US008877454B2

(12) United States Patent (10) Patent No.: US 8,877,454 B2 Nishimura et al. (45) Date of Patent: Nov. 4, 2014

(54) APPARATUS FOR AUTO-PRETREATING (56) References Cited CHAN FOREIGN PATENT DOCUMENTS (75) Inventors: Shinichiro Nishimura, Hokkaido (JP); Yasuro Shinohara, Hokkaido (JP); JP 200529 1958 10/2005 Yoshiaki Miura, Hokkaido (JP): WO WO-2006.112771 10, 2006 Hiroshi Yamazaki, Tokyo (JP); Michio WO WO2007099856 9, 2007 Horiuchi, Tokyo (JP); Hiroaki Motoki, OTHER PUBLICATIONS Tokyo (JP); Toshiharu Kuroda, Tokyo Uematsu et al. “High throughput quantitative glycomics and (JP); Yoko Kita, Hyogo (JP); Mika glycoform-focused proteomics of murine dermis and epidermis'. Nakano, Hyogo (JP) Molecular & Cellular Proteomics, 2005, 4:1977-1989.* Shimaoka et al "One-pot Solid-phase glycoblotting and probing by (73) Assignees: National University Corporation transoximization for high-throughput glycomics and glycoproteom Hokkaido University, Sapporo-Shi, ics', Chem. Eur, J., 2007, 13:1664-1673.* Schlosser etal "Combination of solid-phase affinity capture on mag Hokkaido (JP); Shionogi & Co., Ltd., netic beads and mass spectrometry to study non-covalent interac Chuo-Ku, Osaka (JP) tions: example of minor groove binding drugs'. Rapid Communica tions in Mass Spectrometry, 2005, 19:3307-3314.* (*) Notice: Subject to any disclaimer, the term of this Powell et al. "Stabilization of sialic acids in N-linked oligosac patent is extended or adjusted under 35 charides and gangliosides for analysis by positive ion matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun, in U.S.C. 154(b) by 926 days. mass spectrometry, 1996, 10:1027-1032.* Kita et al., “Quantitative glycomics of human whole serum (21) Appl. No.: 12/681,573 glycoproteins based on the standardized protocol for liberating N-glycans.” Molecular and Cellular Proteomics, 6(8): 1437-1445 (22) PCT Fled : Oct. 3, 2008 (2007). Miura et al., “Rapid and simple solid-phase esterification of Sialic PCT NO.: PCT/UP2O08/O68111 acid residues for quantitative glycomics by mass spectrometry.” (86) Chemistry A European Journal, 13(17);4797-4804 (2007). S371 (c)(1), Yu et al., "A rapid sample preparation method for mass spectrometric characterization of N-Linked glycans. Rapid Communications in (2), (4) Date: Dec. 3, 2010 Mass Spectrometry, 19(16):2331-2336 (2005). Nishimura et al., “High-throughput protein glycomics: Combined (87) PCT Pub. No.: WO2O09/044900 Use of Chemoselective Glycoblotting and MALDI-TOF/TOF Mass PCT Pub. Date: Apr. 9, 2009 Spectrometry.” Angew. Chem. Int. Ed., 44(1):91-96 (2005). (65) Prior Publication Data * cited by examiner US 2011/011 1443 A1 May 12, 2011 Primary Examiner — Bin Shen (74) Attorney, Agent, or Firm — Ropes & Gray LLP. James (30) Foreign Application Priority Data F. Haley, Jr.; Brian M. Gummow Oct. 5, 2007 (JP) ...... 2007-26268O (57) ABSTRACT Oct. 5, 2007 (JP) ...... 2007-262.771 To provide an autoanalyzer for analyzing a Sugar chain con tained in a biological sample, in particular, serum. Namely, it (51) Int. C. is intended to provide a method of analyzing a Sugar chain in (2006.01) a sample, which comprises the following steps: A) the Sugar GOIN33/68 (2006.01) chain-releasing step of releasing the Sugar chain in the GOIN3O/72 (2006.01) sample; B) the detection sample-preparing step of preparing HOIJ 49/00 (2006.01) the released Sugar chain for detection; and, in the case of (52) U.S. C. conducting mass spectrometry using a plate, C) the step of CPC ...... GOIN33/6848 (2013.01); G0IN 30/7233 forming a plate for the mass spectrometry having the captured (2013.01); G0IN 2400/00 (2013.01); HOIJ Sugar chain dotted thereon which comprises the step of pro 49/00 (2013.01) viding the tagged Sugar chain sample solution obtained in the USPC .... step B) on a collection plate; and, if required, the step of ------435/23: 435/4; 435/22 conducting an operation in a solid phase Support-enclosed (58) Field of Classification Search plate to form the plate for mass spectrometry; and D) the step CPC ...... ------GO1N3O/7233 of analyzing the Sugar chain to be assayed.

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Fig.7 Injection from plate for recovery #1 Injection to MALDI plate to MALDPLATE after SPE treatment Plate for mixindi:1 Plate for mixing #2 (6) 2 8) 20 pil injection of free sugar 23) 2 pil injection of free sugar chain-containind filtrate chain-containing filtrate 30) 420 ul injection of the mixed solution to SPE plate

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L------3hirdYa time?- > - Yes 22) 50 injection of 50 mMDTT Heat retention, 60°C, 5 minutes Heat retention, room temperature, 15 minutes Suction recovery plate for recoverwif Heat retention, 100C, END US 8,877,454 B2 1. 2 APPARATUS FOR AUTO-PRETREATING residues for quantitative glycomics by mass spectrometry. SUGAR CHAN Miura Y. ShinoharaY. Furukawa J. Nagahori N. Nishimura S. TECHNICAL FIELD DISCLOSURE OF THE INVENTION The present invention relates to an apparatus for Sugar chain analysis. The present invention can be applied to the Problem to be Solved by the Invention diagnosis of a disease, or the like. More particularly, the present invention relates to an automatic Sugar chain pretreat However, conventionally, Such processes have been carried ment apparatus. 10 out mostly by hand, and thus these processes have required extensive time and labor, while it has been difficult to carry BACKGROUND ART out purification with high accuracy. The present invention was made under Such circumstances, Pretreatment methods for quantitatively analyzing a Sugar and is intended to provide an automatic Sugar chain pretreat chain (Patent Document 1: Non-Patent Document 1). There 15 ment apparatus which allows purification to be carried out at has been reported a method of detecting a Sugar chain rapidly high speed with high accuracy by automating the treatment with high sensitivity in order to rapidly analyze the Sugar processes. chain, by selectively capturing only a Sugar chain released That is, it is an object of the present invention to provide an from a glycoprotein, immobilizing the Sugar chain on beads, auto-analyzing apparatus for analyzing a Sugar chain con and washing the bead (Non-Patent Document 2). However, an tained in a blood serum sample. It is an object to make it apparatus or pretreatment apparatus that automatically per possible by the auto-analyzing apparatus to automatically forms a Sugar chain analysis using a large amount of a sample analyze a large amount of sample simultaneously. In order to Such as a blood serum sample, is not known yet. release a Sugar chain quantitatively from a glycoprotein, cum In the conventional art, purification of a Sugar chain con berSome pretreatments such as a use of a solubilizing agent, tained in a biologically derived mixture of a body fluid such as 25 reductive alkylation and digestion by trypsin, are required. blood (serum) and a cell/tissue extract, is carried out accord Furthermore, a Sugar chain that has been quantitatively ing to the following steps. released can be subjected to a rapid analysis of the Sugar chain A. A glycoprotein in a sample is reductively alkylated in by performing chemoselective capturing and thereby easily the presence of a soluble product. removing troublesome foreign matters. However, it is diffi B. A proteolytic enzyme is added thereto to digest the 30 cult to treat multiple test samples all at a time. Thus, it is protein moiety with the enzyme, and then the system is heated intended to optimize the various processes described above to deactivate the enzyme. and to establish a system adaptable to automatic analysis. C. An enzymatic treatment or a chemical treatment which releases a Sugar chain from a peptide is carried out to release Means for Solving Problem the Sugar chain. 