Department of Pharmaceutical SciencesAssociate Professor

Last Updated :2024/07/23

■Researcher basic information

Research Keyword

  • Microchip electrophoresis   Capillary electrophoresis   

Research Field

  • Life sciences / Pharmaceuticals - analytical and physicochemistry / Analytical chemistry



  • 2024/04 - Today  Kindai UniversityFaculty of Pharmacy Department of Pharmaceutical Sciences准教授
  • 2018/04 - 2024/03  Kindai UniversityFaculty of Pharmacy, Department of Pharmaceutical Sciences講師
  • 2013/04 - 2018/03  Kindai UniversityFaculty of Pharmacy, Department of Pharmaceutical Sciences助教

■Research activity information


  • 2020/11 クロマトグラフィー科学会 2020年度クロマトグラフィー科学会奨励賞
    受賞者: 山本佐知雄
  • 2020/07 The Japan Society for Analytical Chemistry Outstanding reviewer award in Analytical Sciences
    受賞者: Sachio Yamamoto
  • 2019/03 日本薬学会 平成31年度 物理系薬学部会奨励賞
    受賞者: 山本 佐知雄
  • 2018/08 第31回バイオメディカル分析科学シンポジウム星野賞 研究奨励賞
    受賞者: 山本 佐知雄
  • 2012/01 日本薬学会近畿支部 平成23年度日本薬学会近畿支部奨励賞
     光重合性アクリルアミドを利用したマイクロチップ電気泳動における新 規オンライン濃縮電気泳動法の開発 
    受賞者: 山本 佐知雄


  • Sachio Yamamoto; Naho Kato; Miki Wada; Mitsuhiro Kinoshita
    Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 39 (7) 1041 - 1046 2023/07 [Refereed][Invited]
    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.
  • 山本 佐知雄; 宮脇 直久; 川上 夏海; 木下 充弘; 鈴木 茂生
    分析化学 (公社)日本分析化学会 71 (6) 333 - 339 0525-1931 2022/06 [Refereed]
    8-Aminopyrene-1,3,6-trisulfonic acid(APTS)は三つのスルホン酸基を有している蛍光試薬であり、その高い電気泳動移動度を利用することでAPTS標識化糖鎖がキャピラリー電気泳動(CE)で分析されている。しかしながらCE単独で糖鎖の構造解析を達成することは困難である。またAPTSは親水性が非常に高いため、一般的な逆相クロマトグラフィーや親水性相互作用クロマトグラフィー(HILIC)で分離が困難であった。そこで著者らは、官能基にテトラゾール基を有するポリマーが結合したDCpak PTZカラムの試作品を使用してHILICモードを利用したAPTS標識化糖鎖の分離方法を開発した。しかしながら開発した方法では再現性、構造の類似した糖鎖の分離能が低く、溶出時間も1時間程度を要した。特にシアル酸含有糖鎖においては、成分ごとの分離がほとんどできなかった。そこで本研究では市販のDCpak PTZカラムを利用し、糖鎖の分離方法を再検討した。また、DCpak PTZカラムで分離した糖鎖に関して分取、脱塩した試料をCEの試料に添加してがん細胞のピークの同定を行った。(著者抄録)
  • Sachio Yamamoto; Shoko Yano; Mitsuhiro Kinoshita; Shigeo Suzuki
    Gels (Basel, Switzerland) 7 (4) 2021/12 [Refereed][Invited]
    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.
  • Mitsuhiro Kinoshita; Kazuki Nakajima; Sachio Yamamoto; Shigeo Suzuki
    Analytical and bioanalytical chemistry 413 (19) 4727 - 4738 2021/08 [Refereed]
    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.
