BACKGROUND: N-Glycosylation is one of the most important post-translational modifications in proteins. As the N-glycan profiles in biological samples are diverse and change according to the pathological condition, various profiling methods have been developed, such as liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry. However, conventional analytical methods have limitations in sensitivity and/or resolution, hindering the discovery of minor but specific N-glycans that are important both in the basic glycobiology research and in the medical application as biomarkers. Therefore, a highly sensitive and high-resolution N-glycan profiling method is required. RESULTS: In this study, we developed a novel two-dimensional (2D) separation system, which couples hydrophilic interaction liquid chromatography (HILIC) with capillary gel electrophoresis (CGE) via large-volume dual preconcentration by isotachophoresis and stacking (LDIS). Owing to the efficient preconcentration efficiency of LDIS, limit of detection reached 12 pM (60 amol, S/N = 3) with good calibration curve linearity (R2 > 0.999) in the 2D analysis of maltoheptaose. Finally, 2D profiling of N-glycans obtained from standard glycoproteins and cell lysates were demonstrated. High-resolution 2D profiles were successfully obtained by data alignment using triple internal standards. N-glycans were well distributed on the HILIC/CGE 2D plane based on the glycan size, number of sialic acids, linkage type, and so on. As a result, specific minor glycans were successfully identified in HepG2 and HeLa cell lysates. SIGNIFICANCE AND NOVELTY: In conclusion, the HILIC/CGE 2D analysis method showed sufficient sensitivity and resolution for identifying minor but specific N-glycans from complicated cellular samples, indicating the potential as a next-generation N-glycomics tool. Our novel approach for coupling LC and CE can also dramatically improve the sensitivity in other separation modes, which can be a new standard of 2D bioanalysis applicable not only to glycans, but also to other diverse biomolecules such as metabolites, proteins, and nucleic acids.

Efficient enzymatic digestion methods are critical for the characterization and identification of glycans. Glycan hydrolysis enzymes are widely utilized for the identification of glycoprotein or glycolipid glycans. The commonly utilized in solution glycan hydrolysis methods require several hours of incubation with enzymes for complete removal of their target monosaccharides. To develop an efficient and simple method for the rapid release of monosaccharides from glycoprotein glycans, we fabricated exoglycosidase-impregnated acrylamide gels in an automatic pipette tip. Our automated enzymatic reactors are based on the simple photochemical copolymerization of monomers comprising acrylamide and methylene-bis-acrylamide-containing enzymes with an azobis compound functioning as the photocatalytic initiator. After filling the tip of the automatic pipette with these acrylamide solutions, polymerization of the acrylamide gel solution was performed by irradiation with a LED. The immobilized enzymes maintained their activities in the pipette tips and their action was completed by fully automatic pipetting for 10 to 30 min. We utilized 8-aminopyrene-1, 3, 6-trisulfonic acid (APTS)-labeled glycans as a substrate and measured by capillary electrophoresis (CE) before and after enzymatic digestion. We demonstrated that this method exhibited quantitative enzymatic and specific cleavage of monosaccharides from glycoprotein glycans.

An improved method for the online preconcentration, derivatization, and separation of phosphorylated compounds was developed based on the affinity of a Phos-tag acrylamide gel formed at the intersection of a polydimethylsiloxane/glass multichannel microfluidic chip toward these compounds. The acrylamide solution comprised Phos-tag acrylamide, acrylamide, and N,N-methylene-bis-acrylamide, while 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] was used as a photocatalytic initiator. The Phos-tag acrylamide gel was formed around the channel crossing point via irradiation with a 365 nm LED laser. The phosphorylated peptides were specifically concentrated in the Phos-tag acrylamide gel by applying a voltage across the gel plug. After entrapment of the phosphorylated compounds in the Phos-tag acrylamide gel, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was introduced to the gel for online derivatization of the concentrated phosphorylated compounds. The online derivatized DTAF-labeled phosphorylated compounds were eluted by delivering a complex of phosphate ions and ethylenediamine tetraacetic acid as the separation buffer. This method enabled sensitive analysis of the phosphorylated peptides.