35 D. Polymer beads displaying a functional group capable of The inventors of the present invention made a thorough capturinga Sugar chain are contacted with a sample which has investigation, and as a result, they found an apparatus that can been finished with the treatment of item C., and thereby the automatically prepare a sample for mass spectrometry when released Sugar chain is captured by the functional group of a blood serum sample is applied. Thereafter, by performing polymer beads. 40 mass spectrometry, an automatic analysis of Sugar chain was E. The sample is washed and filtered to remove those made possible. The inventors have been able to develop an impurities that are not captured by the functional group of the apparatus which is capable of carrying out all processes polymer beads. including from pretreatment to capturing of a Sugar chain, F. The carboxylic acid of a sialic acid residue of the sugar modification of Sialic acid, and to release of the Sugar chain. chain captured by the functional group of the polymer beads, 45 The present invention provides the following. is protected by methyl esterifying the functional group. (1) A method for analyzing a Sugar chain in a sample, the G. The Sugar chain captured by the functional group of the method comprising the following steps: polymer beads is subjected to a reduction of the hydrazone A) a Sugar chain releasing step of releasing a Sugar chain in bond to stabilize the binding thereof with the polymer beads. a sample, the step comprising the following steps: H. The sugar chain is released from the polymer beads 50 A-1) a step of providing the sample on a plate for reaction; through a hydrazone-oxime exchange reaction, by cleaving A-2) a step of adding a solubilizing agent to the sample to the disulfide bond included in Sugar chain capturing mol thereby place the sample under a reaction condition; ecules on beads, or by dispensing an aminooxy-containing A-3) a step of adding a reducing agent to the sample to compound. In the case of the latter, the treatment of item G. is thereby place the sample under a reaction condition; not carried out. 55 A-4) a step of adding an —SH protecting agent to the I. The released sugar chain is recovered in a filtrate. sample to thereby place the sample under a reaction J. A. MALDI matrix is added to the Sugar chain-containing condition; filtrate, and then the mixture is added dropwise on a MALDI A-5) a step of adding a proteolytic enzyme to the sample to plate. thereby place the sample under a reaction condition; Patent Document 1: WO 2004/058687 60 A-6) a step of deactivating the proteolytic enzyme; and Non-patent Literature 1: Mol. Cell. Proteomics. 2007: A-7) a step of adding a Sugar chain releasing enzyme to the 6:1437-45. Quantitative glycomics of human whole serum sample to thereby release the Sugar chain; glycoproteins based on the standardized protocol for lib B) a detection sample preparing step of preparing the erating N-glycans. Kita Y. Miura Y. Furukawa J, Nakano released Sugar chain for use in detection, the step comprising M, ShinoharaY, Ohno M, Takimoto A, Nishimura S. 65 the following steps: Non-patent Literature 2: Chem. Euro. J. 2007: 13:4797-804. B-1) a step of contacting the sample prepared in the step Rapid and simple solid-phase esterification of Sialic acid (A) with a Sugar chain-capturing bead to thereby place US 8,877,454 B2 3 4 the sample under the conditions allowing the released when subjecting the tagged Sugar chain sample solution to Sugar chain in the sample to bind to the bead, and thus MALDI-TOF MS, comprising the following step (C-7): producing a captured Sugar chain Sample; C-7) a step of mixing the tagged Sugar chain sample solu B-2) a step of adding a protein denaturing agent to the tion with a matrix for mass spectrometry, and dotting the mixture on a plate for determination; and captured Sugar chain sample to thereby place the cap D) a step of conducting an analysis of the Sugar chain to be tured Sugar chain sample under a reaction condition; determined. B-3) a step of Washing the captured Sugar chain sample, (2) The method according to item (1), the preceding steps and then discarding the residual washing liquid by Suc are further characterized by at least any one of the following: tion; A) in the Sugar chain releasing step of releasing a Sugar B-4) a step of adding a salt releasing agent for the Sugar 10 chain in a sample to prepare a Sugar chain sample for analysis: chain capturing agent on beads to the captured Sugar A-1) the sample is a body fluid, a cell extract or a tissue chain sample, and then discarding the salt releasing eXtract, agent by Suction; A-2) the solubilizing agent is 1-propanesulfonic acid, 2-hydroxy-3-lauramide (PHL), 1-propanesulfonic acid, B-5) a step of adding a protective agent to the captured 15 2-hydroxy-3-myristamide (PHM), 2-hydroxy-3-sulfo Sugar chain sample to thereby place the captured Sugar propyl laurate (HSD) or an equivalent thereto, or chain under a reaction condition; the reaction condition is at 25°C. to 42°C.; B-6) a step of adding an acid to the captured Sugar chain A-3) the reducing agent is dithiothreitol (DTT), TCEP sample, and discarding the acid by Suction; (Tris(2-carboxyethyl)phosphine hydrochloride solu B-7) a step of adding an organic reaction solvent to the tion, 0.5 M), or an equivalent thereto, or captured Sugar chain Sample: the reaction condition is at room temperature to 80°C.; A-4) the -SH protecting agent is iodoacetamide (IAA) or B-8) a step of removing moisture and solvents in the bead; an equivalent thereto, or B-9) a step of adding an alkyl esterifying agent to the the reaction condition is at 20 to 37° C. in the dark; captured Sugar chain sample to thereby place the cap A-5) the proteolytic enzyme is trypsin, chymotrypsin oran tured Sugar chain sample under a reaction condition, and 25 equivalent thereto, or alkylating the carboxylic acid of Sialic acid; the reaction condition is at 25 to 42°C.; B-10) a step of adding the organic reaction solvent to the A-6) the condition for deactivating comprises heating to captured Sugar chain sample, and discarding the organic 65° C. or higher; A-7) the Sugar chain releasing enzyme is peptide-N-gly reaction solvent by Suction; 30 cosidase F. peptide-N4-(acetyl-3-glucosaminyl)-aspar B-11) a step of washing the captured Sugar chain sample, agine amidase (PNGaseF), Endo H or an equivalent and subsequently discarding the residual washing liquid thereto, or by Suction; the reaction conditions for the Sugar chain releasing B-12) a step of releasing a Sugar chain sample from the enzyme are at 25°C. to 42°C.; captured Sugar chain sample, wherein when an analysis 35 with regard to the step (B), requiring tagging is conducted, the Sugar chain in the B-1) the bead is a bead or magnetic bead having a Sugar captured Sugar chain sample is tagged with a labeling chain capturing group which includes an aminooxy reagent and is released from the bead; and group, an N-alkylaminooxy group, a hydrazide group, B-13) a step of dissolving the released Sugar chain sample an azide group, a thiosemicarbazide group, a cysteine to produce a Sugar chain sample solution; 40 residue or a derivative thereof bound thereto, or the condition in which the released Sugar chain in the C) when performing mass spectrometry using a plate, a sample binds to the bead is at 25 to 80°C.; step of producing a plate for mass spectrometry having the B-2) the denaturing agent is guanidine hydrochloride, urea, captured Sugar chain dotted thereon, the step comprising: Sodium dodecyl Sulfate or an equivalent thereto, or C-1) a step of disposing the tagged Sugar chain sample 45 the reaction condition involves adding at room tempera solution obtained in the step (B) on a plate for recovery: ture, and maintaining the temperature to allow the bead to and the step also optionally comprising the steps (C-2) to sufficiently swell (from 10 seconds to 5 