  • Sachio Yamamoto; Kazuhito Maetani; Gai Tatsumi; Fuka Okada; Mitsuhiro Kinoshita; Shigeo Suzuki
    Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 2021/04 [Refereed]
  • Maho HIOKI; Hiroshi KOBAYASHI; Mitsuhiro KINOSHITA; Sachio YAMAMOTO; Shigeo SUZUKI
    CHROMATOGRAPHY The Society for Chromatographic Sciences 1342-8284 2021/04
  • Sachio YAMAMOTO
    CHROMATOGRAPHY The Society for Chromatographic Sciences 42 (1) 29 - 36 1342-8284 2021/02 [Refereed][Invited]
    CHROMATOGRAPHY The Society for Chromatographic Sciences 42 (1) 37 - 42 1342-8284 2021/02 [Refereed]
  • Mitsuhiro Kinoshita; Yumi Nakatani; Keita Yamada; Sachio Yamamoto; Shigeo Suzuki
    Journal of pharmaceutical and biomedical analysis 195 113875 - 113875 2021/02 
    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.
  • Sachio Yamamoto; Naonori Utamura; Mitsuhiro Kinoshita; Shigeo Suzuki
    Bunseki Kagaku Japan Society for Analytical Chemistry 70 (1) 39 - 44 0525-1931 2021 
    Non-aqueous capillary electrophoresis (NACE) has been developed as a powerful analytical method for a group of compounds having both a weak charge and high hydrophobicity, including pharmaceuticals. Gangliosides bound to lipoproteins are present in serum, and the content of gangliosides is considered to reflect various pathological conditions, such as cancer. Since gangliosides are present only slightly in the living body, capillary electrophoresis is suitable for separation. However, Capillary electrophoresis using an aqueous buffer cannot separate samples with hydrophilic and hydrophobic moieties in the ganglioside. In this study, we used an ammonium borate buffer containing 1-propanol solution for controlling the polarity of the buffer solution to suppress ganglioside micellization and to separate each component. We applied the developed NACE method to the analysis of gangliosides derived from mouse brain.
  • Mitsuhiro Kinoshita; Sachio Yamamoto; Shigeo Suzuki
    ACS Omega American Chemical Society (ACS) 5 (30) 18608 - 18618 2470-1343 2020/08 [Refereed]
  • Yuka Kishimoto; Fuka Okada; Tomohiro Maesako; Sachio Yamamoto; Mitsuhiro Kinoshita; Takao Hayakawa; Shigeo Suzuki
    Journal of Chromatography A Elsevier BV 1625 461194 - 461194 0021-9673 2020/08 [Refereed]
  • Mitsuhiro Kinoshita; Ai Saito; Sachio Yamamoto; Shigeo Suzuki
    Journal of pharmaceutical and biomedical analysis 186 113267 - 113267 2020/07 [Refereed]
    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.
  • Sachio Yamamoto; Maki Ueda; Masataka Kasai; Yusuke Ueda; Mitsuhiro Kinoshita; Shigeo Suzuki
    Journal of pharmaceutical and biomedical analysis 179 112995 - 112995 2020/02 [Refereed]
    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.
  • Naonori UTAMURA; Sachio YAMAMOTO; Mitsuhiro KINOSHITA; Shigeo SUZUKI
    BUNSEKI KAGAKU Japan Society for Analytical Chemistry 68 (11) 859 - 864 0525-1931 2019/11 [Refereed]
  • Yamamoto S; Fujiwara H; Maruyama K; Tanaka Y; Kinoshita M; Suzuki S
    Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 35 (3) 295 - 300 0910-6340 2019/03 [Refereed]
  • Mitsuhiro Kinoshita; Sachio Yamamoto,; Shigeo Suzuki
    Electrophoresis Letters Japanese Electrophoresis Society 63 (2) 47 - 54 2189-2628 2019 [Refereed]
  • Novel separation mode of non-aqueous capillary electrophoresis based on excess adsorption
    Naonori Utamura; Sachio Yamamoto; Mitsuhiro Kinoshita; Shigeo Suzuki
    Bunseki Kagaku 69 2019 [Refereed]
  • Yamamoto S; Okada F; Kinoshita M; Suzuki S
    The Analyst 143 (18) 4429 - 4435 0003-2654 2018/09 [Refereed]
  • 全自動マイクロチップ電気泳動装置MCE-202”MultiNA”を用いた糖鎖の迅速解析
    木下 充弘; 山本万莉; 御子柴柚子; 松本和樹; 山本佐知雄; 鈴木茂生; 曽我部有司
    74 (3・4) 193 - 204 2018/03
  • Sachio Yamamoto; Mitsuhiro Kinoshita; Toru Ikegami; Shigeo Suzuki
    Journal of Chromatography A Elsevier B.V. 1566 44 - 50 1873-3778 2018 [Refereed]
