucin-type glycans were analyzed by a combination of high-performance liquid chromatography and mass spectrometry techniques, and glycosaminoglycans were analyzed by capillary electrophoresis as fluorescent-labeled unsaturated disaccharides after digestion with specific eliminases followed by fluorescent labeling. Ten cancer cell lines, including blood cancer cells as well as epithelial cancer cells, were used to assess the method. The results clearly revealed that both mucin-type glycans and glycosaminoglycans showed quite interesting profiles. Thus, the current technique will be a powerful tool for discovery of glycan markers of diseases.

Structural characterization of monosialo-, disialo- and trisialo-gangliosides by negative ion AP-MALDI-QIT-TOF mass spectrometry with MS(n) switching.

The atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) is a quite convenient soft ionization for biomolecules, keeping analytes atmospheric conditions instead of high vacuum conditions. In this study, an AP-MALDI ion source has been coupled to a quadrupole ion trap time-of-flight (QIT-TOF) mass spectrometer, which is able to perform MS(n) analysis. We applied this system to the structural characterization of monosialogangliosides, GM1 (NeuAc) and GM2 (NeuAc), disialogangliosides, GD2 (NeuAc, NeuAc), GD1a (NeuAc, NeuAc) and GD1b (NeuAc, NeuAc) and trisialoganglioside GT1a (NeuAc, NeuAc, NeuAc). In this system, the negative ion mass spectra of MS, MS(2) and MS(3), a set of three mass spectra, were able to measure within 2 s per cycle. Thus, obtained results demonstrate that the negative ion mode MS, MS(2) and MS(3) spectra provided sufficient information for the determination of molecular weights, oligosaccharide sequences and ceramide structures, and indicate that the AP-MALDI-QIT-TOF mass spectrometry keeping analytes atmospheric conditions with MS(n) switching is quite useful and convenient for structural analyses of various types of sialic acid-containing GSLs, gangliosides.

Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

Application of partial-filling capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in a therapeutic antibody.

Oligosaccharides in therapeutic recombinant antibodies play important roles in regulation of various biological functions. To monitor the glycosylation profiles of antibody pharmaceuticals in the manufacturing process, a highly sensitive and specific method is required. We extended partial-filling techniques using lectins and exoglycosidases in capillary electrophoresis for the characterization of 8-aminopylene-1,3,6-trisulfonic acid labeled N-linked oligosaccharides derived from the therapeutic antibody rituximab. In the lectin-filling method, Galb1­4GlcNAc-specific Erythrina cristagali agglutinin, a1, 6-linked Fuc-specific Aleuria aurantia lectin and Neu5Aca2­3Gal-specific Maackia amurensis lectin were used. The oligosaccharides migrated through the lectin plug during separation; the changes in separation profiles were observed according to the interaction with the lectins. The glycosidase-filling method allowed rapid digestion as suggested by the electropherograms. Partial-filling CE methods can avoid tedious hands-on procedures such as overnight incubation and optimization reaction condition with lectins and exoglycosidases. Combination of these partial-filling capillary electrophoresis methods makes the characterization of oligosaccharide profiles of therapeutic antibodies easier and faster.

A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals.

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix. Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.

Determination of sulfate ester content in sulfated oligo- and poly-saccharides by capillary electrophoresis with indirect UV detection.

Carbohydrates having sulfate groups such as glycosaminoglycans and chemically synthesized sucrose sulfate show interesting and important biological activities. We adapted CE with indirect UV detection technique to the determination of sulfate ester in sulfated carbohydrates, which were previously hydrolyzed with HCl. The liberated sulfate ion was analyzed using a background electrolyte consisting of triethanolamine-buffered chromate with hexamethonium bromide. Sulfate contents of glucose 3-sulfate and sucrose octasulfate used as a model were in good agreement with theoretical values (accuracy, 95.9-96.7 and 97.4-101.9%, respectively), and relative standard deviation values run-to-run were 0.977 and 1.90%, respectively. We applied the method to the determination of the sulfate contents of some glycosaminoglycan samples and showed that the contents were in good agreement with those calculated from sulfur content.

Hyphenated technique for releasing and MALDI MS analysis of O-glycans in mucin-type glycoprotein samples.