minutes); (C-6): B-3) the washing is performed using water; C-2) a step of disposing the tagged Sugar chain sample B-4) the Sugar chain capturing agent on the bead is an Solution from the plate for recovery and the organic 50 aminooxy group, an N-alkylaminooxy group, a Solvent, on a plate for mixing so as to obtain a concen hydrazide group, an azide group, a thiosemicarbazide tration at which the Sugar chain adsorbs to a solid phase; group, a cysteine residue or a derivative thereof, and the C-3) a step of providing a solid phase carrier-enclosed salt releasing agent is triethylamine or an equivalent plate; thereto in the case of hydrazide, and is triethylamine or 55 an equivalent thereto in the case of an N-alkylaminooxy C-4) a step of activating the solid phase carrier-enclosed group; plate according to the phase of the Solid phase carrier B-5) the protective agent is acetic anhydride, Succinic enclosed plate, and washing the Solid phase carrier-en anhydride or another acid anhydride, or an equivalent closed plate: thereto, or C-5) a step of adding the tagged Sugar chain sample solu 60 the reaction condition uses acetic anhydride/methanol at tion to the Solid phase carrier-enclosed plate, and con 15 to 37° C.; ditioning the tagged Sugar chain sample solution to a B-6) the acid is hydrochloric acid or another inorganic Solvent having a polarity appropriate for the phase of the acid, or an equivalent acid at pH2 to 3: Solid phase carrier-enclosed plate; B-7) the step comprises a step for replacing with a hydro C-6) a step of recovering the tagged Sugar chain sample 65 philic organic solvent before replacing with the organic Solution by Suction from the Solid phase carrier-enclosed reaction solvent, and the hydrophilic organic solvent is a plate to a second plate for recovery; and lower alcohol Such as methanol or ethanol, acetonitrile, US 8,877,454 B2 5 6 oracetone, while the organic reaction solvent is dioxane, when the beads are magnetic beads, the magnetic beads are acetonitrile, tetrahydrofuran or an equivalent thereto; beads having a modifiable functional group, a hydrazide B-8) the step of removing the solvent and the moisture in group or an aminooxy group. the bead comprises wiping of the bottom with a filter (3) The method according to item (1), further characterized paper, a blotting paper, a gauze, a towel, a hand towel, a 5 by at least one step among comprising the following: tissue paper or a cotton sheet; A) a Sugar chain releasing step of releasing a Sugar chain in B-9) the alkyl esterifying agent is methyl-p-tolyl-triazene a sample, the step comprising the following steps: (MTT), ethyl-p-tolyl-triazene (ETT), butyl-p-tolyl-tria A-1) a step of providing blood serum as a sample on a filter Zene (BTT) or an equivalent thereto, or plate; the reaction condition uses 100 mMMTT/dioxane at 20 to 10 A-2) a step of adding 1-propanesulfonic acid, 2-hydroxy 80° C. for 30 minutes to 5 hours: 3-lauramide (PHL) or 1-propanesulfonic acid, 2-hy B-10) the organic reaction solvent is dioxane, acetonitrile, droxy-3-myristamide (PHM)/ammonium bicarbonate, tetrahydrofuran or an equivalent thereto; and allowing the mixture to react for 5 to 60 minutes (for B-11) the washing is performed using at least one selected 15 example, 10 min.) at 25 to 42°C. (for example, 37°C.); from the group consisting of methanol, a NaCl Solution A-3) a step of adding dithiothreitol (DTT) to the sample, and water, allowing the mixture to react for 10 to 60 (for example, B-12) the tagging is carried out, Such that the tagging is 30 min.) minutes at 50 to 80° C. (for example, 60° C.), performed using a chromophore capable of absorbing and then cooling the reaction mixture to room tempera ultraviolet and visible rays, a tag having a structure 2O ture; emitting fluorescence, an affinity tag having a molecule A-4) a step of adding iodoacetamide (IAA), and allowing capable of interacting with another molecule, a tag hav the mixture to react for 0.5 to 2 hours (for example, 1 h) ing a functional group capable of specifically reacting at room temperature in the dark; with a functional group, a tag having a functional group A-5) a step of adding trypsin to the sample, and allowing in a hydrophobic structure, or a tag having a metal ion 25 the mixture to react for 30 to 120 minutes (for example, ligand, and the tagging is conducted by adding acetic 60 min.) at 25 to 42°C. (for example, 37° C): acid, acetonitrile, an acetate buffer or an equivalent A-6) a step of heating the sample to 80 to 100° C. (for thereto; and example, 90° C.) for 1 to 10 minutes (for example, 5 B-13) the dissolving of the tagged captured Sugar chain min.), and then cooling the sample to room temperature; sample is performed using water, an aqueous Solution or 30 and an equivalent thereto; A-7) a step of adding PNGaseF, and allowing the mixture C) in the step of producing a plate for mass spectrometry to react for 6 to 24 hours (for example, 12h) at 25 to 42° having the captured Sugar chain sample dotted thereon; C. (for example, 37° C.); C-1) the disposing on the plate for recovery is conducted B) a detection sample preparing step of preparing the under the conditions of removing the reagent for tag 35 released Sugar chain for use in detection, the step comprising ging: the following steps: C-2) the concentration at which the Sugar chain adsorbs to B-1) a step of contacting the captured Sugar chain sample the solid phase is 80 to 90% in an organic solvent; prepared in the step (A) with beads for capturing Sugar C-3) the solid phase carrier-enclosed plate is of multi-well chains, to thereby allow binding at 40 or higher (for type and includes a Surface of a resin or membrane 40 example, 80° C.), and thus producing a captured Sugar suitable for solid phase extraction; chain sample; C-4) when the Solid phase carrier-enclosed plate is in nor B-2) a step of adding guanidine hydrochloride to the cap mal phase mode, washing is conducted sequentially tured Sugar chain sample to thereby place the captured with water and acetonitrile, and when the solid phase Sugar chain sample under a reaction condition, and then carrier-enclosed plate is in reverse phase mode, washing 45 discarding the reaction liquid by Suction; is conducted sequentially with a lower alcohol Such as B-3) a step of washing the captured Sugar chain sample methanol and water; with water, and then discarding the water by Suction; C-5) the solvent having an opposite polarity is a hydropho B-4) a step of washing the captured Sugar chain sample bic organic solvent in the case of the normal phase mode, with triethylamine, and then discarding the triethy and is a hydrophilic solvent in the case of the reverse 50 lamine by Suction; phase mode; B-5) a step of adding acetic anhydride to the captured Sugar C-6) the second plate for recovery is of multi-well type and chain sample to thereby place the captured Sugar chain includes a surface of a resin or membrane suitable for sample under the reaction conditions of using 10% ace Solid phase extraction; and tic anhydride/methanol at room temperature for 10 min C-7) the matrix for mass spectrometry is 2,5-dihydroxy 55 utes to 2 hours (for example, 30 min.), and then discard benzoic acid or an equivalent thereto, and the dotting of ing the acetic anhydride by Suction; the tagged Sugar chain sample solution on the matrix for B-6) a step of adding hydrochloric acid to the captured mass spectrometry is conducted in mixture or in Sugar chain sample, and discarding the hydrochloric sequence, and is optionally diluted; acid by Suction; D) the analysis of the sugar chain to be determined is 60 B-7) a step of adding methanol to the captured Sugar chain conducted by high performance liquid chromatography sample, discarding the methanol by Suction, and then (HPLC), liquid chromatography-electrospray ionization adding dioxane to the captured