    8-Aminopyrene-1,3,6-trisulfonate (APTS) is one of the most frequently used reagent in capillary electrophoresis. Three sulfonate groups in APTS generate fast electrophoretic mobilities of derivatized glycans, therefore very suitable for CE-LIF applications. However, these groups also make separation with partition chromatography difficult. A novel column for hydrophilic interaction liquid chromatography (HILIC) with an anionic tetrazole functionalized polymer-based silica was examined for the separation of APTS-labeled glycans derived from specific glycoproteins. This separation mode has enhanced capability for the size resolution of neutral and acidic oligosaccharides. In addition, specific glycan isomers, which are usually difficult to separate with HILIC methods, were also separated. IgG-derived complex-type glycans that have an isomeric pair of monogalactosylated glycans, as well as differences in the number of galactose residues, are separable using this mode. We also utilized this column for the fractionation of APTS-labeled glycans from porcine thyroglobulin and examined their migration times with CE by co-migration with a mixture of glycoprotein glycans. Combinational modes of HILIC and anionic repulsion show promise for the separation and preparation of glycoprotein-derived glycans labeled with APTS.
  • Noriaki Nishida; Yasuko Kokaji; Sachio Yamamoto; Mitsuhiro Kinoshita; Shigeo SuzuKi
    BUNSEKI KAGAKU JAPAN SOC ANALYTICAL CHEMISTRY 66 (12) 909 - 917 0525-1931 2017/12 [Refereed]
    Compared to N-linked oligosaccharides, O-linked oligosaccharides were rarely reported for their analysis due to the absence of glycanases having wide substrate specificity to release all O-linked oligosaccharides. Therefore, O-linked oligosaccharides have often been released by chemical methods, such as hydrazinolysis and beta-elimination under alkaline conditions, but the liberated oligosaccharides released by these methods are susceptible to alkaline conditions which cause epimerization and degradation, called peeling reaction and the hydrolysis of N-acetyl groups. To compare the releasing efficiency and the degradation rate of previously reported releasing methods, we developed a novel HPLC method for the analysis of O-linked oligosaccharides: oligosaccharides released from glycoproteins are labeled quantitatively with 7-amino-4-methylcoumarin (AMC) and separated by reversed-phase HPLC, and then fluorimetrically detected with high sensitivity. In the releasing methods, we found that 25 % ammonia is most optimal, and can be applied to the release of N-linked oligosaccharides. We established a method that could be applied to the rapid and simple and nonspecific release of N-linked oligosaccharides. Various AMC-labeled N-linked oligosaccharides released from glycoproteins were used as samples, and were separated by reversed-phase HPLC and fluorimetrically detected with high sensitivity for comparing with the enzymatic releasing method using PNGase F.
  • Sachio Yamamoto; Miyuki Himeno; Masaya Kobayashi; Miki Akamatsu; Ryosuke Satoh; Mitsuhiro Kinoshita; Reiko Sugiura; Shigeo Suzuki
    ANALYST ROYAL SOC CHEMISTRY 142 (18) 3416 - 3423 0003-2654 2017/09 [Refereed]
    A method was developed for the specific entrapment and separation of phosphorylated compounds using a Phos-tag polyacrylamide gel fabricated at the channel crossing point of a microfluidic electrophoresis chip. The channel intersection of the poly(methyl methacrylate)-made microchip was filled with a solution comprising acrylamide, N, N-methylene-bis-acrylamide, Phos-tag acrylamide, and 2,2'-azobis [2-methyl-N-(2-hydroxyethyl) propionamide], which functioned as a photocatalytic initiator. In situ polymerization at the channel crossing point was performed by irradiation with a UV LED laser beam. The fabricated Phos-tag gel (100 x 100 x 30 mu m) contains ca. 20 fmol of the Phos-tag group and therefore could entrap phosphorylated compounds at the femtomolar level. The electrophoretically trapped phosphorylated compounds were released from the gel by switching the voltage to deliver high concentrations of phosphate and EDTA in a background electrolyte. The broad sample band eluted from the gel was effectively reconcentrated at the boundary of a pH junction generated by sodium ions delivered from the outlet reservoir. The reconcentrated sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, the phosphorylated compounds were concentrated by a factor of 100-fold, and the peak resolution was comparable to that obtained by pinched injection. This method was successfully utilized to preconcentrate and analyze phosphorylated peptides in a complex peptide mixture.