We developed an automatic apparatus for the release of O-glycans from mucin-type glycoproteins and proteoglycans (Matsuno, Y.-k.; Yamada, K.; Tanabe, A.; Kinoshita, M.; Maruyama, S.-z.; Osaka, Y.-s.; Masuko, T.; Kakehi, K. Anal. Biochem. 2007, 363, 245-257. Yamada, K.; Hyodo, S.; Matsuno, Y. K.; Kinoshita, M.; Maruyama, S. Z.; Osaka, Y. S.; Casal, E.; Lee, Y. C.; Kakehi, K. Anal. Biochem. 2007, 371, 52-61). The method allows rapid release of O-glycans as the reducing form within 10 min. In the present study, we connected the device to a MALDI-TOF MS spotter and achieved routine analysis of O-glycans in biological samples for clinical use after in situ derivatization of the released O-glycans with phenylhydrazine. We applied the method to the analysis of O-glycans expressed on MKN45 cells derived from human stomach cancer cells and found that MKN45 cells expressed characteristic trisialo-polylactosamine-type glycans as reported previously (Yamada, K.; Kinoshita, M.; Hayakawa, T.; Nakaya, S.; Kakehi, K. J. Proteome Res. 2009, 8, 521-537). We also applied the method to the analysis of O-glycans in serum samples. The present technique is the first attempt to use MS measurement for routine clinical diagnostic works.

Definitive evidence that a single N-glycan among three glycans on inducible costimulator is required for proper protein trafficking and ligand binding.

Glycosylation is a widespread post-translational modification found in glycoproteins. Glycans play key roles in protein folding, quality control in the endoplasmic reticulum (ER) and protein trafficking within cells. However, it remains unclear whether all positions of protein glycosylation are involved in glycan functions, or if specific positions have individual roles. Here we demonstrate the integral involvement of a specific N-glycan from amongst the three glycans present on inducible costimulator (ICOS), a T-cell costimulatory molecule, in proper protein folding and intracellular trafficking to the cell surface membrane. We found that glycosylation-defective mutant proteins lacking N-glycan at amino-acid position 89 (N89), but not proteins lacking either N23 or N110, were retained within the cell and were not detected on the cell surface membrane. Additional evidence suggested that N89 glycosylation was indirectly involved in ICOS ligand binding. These data suggest that amongst the three putative ICOS glycosylation sites, N89 is required for proper ICOS protein folding in the ER, intracellular trafficking and ligand binding activity. This study represents a substantial contribution to the current mechanistic understanding of the necessity and potential functions of a specific N-glycan among the multiple glycans of glycoproteins.

Determination of Tn antigen released from cultured cancer cells by capillary electrophoresis.

An incomplete elongation of O-glycans in mucins has been found in epithelial cancers, leading to the expression of shorter carbohydrate structures such as Tn antigen (GalNAc-O-Ser/Thr), which has been reported to be one of the most specific human cancer-associated structures. However, there have been no appropriate physicochemical methods for the determination of Tn antigen in biological samples. In the present paper, we developed a capillary electrophoresis method for the determination of Tn antigen, and applied the method to the analysis of the expressed Tn antigen on some leukemia and epithelial cancer cells.

Capillary electrophoresis-electrospray ionization mass spectrometry for rapid and sensitive N-glycan analysis of glycoproteins as 9-fluorenylmethyl derivatives.

It is well known that most protein therapeutics such as monoclonal antibody pharmaceuticals and other biopharmaceuticals including cancer biomarkers are glycoproteins, and thus the development of high-throughput and sensitive analytical methods for glycans is essential in terms of their determination and quality control. We previously reported a novel alternative labeling method for glycans involving 9-fluorenylmethyl chloroformate (Fmoc-Cl) instead of the conventional reductive amination procedure. The derivatives were analyzed by high-performance liquid chromatography (HPLC) (Kamoda S, Nakano M, Ishikawa R, Suzuki S, Kakehi K. 2005. Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: A method which can recover free oligosaccharides after analysis. J Proteome Res. 4:146-152). This method was rapid and simple; however, it was time-consuming in terms of analysis by HPLC and did not provide so much information such as the detailed structures and mass numbers of glycans. Here we have developed a high-throughput and highly sensitive method. It comprises three steps, i.e., release of glycans, derivatization with Fmoc, and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI MS) analysis. We analyzed several glycoproteins such as fetuin, alpha1 acid glycoprotein, IgG, and transferrin in order to validate this method. We were able to analyze the above glycoproteins with the three-step procedure within only 5 h, which provided detailed N-glycan patterns. Moreover, the MS/MS analysis allowed identification of the N-glycan structures. As novel applications, the method was employed for the analysis of N-glycans derived from monoclonal antibody pharmaceuticals and also from alpha-fetoprotein; the latter is known as one of the tumor markers of hepatocellular carcinomas. We were able to easily and rapidly determine the detailed structures of the N-glycans. The present method is very useful for the analysis of large numbers of samples such as a routine analysis.