Sugar chain sample: mass spectrometry (LC-ESIMS), matrix assisted laser des B-8) a step of wiping the bottom with a cotton sheet: orption ionization Time-of-Flight (MALDI-TOF), or an B-9) a step of adding methyl-p-tolyl-triazene (MTT) to the equivalent thereto, while when using a coloring reagent or 65 captured Sugar chain sample, and allowing the mixture biotin in the tagging, a step of removing any excess coloring to react for 30 to 120 minutes (for example, 60 min.) at reagent is carried out as necessary, and 60° C. or higher (for example, 80° C.); US 8,877,454 B2 7 8 B-10) a step of adding dioxane to the captured Sugar chain B) a detection sample preparing step of preparing the sample, and discarding the dioxane by Suction; release Sugar chain for use in detection, the step comprising B-11) a step of Washing the captured Sugar chain sample the following steps: sequentially with methanol, a NaCl solution and water, B-1) a step of contacting the sample prepared in the step and then discarding the water by Suction; (A) with beads to thereby place the sample under the B-12) a step of adding acetic acid and acetonitrile to the conditions allowing the released Sugar chain in the captured Sugar chain sample, and tagging the Sugar sample to bind to the bead, and thus producing a cap chain in the captured Sugar chain sample using aminoOX tured Sugar chain sample: ytryptophanyl arginine methyl ester/water, O-benzylhy B-2) a step of adding a protein denaturing agent to the droxylamine hydrochloride/water, or anthraniloyl 10 captured Sugar chain sample to thereby place the cap hydrazine/water, and tured Sugar chain sample under a reaction condition; B-13) a step of adding water to the tagged captured Sugar B-3) a step of Washing the captured Sugar chain sample, chain sample to produce a tagged Sugar chain sample and then discarding the residual washing liquid by Suc Solution; tion; 15 B-4) a step of adding a salt releasing agent for the Sugar C) a step of producing a plate for mass spectrometry having chain capturing agent on beads to the captured Sugar the tagged captured Sugar chain sample dotted thereon, the chain sample, and then discarding the salt releasing step comprising: agent by Suction; C-1) a step of disposing the tagged Sugar chain sample B-5) a step of adding a protective agent to the captured solution obtained in the step (B) on a plate for recovery: Sugar chain sample to thereby place the captured Sugar C-2) a step of disposing the tagged Sugar chain sample chain sample under a reaction condition; solution from the plate for recovery and acetonitrile on a B-6) a step of adding an acid to the captured Sugar chain plate for mixing, so as to achieve a final concentration of sample, and discarding the acid by Suction; acetonitrile of 80 to 90%; B-7) a step of adding an organic reaction solvent to the C-3) a step of providing a solid phase carrier-enclosed plate 25 captured Sugar chain Sample: which is in normal phase mode; B-8) a step of removing the solvent and the moisture in the C-4) a step of washing the solid phase carrier-enclosed bead; plate sequentially with water and acetonitrile, and dis B-9) a step of adding an alkyl esterifying agent to the carding water and acetonitrile by Suction; captured Sugar chain sample to thereby place the cap C-5) a step of adding the tagged Sugar chain sample solu 30 tured Sugarchain sample under a reaction condition, and tion to the Solid phase carrier-enclosed plate, discarding alkylating the carboxylic acid of Sialic acid; the liquid, washing the plate with acetonitrile, and add B-10) a step of adding the organic reaction solvent to the ing 1 to 20% (for example, 5%) acetonitrile thereto; captured Sugar chain sample, and discarding the organic reaction solvent by Suction; C-6) a step of recovering the bead by suction from the solid 35 B-11) a step of washing the captured Sugar chain sample, phase carrier-enclosed plate to the second plate for and then discarding the residual washing liquid by Suc recovery; and tion; C-7) a step of adding 2,5-dihydroxybenzoic acid in 20 to B-12) a step of releasing a Sugar chain sample from the 40% (for example, 30%) acetonitrile, to the tagged sugar captured Sugar chain sample, wherein when an analysis chain sample solution, and mixing and dotting the mix 40 requiring tagging is performed, the Sugar chain in the ture; and captured Sugar chain sample is tagged with a labeling D) a step of performing mass spectrometry by MALDI reagent and is released from the bead; and TOF MIS. B-13) a step of dissolving the released Sugar chain sample (4) The method according to item (3), wherein the Sugar to produce a Sugar chain sample solution. chain-capturing bead are magnetic beads, and separation is 45 (6) The method according to item (5), further characterized conducted by means of a magnetic field instead of the dis by any of the features of item (2), (3) or (4). carding by Suction. (7) A Sugar chain analyzing apparatus, comprising the fol (5) A method for preparing a pretreatment sample for ana lowing units: lyzing a Sugar chain in a sample, the method comprising the 1) a plate mounting stand, which is optionally heatable following steps: 50 and/or movable; 2) a reagent storing unit that stores one or a plurality of A) a Sugar chain releasing step of releasing a Sugar chain in prepared reagents at a temperature required for storage, a sample, the step comprising the following steps: wherein the reagent storing unit is a storage connected to a A-1) a step of providing the sample on a plate for reaction; rack or a valve, and the reagent storing unit stores one or A-2) a step of adding a solubilizing agent to the sample to 55 several reagents selected from the group consisting of a solu thereby place the sample under a reaction condition; bilizing agent, a reducing agent, an —SH protecting agent, a A-3) a step of adding a reducing agent to the sample to proteolytic enzyme, a Sugar chain releasing enzyme, a Sugar thereby place the sample under a reaction condition; chain-capturing bead, a protein denaturing agent, a washing A-4) a step of adding an —SH protecting agent to the liquid, a salt releasing agent for a Sugar chain capturing agent, 60 a protective agent, an acid, an organic reaction solvent, an sample to thereby place the sample under a reaction alkyl esterifying agent, a tagging agent, a solvent for the condition; tagged Sugar chain sample, an organic solvent for adsorbing A-5) a step of adding a proteolytic enzyme to the sample to to a solid phase, a solvent for acclimation, and a matrix for thereby place the sample under a reaction condition; mass spectrometry; A-6) a step of deactivating the proteolytic enzyme; and 65 3) a nozzle and/or a valve for dispensing each reagent from A-7) a step of adding a Sugar chain releasing enzyme to the the reagent storing unit; sample to release the Sugar chain; and 4) a plate moving unit; US 8,877,454 B2 10 5) optionally, a Suction discarding unit or a magnetic field a row along the longitudinal direction and above the installa generating unit: tion space for the casing base; 6) optionally, a shaking/stirring unit; a filter plate moving mechanism that retains a filter plate 7) a plate storing unit; and and moves the filter plate to each of the first low-pressure 8) a unit that performs mass spectrometry. recovering device, the first Suction discarding device and the (8) The apparatus according to item (7), characterized in second constant-temperature bath in sequence, as well as a that the features of any one of items (2) to (4) are manifested. second low-pressure recovering device, being in a frame (9) An automatic Sugar chain pretreatment apparatus, com form, which depressurizes while having an SPE plate prising the following units: mounted on the upper opening, and receives the liquid that 1) a plate mounting stand, which is heatable and/or mov 