  • Maki Yamagami; Yurie Matsui; Takao Hayakawa; Sachio Yamamoto; Mitsuhiro Kinoshita; Shigeo Suzuki
    JOURNAL OF CHROMATOGRAPHY A ELSEVIER SCIENCE BV 1496 157 - 162 0021-9673 2017/05 [Refereed]
    An online exoglycosidase digestion was combined with a plug-plug kinetic mode of capillary electrophoresis (CE) for the analysis of glycoprotein-derived oligosaccharides. An exoglycosidase solution and a solution of glycoprotein glycans derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were introduced to a neutrally coated capillary previously filled with electrophoresis buffer solution containing 0.5 w/v% hydroxypropylcellulose. After immersion of both ends of the capillary in the buffer solutions, a negative voltage was applied for analysis. An APTS group of an oligosaccharide derivative has triply negative charges, which forced saccharide derivatives to anode with fast mobility and pass through the enzyme plug, which are detected at the anodic end. If the terminal monosaccharides of APTS-labeled oligosaccharides are released by the action of an exoglycosidase, the migration times of the oligosaccharides shift to those of digested oligosaccharides. We examined beta-galactosidase, alpha-mannosidase, beta-N-acetylhexosaminidase, alpha-neuraminidase, and alpha-fucosidase, and found only beta-galactosidase and alpha-neuraminidase showed good reactivity toward APTS-labeled oligosaccharides; the reaction was completed by injecting a 3.6 cm long plug of 200 and 50 mU/mL concentration of exoglycosidases. In contrast, other exoglycosidases could not react with APTS labeled oligosaccharides at a concentration up to 5 U/mL. The beta-N-acetylhexosaminidase reaction was successively followed by the electrophoretic mobility of APTS oligosaccharides and stopped for 10 min when saccharide derivatives were achieved in the enzyme plug. The reaction of alpha-fucosidase and alpha-mannosidase was completed by decreasing the electrophoretic voltage to -2 kV when the APTS oligosaccharides were passing through an exoglycosidase plug. We established the CE conditions for all of the glycosidic linkage analysis of glycoprotein glycans. (C) 2017 Elsevier B.V. All rights reserved.
    CHROMATOGRAPHY The Society for Chromatographic Sciences 38 (1) 23 - 30 1342-8284 2017 [Refereed]
  • Sachio Yamamoto; Mitsuhiro Kinoshita; Shigeo Suzuki
    This review covers the basics and some applications of methodologies for the analysis of glycoprotein glycans. Analytical techniques used for glycoprotein glycans, including liquid chromatography (LC), capillary electrophoresis (CE), mass spectrometry (MS), and high-throughput analytical methods based on microfluidics, were described to supply the essentials about biopharmaceutical and biomarker glycoproteins. We will also describe the MS analysis of glycoproteins and glycopeptides as well as the chemical and enzymatic releasing methods of glycans from glycoproteins and the chemical reactions used for the derivatization of glycans. We hope the techniques have accommodated most of the requests from glycoproteomics researchers. (C) 2016 Elsevier B.V. All rights reserved.