N-Glycosylation of therapeutic antibodies is a critical quality attribute (CQA), and the micro-heterogeneity affects the biological and physicochemical properties of antibodies. Therefore, the profiling of N-glycans on antibodies is essential for controlling the manufacturing process and ensuring the efficacy and safety of the therapeutic antibodies. To monitor N-glycosylation in recombinant proteins, a high-throughput (HTP) methodology for glycan analysis is required to handle bulk samples in various stages of the manufacturing process. In this study, we focused on the HTP methodology for N-glycan analysis using a commercial microchip electrophoresis-based DNA analyzer and demonstrated the feasibility of the workflow consisting of sample preparation and electrophoretic separation. Even if there is a demand to analyze up to 96 samples, the present workflow can be completed in a day without expensive instruments and reagent kits for sample preparation, and it will be a promising methodology for cost-effective and facile HTP N-glycosylation analysis while optimizing the manufacturing process and development for therapeutic antibodies.

Glycoanalytical technology is required for a wide variety of scientific research, including basic glycobiological pharmaceutical, and biomarker research. Although several innovative analytical techniques have been developed for these purposes, quantitative glycan analysis based on electrophoretic separation, has often been impeded by the lack of cost-effective and facile sample preparation approaches. Here, we developed a rapid and facile sample preparation workflow for cost-effective glycan analysis and demonstrated its use with fully automated microchip electrophoresis (ME). Purification of 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans was based on the combination of ion-pair assisted extraction (IPAE) with hydrophilic interaction chromatography-solid phase extraction (HILIC-SPE). Compared to commonly used sample preparation methods, the IPAE/HILIC-SPE method undergoes minimal nonspecific loss and undesirable degradation of N-glycans during the purification step. Furthermore, our method required only 10 min, and the entire workflow, including glycan release, labeling, and concentration processes was completed within 4 h. Although the present system should be improved to enable analysis of more complex mixtures, ME-based separation of APTS-labeled N-glycans offers a fully automated operation including conditioning, sample loading, separation, and can be analyzed with a sample-to-sample throughput of 120 s in parallel processes. The present workflow is easy to implement, does not require expensive reagents and instruments and may be useful for glycoscientists across disciplines.

Analysis of glycans in glycoproteins is often performed by liquid chromatography (LC) separation coupled with fluorescence detection and/or mass spectrometric detection. Enzymatically or chemically released glycans from glycoproteins are usually labeled by reductive amination with a fluorophore reagent. Although labeling techniques based on reductive amination have been well-established as sample preparation methods for fluorometric HPLC-based glycan analysis, they often include time-consuming and tedious purification steps. Here, we reported an alternative fluorescent labeling method based on the synthesis of hydrazone and its reduction using 9-fluorenylmethyl carbazate (Fmoc-hydrazine) as a fluorophore reagent. Using isomaltopentaose and N-glycans from human IgG, we optimized the Fmoc-labeling conditions and purification procedure of Fmoc-labeled N-glycans and applied the optimized method for the analysis of N-glycans released from four glycoproteins (bovine RNase B, human fibrinogen, human α1-acid glycoprotein, and bovine fetuin). The complete workflow for preparation of fluorescent-labeled N-glycans takes a total of 3.5 h and is simple to implement. The method presented here lowers the overall cost of a fluorescently labeled N-glycan and will be practically useful for the screening of disease-related glycans or routine analysis at an early stage of development of biopharmaceuticals.

An efficient deglycosylation process is a key requirement for the identification and characterization of glycosylation during the production and purification of therapeutic antibodies. PNGase F is widely used for the deglycosylation of N-linked glycans. The commonly-used in-solution deglycosylation method is relatively time-consuming and requires several hours up to overnight for complete removal of all N-linked glycans. In order to develop a simple and efficient method for the rapid release of N-linked glycans from glycoproteins, we fabricated trypsin- and PNGase F-impregnated polyacrylamide gels in a commercial 200 μL volume pipette tip. Our enzyme reactor is based on simple photochemical copolymerization of monomers using the following procedure: (1) a pipette tip was filled with a gel solution comprising acrylamide, N,N'-methylene-bis-acrylamide containing PNGase F or trypsin with 2,2-azobis(2-methyl-N-(2-hydroxyethyl) propionamide) as a photocatalytic initiator; and (2) in situ polymerization of gel solution approximately 30 mm from the tip was performed by irradiation with a 365 nm blue LED beam from a distance 10 mm. The fixed enzymes maintained their activities in the polyacrylamide gel and the reaction was completed by 40 iterations of suction and discharge with a pipette (hereafter referred to as manual pipetting times) for 8 min with each enzyme digestion. Capillary electrophoresis (CE) of released glycans labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS) demonstrated quantitative recovery of glycans from selected glycoproteins.