10 has passed through the solid phase body of the SPE plate, into able as necessary; a microplate installed inside the recovering device; a second 2) a reagent storing unit that stores one or a plurality of Suction discarding device, being in a frame form, which prepared reagents at a temperature required for storage, depressurizes and suctions while having an SPE plate wherein the reagent storing unit is a storage connected to a mounted on the upper opening, and discards the liquid that rack or a valve, and the reagent storing unit stores one or 15 has passed through the Solid phase body; and a target plate several reagents selected from the group consisting of a solu receiving stand, which holds a target plate that is dotted on the bilizing agent, a reducing agent, an —SH protecting agent, a surface with the sample that has been finished with the final proteolytic enzyme, a Sugar chain releasing enzyme, a Sugar treatment step, all of these devices being disposed and chain-capturing bead, a protein denaturing agent, a washing arranged in a row along the longitudinal direction and above liquid, a salt releasing agent for a Sugar chain capturing agent, the installation space for the casing base; a protective agent, an acid, an organic reaction solvent, an an SPE plate moving mechanism that retains an SPE plate alkyl esterifying agent, a tagging agent, a solvent for the and moves the SPE plate between the second low-pressure tagged Sugar chain sample, an organic solvent for adsorbing recovering device and the second Suction discarding device; to a solid phase, a solvent for acclimation, and a matrix for a control device that is installed in the casing base, and has mass spectrometry; 25 been inputted with the operation protocol; 3) a nozzle and/or a valve for dispensing each reagent from a microplate having a plurality of wells arranged in a the reagent storing unit; matrix array, which is covered with a sheet while biological 4) a plate moving unit; samples have been injected into each well; a filterplate having 5) optionally, a Suction discarding unit or a magnetic field a plurality of filters arranged in a matrix array; and an SPE generating unit: 30 plate for sample in a trace amount, having a plurality of solid 6) optionally, a shaking/stirring unit; and phase bodies arranged in a matrix array, 7) a plate storing unit. wherein the apparatus is made to operate each of the (10) The apparatus according to item (9), characterized in devices according to the operation protocol inputted to the that the features of any one of items (2) to (4) are manifested. control device. Alternatively, the present invention provides the following 35 (12) An automatic Sugar chain pretreatment apparatus, in connection with the apparatus of item (9). comprising: (11) An automatic Sugar chain pretreatment apparatus, a casing base provided with a cover capable of freely open comprising: ing and closing; and a dispensing head moving mechanism a casing base provided with a cover capable of freely open that is installed inside the casing base and moves a below ing and closing; and a dispensing head moving mechanism 40 described dispensing head in longitudinal and transverse that is installed inside the casing base and moves a dispensing directions; head in longitudinal and transverse directions as described a dispensing head that raises and lowers a plurality of below: dispensing needles arranged in a row, altogether by means of a dispensing head that raises and lowers a plurality of an elevating mechanism; dispensing needles arranged in a row, altogether by means of 45 a first constant-temperature bath installed above the instal an elevating mechanism; lation space for the casing base, which is equipped with a unit a first constant-temperature bath installed above the instal that conducts heating and cooling of a receiving stand that lation space for the casing base, which is equipped with a unit holds a microplate, and is provided with a lid having an inner that conducts heating and cooling of a receiving stand that lid to cover the upper part of the receiving stand; holds a microplate, and is provided with a lid having an inner 50 a reagent rack and a plurality of microplates for mixing, lid to cover the upper part of the receiving stand; which are installed above the installation space for the casing a reagent rack and a plurality of microplates for mixing, base; which are installed above the installation space for the casing a first low-pressure recovering device, being in a frame base; form, which depressurizes while having a filterplate mounted a first low-pressure recovering device, being in a frame 55 on the upper opening, and receives the liquid that has passed form, which depressurizes while having a filterplate mounted through the filter of the filter plate, into a microplate installed on the upper opening, and receives the liquid that has passed inside the recovering device; a first Suction discarding device, through the filter of the filter plate, into a microplate installed being in a frame form, which depressurizes and Suctions inside the recovering device; a first Suction discarding device, while having a filter plate mounted on the upper opening, and being in a frame form, which depressurizes and Suctions 60 discards the liquid that has passed through the filter; a bottom while having a filter plate mounted on the upper opening, and wiper, which is provide with a planar wiping material on the discards the liquid that has passed through the filter; and a upper Surface, and is intended to wipe out the liquid adhering second constant-temperature bath, which is equipped with a to the lower surface of the bottom of the filter plate; and a unit that conducts heating and cooling of a receiving stand second constant-temperature bath, which is equipped with a that holds a filter plate, and is provided with an automatically 65 unit that conducts heating and cooling of a receiving stand opening and closing lid to cover the upper part of the receiv that holds a filter plate, and is provided with an automatically ing stand, all of these devices being disposed and arranged in opening and closing lid to cover the upper part of the receiv US 8,877,454 B2 11 12 ing stand, all of these devices being disposed and arranged in installed at both ends in the length direction of a Supporting a row along the longitudinal direction and above the installa plate that is installed to standalong the longitudinal direction tion space for the casing base; of the casing base, with one of the pulleys being made to be a filter plate moving mechanism that retains a filter plate rotary driven by a step motor, wherein a horizontally moving and moves the filter plate to each of the first low-pressure plate is connected to a belt hung between the two pulleys and recovering device, the first Suction discarding device, the revolved, a vertical moving unit is installed on the horizon bottom wiper and the second constant-temperature bath in tally moving plate, and the vertical moving unit is ascended sequence, as well as a second low-pressure recovering device, and descended by a vertically moving rod that Supports a being in a frame form, which depressurizes while having an receiving frame intended to receive an inner frame at the SPE plate mounted on the upper opening, and receives the 10 liquid that has passed through the solid phase body of the SPE upper end, and slides in the vertical direction along a guide plate, into a microplate installed inside the recovering device; installed vertically inside the Supporting frame, and by a ball a second Suction discarding device, being in a frame form, screw connected to the rotating axis of a motor fixed inside the which depressurizes and suctions while having an SPE plate Supporting frame, and wherein the mechanisms are each con mounted on the upper opening, and