  • Yuto Takeda; Yuka Hayashi; Naonori Utamura; Chise Takamoto; Mitsuhiro Kinoshita; Sachio Yamamoto; Takao Hayakawa; Shigeo Suzuki
    JOURNAL OF CHROMATOGRAPHY A ELSEVIER SCIENCE BV 1427 170 - 176 0021-9673 2016/01 [Refereed]
    Monoamine- and triamine-bonded silica nanoparticles were prepared using 3-aminopropyl-trimethoxysilane and N-1-(3-trimethoxysilylpropyl)diethylenetriamine, respectively, and used as pseudostationary phases for capillary electrochromatography. The amine-bonded silica nanoparticles were tightly adsorbed on the inner wall of a capillary and generated fast electro-osmotic flow (2.59 x 10(-4) cm(2) V-1 s(-1)) toward the anode in an electric field. The electro-osmotic velocities obtained with 20 nm triamine-bonded silica were three to five times larger than those generated by a fused silica capillary and two times faster than those for the commercial cationic polymer-modified capillary. Fast electro-osmotic flow enables rapid analysis. This method was applied to hydrophilic interaction chromatography (HILIC) mode separation of various samples including the size separation of glucose oligomer derivatives and the resolution of four nucleic acid bases. (C) 2015 Elsevier B.V. All rights reserved.
    Chromatography The Society for Chromatographic Sciences 36 (3) 93 - 98 1342-8284 2015/12 [Refereed]
    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.
  • Sachio Yamamoto; Yoko Tamata; Kaori Sejima; Mitsuhiro Kinoshita; Shigeo Suzuki
    ANALYTICAL AND BIOANALYTICAL CHEMISTRY SPRINGER HEIDELBERG 407 (20) 6201 - 6206 1618-2642 2015/08 [Refereed]
    A novel method was developed for d/l-isomeric separation of aldopentoses and aldohexoses as their (S)-(+)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole derivatives using phenylboronate buffer containing sodium dodecyl sulfate as a background electrolyte. The combination of derivatization with a chiral labeling reagent and micellar electrokinetic chromatography with phenylboronate made possible the efficient separation of d/l isomers as well as epimeric isomers of aldopentoses and aldohexoses. Laser-induced fluorescence detection permitted the micromolar-level determination of monosaccharide derivatives. The limit of detection was 105 amol (300 nM), and the repeatabilities of the migration times and peak area responses were 0.8 % and 7.9 % (relative standard deviation; n = 6), respectively. The method was applied to the determination of d/l- galactose in red seaweed.
  • Sachio Yamamoto; Eriko Nagai; Yuki Asada; Mitsuhiro Kinoshita; Shigeo Suzuki
    ANALYTICAL AND BIOANALYTICAL CHEMISTRY SPRINGER HEIDELBERG 407 (5) 1499 - 1503 1618-2642 2015/02 [Refereed]
    A selective separation method using a poly(methylmethacrylate) microchip was developed for 7-amino-4-methylcoumarin-labeled saccharides in a crude reaction mixture. In an alkaline borate buffer, saccharide derivatives formed strong anionic borate complexes. These complexes moved from the cathode to the anode in an electric field and were detected near the anode. Excess labeling reagents and other foreign substances remained at the inlet reservoir. A confocal fluorimetric detection system enabled the determination of monosaccharide derivatives with good linearity between at least 5 and 100 nM, corresponding to 50 fmol to 1 pmol per injection. The lower limit of detection (signal-to-noise=5) was 2 nM. The sensitivity and linear quantitation range were comparable to reported values using fluorometric detection, capillary electrophoresis, or liquid chromatography. Application of the method showed excellent resolution in the analysis of O-linked glycans chemically released from glycoproteins.
    Chromatography クロマトグラフィー科学会 34 (3) 157 - 160 1342-8284 2013/12
  • Sachio Yamamoto; Yumi Nakatani; Shigeo Suzuki
    ANALYTICAL SCIENCES JAPAN SOC ANALYTICAL CHEMISTRY 29 (8) 831 - 835 0910-6340 2013/08 [Refereed]
    An online preconcentration technique, large volume sample stacking with an electroosmotic flow pump, was combined with partial filling affinity capillary electrophoresis (PFACE) to create a highly sensitive analysis of the interaction of glycoprotein-derived oligosaccharides with plant lectins. Oligosaccharides were derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) for use in a blue light emitting diode-induced fluorescence detection capillary electrophoresis system. ANTS-labeled oligosaccharides were delivered to an entire neutrally coated capillary, and lectin solution was then hydrodynamically introduced from the outlet of the capillary as a short plug. When negative voltage was then applied, a low concentration sample solution caused a significant flow by electroosmosis from anode to cathode and the ANTS-labeled oligosaccharides moved quickly towards the anode and concentrated in the lectin phase. Finally, when the electroosmotic flow became negligible, ANTS-labeled saccharides passed through the lectin plug and were detected at the anodic end. The sensitivity was enhanced by a factor of roughly 200 compared to typical hydrodynamic injection (13.8 kPa, 5 s).