In this work, quaternary ammonium group-modified celluloses (QCs) were homogeneously synthesized by reacting cellulose with 3-chloro-2-hydroxypropyltrimethylammonium chloride in NaOH/urea solutions. The structure and solution properties of the QCs were characterized by elemental analysis, FT/IR, ¹H-NMR, and size exclusion chromatography. The results show that water-soluble QCs, with degree of substitution values, defined as the substitution of free hydroxyl groups of cellulose, 0.49– 0.72 and molecular weight 21–66 kDa could be obtained by optimizing the reaction time. The synthesized QCs were tested for protein separation as physically adsorbed coatings in capillary electrophoresis. Among the derivatives studied, quaternary ammonium cellulose showed rapid electroosmotic mobility and effective suppression of protein adsorption. The EOF can be manipulated for various applications by using QCs with different molecular weight. Because the reversed EOF can be obtained over a broad pH range, it is possible to separate basic, neutral, and acidic proteins under physiological conditions. Eight proteins; lysozyme, ribonuclease A, cytochrome C, α-chymotrypsinogen, α-lactalbumin, ovalbumin, transferrin, and myoglobin were baseline separated within 25 min by 25 mM sodium phosphate buffers (pH 7.0) containing 100 µg/mL QC.

高機能化マイクロチップ電気泳動システムによる糖鎖、リン酸化の全自動解析では、ガンなどの疾病によりタンパク質のリン酸化や糖鎖付加などの翻訳後修飾がどのタイミングでどのように変化するのかを解明する分析手段の開発について検討を行っている。目的とする分析法を開発するためには前処理を含む一連の分析操作を一枚のチップ上で電圧印加のみで達成できる条件の開発が必要となるが、特にターゲットとしている糖鎖やリン酸化などの翻訳後修飾はタンパク質重量から換算すると数%以下であることが多いため、本法を開発するためにはオンラインでの高感度検出に係る前処理を高効率に達成することが必要不可欠になる。 本年度はリン酸化ペプチドのオンライン高感度検出に向けてリン酸化化合物を特異的に捕捉することが可能なPhos-tagを含有したアクリルアミドゲルを多分岐のマイクロチップの流路にピンポイントで作製することによりリン酸化ペプチドの特異的濃縮、オンライン標識、分離・検出を電圧印加のみで達成できるシステムの検討を行った。タンパク質にトリプシン消化を実施することにより得られたペプチドを試料として、まずは試料溶液をマイクロチップの流路交差部の一つに作製したPhos-tagアクリルアミドに導入した。この操作によりリン酸化ペプチドのみをPhos-tagゲルに捕捉し、膨大な単純ペプチドを除去することが可能となった。続いて電圧を切り替えてゲルに向かって蛍光試薬を導入することによりゲル中での蛍光標識化を達成した。最後に高濃度のリン酸緩衝液をゲルに向かって導入することでリン酸化化合物を高感度に検出することが可能となった。

For the fast releasing of N-linked glycans from glycoproteins, a simple and efficient method has been developed by fabricating trypsin and peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase (PNGase F)-impregnated polyacrylamide gel onto a commercial pipette tip, respectively. The fixed enzymes maintained their activities on the polyacrylamide gel and the reaction was completed by a few times pipetting operation.Also, a simple and efficient method has been developed to fabricate sample concentrate and derivative on a channel of a commercial polymethylmethacrylate-made microchip The availability of ionic acrylamide gel was demonstrated for the sensitive analysis of some FITC-amino acids under various buffer systems at several different pH. Then, the multi channel microchip was developed for combination of various functions of acrylamide gel, and achieves high-throughput glycans analysis.