discards the liquid that 15 stituted to have an elevating rod connected to the vertically has passed through the Solid phase body; and a target plate moving rod. receiving stand, which holds a target plate that is dotted on the (17) The automatic Sugar chain pretreatment apparatus surface with the sample that has been finished with the final according to item (11), (12), (13), (14), (15) or (16), wherein treatment step, all of these devices being disposed and the second constant-temperature bath is a second constant arranged in a row along the longitudinal direction and above temperature bath having a main body part that is constructed the installation space for the casing base; by including a cartridgeheater built in at the internal center of an SPE plate moving mechanism that retains an SPE plate the receiving stand formed from an aluminum block, and also and moves the SPE plate between the second low-pressure an air circulation path installed across from the internal center recovering device and the second Suction discarding device; to the Surface, and by disposing a Peltier element and a heat a control device that is installed in the casing base, and has 25 sink at the lower part of the receiving stand, wherein an air been inputted with the operation protocol; circulation path which is in communication with the above air a microplate having a plurality of wells arranged in a circulation path is provided between the upper part of the matrix array, which is covered with a sheet while biological receiving stand in the main body part and the filter plate samples have been injected into each well; a filterplate having located thereon, and a duct and a fan are further provided to let a plurality of filters arranged in a matrix array; and an SPE 30 the air discharged out of the filter plate flow again into the plate for sample in a trace amount, having a plurality of solid receiving stand to thereby circulate, while the upper part of phase bodies arranged in a matrix array, the receiving stand in the main body part is covered by a lid. wherein the apparatus is made to operate each of the (A1) devices according to the operation protocol inputted to the A Sugar chain releasing method for releasing a Sugar chain control device. 35 in a sample, the method comprising the following steps: (13) The automatic Sugar chain pretreatment apparatus A) a Sugar chain releasing step of releasing a Sugar chain in according to item (1) or (2), wherein the dispensing head is a a sample, the step comprising the following steps: dispensing head constituted to include a Supporting frame; an A-1) a step of providing the sample on a plate for reaction; elevator stand that slides in the vertical direction along a guide A-2) a step of adding a solubilizing agent to the sample to rod installed on the supporting frame in parallel with the 40 thereby place the sample under a reaction condition; dispensing needles; a driving motor fixed on the Supporting A-3) a step of adding a reducing agent to the sample to frame; an elevator stand moving mechanism that has a ball thereby place the sample under a reaction condition; screw connected to the rotating axis of the driving motor and A-4) a step of adding an —SH protecting agent to the moves the elevator stand in the vertical direction; and dis sample to thereby place the sample under a reaction pensing needles retained in a dispensing needle holder 45 condition; installed on the elevator stand. A-5) a step of adding a proteolytic enzyme to the sample to (14) The automatic Sugar chain pretreatment apparatus thereby place the sample under a reaction condition; according to item (13), wherein a cylinder is fixed vertically A-6) a step of deactivating the proteolytic enzyme; and downward at the lower part of the supporting frame in the A-7) a step of adding a Sugar chain releasing enzyme to the dispensing head, and at the same time, a pressurizing plate is 50 sample to thereby release the Sugar chain. adhered at the tip of the piston rod of the cylinder. (A2) (15) The automatic Sugar chain pretreatment apparatus The method according to item (A1), wherein the preceding according to item (11), (12), (13) or (14), wherein the first steps are further characterized by at least any one of the constant-temperature bath is a first constant-temperature bath following: having a main body part that is constructed by including a 55 A) in the Sugar chain releasing step of releasing a Sugar cartridge heater built in at the internal center of the receiving chain in a sample to prepare a Sugar chain sample for analysis: stand formed from an aluminum block, and also by disposing A-1) the sample is a body fluid, a cell extract or a tissue a Peltier element and a heat sink at the lower part of the eXtract, receiving stand, wherein the upper part of the receiving stand A-2) the solubilizing agent is 1-propanesulfonic acid, in the main body part is covered by a lid which has a silicone 60 2-hydroxy-3-lauramide (PHL), 1-propanesulfonic acid, sheet pasted on the inside and has an inner lid with a built-in 2-hydroxy-3-myristamide (PHM), 2-hydroxy-3-sulfo cartridge heater at the internal center, the inner lid being propyl laurate (HSD) or an equivalent thereto, and elastically supported via a spring. the reaction condition is at 25°C. to 42°C.; (16) The automatic Sugar chain pretreatment apparatus A-3) the reducing agent is dithiothreitol (DTT), TCEP according to item (11), (12), (13), (14) or (15), wherein the 65 (Tris(2-carboxyethyl)phosphine hydrochloride solu filter plate moving mechanism and the SPE plate moving tion, 0.5 M), or an equivalent thereto, and mechanism are moving mechanisms each having a pulley the reaction condition is at room temperature to 80°C.; US 8,877,454 B2 13 14 A-4) the -SH protecting agent is iodoacetamide (IAA) or B-6) a step of adding an acid to the captured Sugar chain an equivalent thereto, and sample, and discarding the acid by Suction; the reaction condition is at 20 to 37°C. in the dark; B-7) a step of adding an organic reaction solvent to the A-5) the proteolytic enzyme is trypsin, chymotrypsin oran captured Sugar chain Sample: equivalent thereto, and B-8) a step of removing the solvent and the moisture in the the reaction condition is at 25 to 42°C.; bead; A-6) the conditions for deactivating include heating to 65° B-9) a step of adding an alkyl esterifying agent to the C. or higher; captured Sugar chain sample to thereby place the cap A-7) the Sugar chain releasing enzyme is peptide-N-gly tured Sugarchain sample under a reaction condition, and cosidase F. peptide-N4-(acetyl-3-glucosaminyl)-aspar 10 alkylating the carboxylic acid of Sialic acid; agine amidase (PNGaseF), Endo H or an equivalent B-10) a step of adding the organic reaction solvent to the thereto, and captured Sugar chain sample, and discarding the organic the reaction conditions for the Sugar chain releasing reaction solvent by Suction; enzyme are at 25° C. to 42° C. 15 B-11) a step of washing the captured Sugar chain sample, (A3) and Subsequently discarding the residual washing liquid The method according to item (A1) or (A2), further com by Suction; prising at least one of the following steps: B-12) a step of releasing a Sugar chain sample from the A) a Sugar chain releasing step of releasing a Sugar chain in captured Sugar chain sample, wherein when an analysis a sample, the step comprising the following steps: requiring tagging is conducted, the Sugar chain in the A-1) a step of providing blood serum as a sample on a filter captured Sugar chain sample is tagged with a labeling plate; reagent and is released from the bead; and A-2) a step of adding 1-propanesulfonic acid, 2-hydroxy B-13) a step of dissolving the released Sugar chain sample 3-lauramide (PHL) or 1-propanesulfonic acid, 2-hy to produce a Sugar chain sample solution. droxy-3-myristamide (PHM)/ammonium bicarbonate, 