  • Sachio Yamamoto
    Microchip electrophoresis is widely used for microfluidics and has been studied extensively over the past decade. Translation of capillary electrophoresis methods from traditional capillary systems to a microchip platform provides rapid separation and easy quantitation of sample components. However, most microfluidic systems suffer from critical scaling problems. One promising solution to this problem is online sample preconcentration of all analytes in a sample reservoir before the separation channel. Herein, the following three techniques for online preconcentration during microchip electrophoresis are proposed: (1) in situ fabrication of an ionic polyacrylamide-based preconcentrator on a simple poly (methyl methacrylate) microfluidic chip for perm-selective preconcentration and capillary electrophoretic separation of anionic compounds, (2) simultaneous concentration enrichment and electrophoretic separation of weak acids on a microchip using an in situ photopolymerized carboxylate-type polyacrylamide gels as the perm-selective preconcentrator, and (3) microchip electrophoresis of oligosaccharides using lectin-immobilized preconcentrator gels fabricated by in situ photopolymerization. These techniques are expected to be powerful tools for clinical and pharmaceutical studies with on-line preconcentration during microchip electrophoresis.
  • Eriko Fukushima; Yuki Yagi; Sachio Yamamoto; Yumi Nakatani; Kazuaki Kakehi; Takao Hayakawa; Shigeo Suzuki
    JOURNAL OF CHROMATOGRAPHY A ELSEVIER SCIENCE BV 1246 84 - 89 0021-9673 2012/07 [Refereed]
    An online preconcentration technique, large-volume sample stacking with an electroosmotic flow pump (LVSEP) was combined with partial filling affinity capillary electrophoresis (PFACE) to realize highly sensitive analysis of the interaction of glycoprotein-derived oligosaccharides with some plant lectins. Oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were delivered to an entire neutrally-coated capillary and then lectin solution was hydrodynamically introduced from the outlet of the capillary as a short plug. A negative voltage was then applied after immersion of both ends of the capillary in 100 mM Tris-acetate buffer. pH 7.0 containing 0.5% hydroxypropylcellulose as electrophoresis buffers. A low concentration of electrolytes in the sample solution causes a significant flow by electroendosmosis from anode to cathode and the APTS-labeled oligosaccharides move quickly towards the anode and concentrate in the lectin phase. Finally, electroosmotic flow becomes negligible when the capillary is filled with the background electrolyte delivered from the anodic reservoir and APTS-labeled saccharides pass through the lectin plug and are detected at the anodic end. If the APTS-labeled oligosaccharides are recognized by the lectin, the migration profiles should be altered. The sensitivity was enhanced by a factor of ca. 900 compared to typical hydrodynamic injection (3.45 kPa, 10 s). By this method, increased residence time of APTS-saccharides in the lectin plug indicates highly efficient interaction with lectins, which differs completely from the results obtained by ordinary lectin PFACE. The run-to-run repeatability (n = 18) of the migration time and peak area was high, with relative standard deviations of less than 0.7% and 6.1%, respectively. (C) 2012 Elsevier B.V. All rights reserved.