25 (B2) and allowing the mixture to react for 5 to 60 minutes at The method according to item (B1), wherein the preceding 25 to 42° C.: steps are further characterized by at least any one of the A-3) a step of adding dithiothreitol (DTT) to the sample, following: allowing the mixture to react for 10 to 60 minutes at 50 B-1) the bead is a bead or magnetic bead having a Sugar to 80°C., and then cooling the reaction mixture to room 30 chain capturing group which includes an aminooxy temperature; group, an N-alkylaminooxy group, a hydrazide group, A-4) a step of adding iodoacetamide (IAA), and allowing the mixture to react for 0.5 to 2 hours at room tempera an azide group, a thiosemicarbazide group, a cysteine ture in the dark; residue or a derivative thereof bound thereto, or A-5) a step of adding trypsin to the sample, and allowing 35 the conditions in which the released Sugar chain in the the mixture to react for 30 to 120 minutes at 25 to 42°C.; sample binds to the bead are at 25 to 80° C.; A-6) a step of heating the sample to 80 to 100° C. for 1 to B-2) the denaturing agent is guanidine hydrochloride, urea, 10 minutes, and then cooling the sample to room tem Sodium dodecyl Sulfate or an equivalent thereto, or perature; and the reaction conditions involve adding at room tempera A-7) a step of adding PNGaseF, and allowing the mixture 40 ture, and maintaining the temperature to allow the bead to to react for 6 to 24 hours at 25 to 42°C. sufficiently swell (from 10 seconds to 5 minutes); (A4) B-3) the washing is performed using water; The method according to any one of items (A1) to (A3), B-4) the Sugar chain capturing agent on the bead is an which is used to prepare a pretreatment sample for an analysis aminooxy group, an N-alkylaminooxy group, a of a Sugar chain in a sample. 45 hydrazide group, an azide group, a thiosemicarbazide (B1) group, a cysteine residue or a derivative thereof, and the A detection sample preparing method for preparing a salt releasing agent is triethylamine or an equivalent released Sugar chain for use in detection, the method com thereto in the case of hydrazide, and is triethylamine or prising the following steps: an equivalent thereto in the case of an N-alkylaminooxy B-1) a step of contacting the sample with a Sugar chain 50 group; capturing bead to thereby place the sample under the B-5) the protective agent is acetic anhydride, Succinic conditions allowing the released Sugar chain in the anhydride or another acid anhydride, or an equivalent sample to bind to the bead, and thus producing a cap thereto, or tured Sugar chain sample: the reaction conditions use acetic anhydride/methanol at B-2) a step of adding a protein denaturing agent to the 55 15 to 37° C.; captured Sugar chain sample to thereby place the cap B-6) the acid is hydrochloric acid or another inorganic tured Sugar chain sample under a reaction condition; acid, or an equivalent acid at pH2 to 3: B-3) a step of Washing the captured Sugar chain sample, B-7) the step includes a step of replacing with a hydrophilic and then discarding the residual washing liquid by Suc organic solvent before replacing with the organic reac tion; 60 tion solvent, and the hydrophilic organic solvent is a B-4) a step of adding a salt releasing agent for the Sugar lower alcohol Such as methanol or ethanol, acetonitrile, chain capturing agent on beads to the captured Sugar or acetone, while the organic reaction solventis dioxane, chain sample, and then discarding the salt releasing acetonitrile, tetrahydrofuran or an equivalent thereto; agent by Suction; B-8) the step of removing the solvent and the moisture in B-5) a step of adding a protective agent to the captured 65 the bead includes wiping of the bottom with a filter Sugar chain sample to thereby place the captured Sugar paper, a blotting paper, a gauze, a towel, a hand towel, a chain under a reaction condition; tissue paper or a cotton sheet; US 8,877,454 B2 15 16 B-9) the alkyl esterifying agent is methyl-p-tolyl-triazene ytryptophanyl arginine methyl ester/water, O-benzylhy (MTT), ethyl-p-tolyl-triazene (ETT), butyl-p-tolyl-tria droxylamine hydrochloride/water, or anthraniloyl Zene (BTT) or an equivalent thereto, or hydrazine/water, and the reaction conditions use 100 mMMTT/dioxane at 20 to B-13) a step of adding water to the tagged captured Sugar 80° C. for 30 minutes to 5 hours: 5 chain sample to produce a tagged Sugar chain sample B-10) the organic reaction solvent is dioxane, acetonitrile, Solution. tetrahydrofuran or an equivalent thereto; (B4) B-11) the washing is performed using at least one selected The method according to item (B3), wherein the sugar from the group consisting of methanol, a NaCl Solution chain-capturing bead are magnetic beads, and separation is and water, 10 conducted by means of a magnetic field instead of the dis B-12) the tagging is carried out, Such that the tagging is carding by Suction. performed using a chromophore capable of absorbing (B5) ultraviolet and visible rays, a tag having a structure The method according to any one of items (B1) to (B4), emitting fluorescence, an affinity tag having a molecule 15 which is used for preparing a pretreatment sample for an capable of interacting with another molecule, a tag hav analysis of a Sugar chain in a sample. ing a functional group capable of specifically reacting (C1) with a functional group, a tag having a functional group A method for producing a plate for mass spectrometry in a hydrophobic structure, or a tag having a metal ion having a captured Sugar chain sample dotted thereon for ligand, and the tagging is conducted by adding acetic performing mass spectrometry using a plate, the method.com acid, acetonitrile, an acetate buffer or an equivalent prising the following steps: thereto; and C-1) a step of disposing a tagged Sugar chain sample solu B-13) the dissolving of the tagged captured Sugar chain tion on a plate for recovery; and, optionally comprising sample is performed using water, an aqueous Solution or the steps (C-2) to (C-6): an equivalent thereto. 25 C-2) a step of disposing the tagged Sugar chain sample (B3) Solution from the plate for recovery and the organic The method according to item (B1) or (B2), further com Solvent, on a plate for mixing so as to obtain a concen prising at least one of the following steps: tration at which the Sugar chain adsorbs to a solid phase; B-1) a step of contacting the captured Sugar chain sample C-3) a step of providing a solid phase carrier-enclosed prepared in the step (A) with beads for capturing Sugar 30 plate; chain, to thereby allow binding at 40 to 80°C., and thus C-4) a step of activating the solid phase carrier-enclosed producing a captured Sugar chain Sample; plate according to the phase of the Solid phase carrier B-2) a step of adding guanidine hydrochloride to the cap enclosed plate, and washing the Solid phase carrier-en tured Sugar chain sample to thereby place the captured closed plate; Sugar chain sample under a reaction condition, and then 35 C-5) a step of adding the tagged Sugar chain sample solu discarding the reaction liquid by Suction; tion to the Solid phase carrier-enclosed plate, and con B-3) a step of Washing the captured Sugar chain sample ditioning the tagged Sugar chain sample solution to a with water, and then discarding the water by Suction; Solvent having a polarity appropriate for the phase of the B-4) a step of Washing the captured Sugar chain sample 40 Solid phase carrier-enclosed plate; with triethylamine, and then discarding the triethy C-6) a step of recovering the tagged Sugar chain sample lamine by Suction; Solution by Suction from the Solid phase carrier-enclosed B-5) a step of adding acetic anhydride to the captured Sugar plate to a second plate for recovery; and chain sample to thereby place the captured Sugar chain when subjecting the tagged Sugar chain sample