  • Sachio Yamamoto; Sho Suzuki; Shigeo Suzuki
    ANALYST ROYAL SOC CHEMISTRY 137 (9) 2211 - 2217 0003-2654 2012 [Refereed]
    A lectin-impregnated gel was fabricated at the channel crossing point in a microfluidic chip made from polymethyl methacrylate (PMMA). The acrylamide containing lectin was photopolymerized to form a round gel (radius 60 mu m) by irradiation with an argon laser, which was also used for fluorometric detection. This gel was applied to specific concentration, elution, and electrophoretic separation of fluorescent-labeled oligosaccharides. Because the lectin in the polyacrylamide gel was mechanically immobilized, it maintained its activity. The lectin was used to trap up to a few tens of femtomoles of specific oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid with 2 min by a factor >800, and the amount trapped corresponded to ca. 70% of lectin in the gel. The trapped oligosaccharides were released from the gel by lowering the pH with an acidic background electrolyte. The oligosaccharides that eluted as a broad band were concentrated by transient isotachophoresis stacking using concentrated sodium borate buffer (pH 11.0). The stacked sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, resolution of the saccharides was good, and was similar to that obtained by pinched injection. The method was applied to preconcentration and analysis of oligosaccharides derived from some glycoproteins.
  • Yuki Yagi; Sachio Yamamoto; Kazuaki Kakehi; Takao Hayakawa; Yukihito Ohyama; Shigeo Suzuki
    ELECTROPHORESIS WILEY-BLACKWELL 32 (21) 2979 - 2985 0173-0835 2011/11 [Refereed]
    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, Gal beta 1-4GlcNAc-specific Erythrina cristagali agglutinin, alpha 1, 6-linked Fuc-specific Aleuria aurantia lectin and Neu5Ac alpha 2-3Gal-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.
  • Sachio Yamamoto; Yuki Watanabe; Noriaki Nishida; Shigeo Suzuki
    JOURNAL OF SEPARATION SCIENCE WILEY-V C H VERLAG GMBH 34 (20) 2879 - 2884 1615-9306 2011/10 [Refereed]
    A method for the simultaneous concentration and separation of weak acids using an acidic polyacrylamide gel, fabricated in the microfluidic channel of a commercial poly(methyl methacrylate)-made microchip, is reported. This approach is based on simple photochemical copolymerization for the fabrication of a permselective preconcentrator. The intersection of the poly(methyl methacrylate)-made microchip was filled with a gel solution comprising acrylamide, N,N'-methylene-bis-acrylamide, and 2-acrylamidoglycolic acid, with riboflavin as a photocatalytic initiator. In situ polymerization, near the cross of the sample outlet channel, was performed by irradiation with an argon ion laser beam that is also used as the light source for fluorimetric detection. The electrokinetic properties, combined with electrostatic repulsion between sample components and the anionic groups on the polyacrylamide gel, enable the entrapment and concentration of weak acids at the interface of the cathodic side of the gel plug. This method displays concentration factors of up to 10(5) within 3 min. The effectiveness of the ionic preconcentrator was demonstrated by the sensitive analysis of fluorescein isothiocyanate-labeled amino acids.
  • Sachio Yamamoto; Chikayo Shinohara; Eriko Fukushima; Kazuaki Kakehi; Takao Hayakawa; Shigeo Suzuki
    JOURNAL OF CHROMATOGRAPHY A ELSEVIER SCIENCE BV 1218 (29) 4772 - 4778 0021-9673 2011/07 [Refereed]
    Partial-filling affinity capillary electrophoresis has been applied to the simultaneous analysis of interactions between glycoprotein oligosaccharides and certain plant lectins. A lectin solution and a mixture of glycoprotein-derived oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid were introduced to a neutrally coated capillary in this order, and separated by application of a negative voltage. Interaction of a lectin with each oligosaccharide in the mixture was observed as the specific retardation or dissipation of peaks, in addition to the size/charge separation of oligosaccharides by zone electrophoresis in the remainder (approximate to 90%) of the capillary. The strength of the interaction with lectin was controlled by introducing an appropriate volume of lectin solution. Application of various specificities of lectins indicated characteristic migration profiles of the oligosaccharides. Moreover, sequential injection of four lectins (Maachia amurensis mitogen, Sambucus sieboldiana agglutinin, Erythrina cristagalli agglutinin, Aleuria aurantia lectin) induced complete dissipation of complex-type oligosaccharides and enabled specific determination of the presence of high-mannose oligosaccharides without the interference or alteration of the electropherogram in porcine thyroglobulin. This method was also applied to determine the binding constants of ovalbumin-derived oligosaccharides to wheat germ agglutinin. (C) 2011 Elsevier B.V. All rights reserved.
    検査と技術 38 (12) 1138 - 1145 0301-2611 2010/11 [Refereed]
  • Sachio Yamamoto; Shingo Hirakawa; Shigeo Suzuki
    ANALYTICAL CHEMISTRY AMER CHEMICAL SOC 80 (21) 8224 - 8230 0003-2700 2008/11 [Refereed]
    A simple and efficient method was developed for fabrication of an anionic sample preconcentrator on a channel of a commercial poly(methyl methacrylate) (PMMA)-made microchip using no photolithography or etching technique. The originality of our preconcentrator is based on simple photochemical copolymerization of monomers using the following procedure: All channels of the PMMA-made microchip were filled with gel solution comprising acrylamide, N,N'-methylene-bisacrylamide, and 2-acrylamide-2-methylpropanesulfonic acid with riboflavin as a photocatalytic initiator. In situ polymerization near the cross of the sample outlet channel was performed by irradiation with an argon ion laser beam, which is also used as the light source for fluorometric detection. The electrokinetic property and electric repulsion between sample components and anionic groups on the polyacrylamide gel layer produce, trap, and concentrate anions within a few minutes at the interface of the cathodic side of the gel layer. This method displays concentration factors as high as 10(5). The availability of ionic preconcentrator was demonstrated by applying sensitive analysis of oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonate and some glycoproteins labeled with fluorescein isothiocyanate under various buffer systems.
    生物物理化学 日本電気泳動学会 52 (3) 127 - 131 0031-9082 2008/09


Lectures, oral presentations, etc.

Research Themes

  • 日本学術振興会:科学研究費助成事業
    Date (from‐to) : 2023/04 -2027/03 
    Author : 川井 隆之; 山本 佐知雄
  • 日本学術振興会:科学研究費助成事業
    Date (from‐to) : 2023/04 -2026/03 
    Author : 山本 佐知雄
  • 高速全自動マイクロチップ電気泳動シス テムによる糖タンパク質糖鎖を指標とし た新規臨床検査法の開発
    公益財団法人 黒住医学研究振興財団:第31回研究助成金
    Date (from‐to) : 2023/11 -2024/10 
    Author : 山本佐知雄
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2020/04 -2023/03 
    Author : 山本 佐知雄
    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。
  • 高機能化マイクロチップを用いる糖鎖の全自動高速構造解析法の開発
    Date (from‐to) : 2019/09 -2021/09 
    Author : 山本佐知雄
  • 高機能化マイクロチップを用いるバイオ医薬品の超高速分析システムの開発
    Date (from‐to) : 2017/04 -2020/03 
    Author : 山本 佐知雄
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015 -2016 
    Author : YAMAMOTO Sachio
    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.


  • 2022/04 -2023/03  高機能化マイクロチップ電気泳動システムによる生体内微量 糖鎖の網羅的高感度解析システムの確立 
    近畿大学学内研究助成金 奨励研究助成金 SR09 研究内容:電気泳動用マイクロ流体デバイスは生体成分中に微量にしか含まれない糖鎖を分離・検出するために有用であるが,実際には測定用の試料調製に一週間程度の時間を要する。本研究計画では前処理を含む一連の分析操作が特に困難な糖鎖の構造と,その機能を明らかにするために,アクリルアミドゲル層と3次元型のマイクロチップを用いて糖鎖分析に必要な前処理を含む一連の工程を流路中で行うことにより真のハイスループット分析を実現する。
  • 2019/04 -2020/03  機能性アクリルアミドゲルと高機能化マイクロチップを用いる糖鎖高速高感度分析システム法の開発 
    近畿大学学内研究助成金 研究種目:奨励研究助成金 課題番号:SR08 研究内容:バイオ医薬品の品質管理や臨床現場でも使用することが可能なマイクロチップ分析システムの開発を目的とし実験を行った。迅速自動解析を目的とし,濃縮,特異的抽出および粗分画,標識化などの一連の分析操作をチップ上に集約させるためこれらの前処理操作を電圧印加のみで達成できるシステムを構築した。