solution to sample under the reaction conditions of using 10% ace 45 MALDI-TOF MS, comprising the following step (C-7): tic anhydride/methanol at room temperature for 10 min C-7) a step of mixing the tagged Sugar chain sample solu utes to 2 hours, and then discarding the acetic anhydride tion with a matrix for mass spectrometry, and dotting the by Suction; mixture on a plate for determination. B-6) a step of adding hydrochloric acid to the captured (C2) Sugar chain sample, and discarding the hydrochloric 50 The method according to item (C1), wherein the preceding acid by Suction; steps are associated with at least any one of the following B-7) a step of adding methanol to the captured Sugar chain conditions: sample, discarding the methanol by Suction, and then C-1) the disposing on the plate for recovery is conducted adding dioxane to the captured Sugar chain sample: under the conditions of removing the reagent for tag B-8) a step of wiping the bottom with a cotton sheet; 55 B-9) a step of adding methyl-p-tolyl-triazene (MTT) to the ging: captured Sugar chain sample, and allowing the mixture C-2) the concentration at which the Sugar chain adsorbs to to react for 30 to 120 minutes (for example, 60 min.) at the solid phase is 80 to 90% in an organic solvent; 60° C. or higher; C-3) the solid phase carrier-enclosed plate is of multi-well B-10) a step of adding dioxane to the captured Sugar chain 60 type and includes a Surface of a resin or membrane sample, and discarding the dioxane by Suction; suitable for solid phase extraction; B-11) a step of Washing the captured Sugar chain sample C-4) when the solid phase carrier-enclosed plate is in nor sequentially with methanol, a NaCl solution and water, mal phase mode, washing is conducted sequentially and then discarding the water by Suction; with water and acetonitrile, and when the solid phase B-12) a step of adding acetic acid and acetonitrile to the 65 carrier-enclosed plate is in reverse phase mode, washing captured Sugar chain sample, and tagging the Sugar is conducted sequentially with a lower alcohol Such as chain in the captured Sugar chain sample using aminoOX methanol and water; US 8,877,454 B2 17 18 C-5) the solvent having an opposite polarity is a hydropho B-8) cotton sheet; bic organic solvent in the case of the normal phase mode, B-9) methyl-p-tolyl-triazene (MTT): and is a hydrophilic solvent in the case of the reverse B-10) dioxane: phase mode; B-11) methanol, a NaCl solution and water; C-6) the second plate for recovery is of multi-well type and 5 B-12) acetic acid and acetonitrile, and aminooxytryp includes a surface of a resin or membrane suitable for tophanyl-arginine methyl ester/water, O-benzylhydroxy Solid phase extraction; and lamine hydrochloride/water or anthraniloyl hydrazine/water; B-13) water; C-7) the matrix for mass spectrometry is 2,5-dihydroxy C-1) a plate for recovery: benzoic acid or an equivalent thereto, and the dotting of C-2) acetonitrile and a plate for mixing; the tagged Sugar chain sample solution on the matrix for 10 C-3) a solid phase carrier-enclosed plate; mass spectrometry is conducted in mixture or in C-4) water and acetonitrile; sequence, and is diluted as necessary. C-5) acetonitrile and 1 to 20% acetonitrile; (C3) C-6) a second plate for recovery; and The method according to item (C1) or (C2), further com C-7) 2.5-dihydroxybenzoic acid in 20 to 400 acetonitrile. prising at least one of the following steps: 15 (18A) A kit for producing a plate for a reaction comprising C) a step of producing a plate for mass spectrometry having a Sugar chain derived from a sample and a Sugar chain-cap the tagged captured Sugar chain sample dotted thereon, the turing bead, the kit comprising the means for realizing any step comprising: operations of items (1) to (5). C-1) a step of disposing the tagged Sugar chain sample (19) A plate for analysis comprising a Sugar chain derived solution obtained in the step (B) on a plate for recovery: from a sample and a Sugar chain-capturing bead, C-2) a step of disposing the tagged Sugar chain sample wherein the Sugar chain-capturing beadbound by the Sugar solution from the plate for recovery and acetonitrile on a chain released from the sample are dotted on at least one well plate for mixing, so as to achieve a final concentration of of the plate, and the Sugar chain is tagged with aminooxytryp acetonitrile of 80 to 90%; tophanyl-arginine methyl ester/water, O-benzylhydroxy C-3) a step of providing a solid phase carrier-enclosed plate 25 lamine hydrochloride/water or anthraniloyl hydrazine/water. (20) A plate for analyzing a Sugar chain in a sample, which is in normal phase mode; wherein the plate has at least one compartment for analysis, C-4) a step of washing the solid phase carrier-enclosed and a Sugar chain-capturing bead having been dispensed in plate sequentially with water and acetonitrile, and dis advance to said at least one compartment. carding water and acetonitrile by Suction; Therefore, these and other advantages of the present inven C-5) a step of adding the tagged Sugar chain sample solu 30 tion will be made clear to the readers when they read the tion to the Solid phase carrier-enclosed plate, discarding following detailed description with reference to the appended the liquid, washing the plate with acetonitrile, and add drawings. ing 1 to 20% acetonitrile thereto; C-6) a step of recovering the bead by suction from the solid Effect of the Invention phase carrier-enclosed plate to the second plate for 35 recovery; and It was made successful by the present invention to provide C-7) a step of adding 2,5-dihydroxybenzoic acid in 20 to an auto-analyzing apparatus for analyzing a Sugar chain con 40% acetonitrile, to the tagged Sugar chain sample solu tained in a sample Such as a blood serum sample. Thereby, it tion, and mixing and dotting the mixture. was made possible to automatically analyze a large amount of (C4) 40 sample simultaneously. The cumbersome pretreatments such The method according to item (C3), wherein the Sugar as a use of a solublizing agent, reductive alkylation and diges chain-capturing bead are magnetic beads, and separation is tion by trypsin, which were required to release a Sugar chain conducted by means of a magnetic field instead of the dis quantitatively from a glycoprotein, were automated. Further carding by Suction. more, a Sugar chain that has been quantitatively released can (C5) 45 be subjected to a rapid analysis of the Sugar chain by perform The method according to any one of items (C1) to (C4), ing chemoselective capturing and thereby easily removing which uses the method for preparing a pretreatment sample troublesome foreign matters, and it was made possible to treat for an analysis of a Sugar chain in a sample. multiple test samples all at a time. The various processes (18) A kit for producing a plate for a reaction comprising a described above were optimized, and a system adaptable to Sugar chain derived from a sample and a Sugar chain-captur 50 automatic analysis was established. ing bead, the kit comprising: Since the present invention can automate the treatment A-1) the sample: processes of Sugar chain purification, the processes can be A-2) 1-propanesulfonic acid, 2-hydroxy-3-lauramide carried out much more efficiently as compared to the conven (PHL) or 1-propanesulfonic acid, 2-hydroxy-3-myristamide tional processes carried out manually, and purification can be (PHM)/ammonium bicarbonate; 55 conducted at high speed with high accuracy. A-3) dithiothreitol (DTT): Since the apparatus of the present invention makes it pos A-4) iodoacetamide (IAA); sible to performan analysis of multiple samples, a search for A-5) trypsin; Sugar chain biomarkers is anticipated, and it is conceived that A-6) aheating unit; the apparatus of the present invention may be used in diag A-7) PNGaseF: 60 nosis and the like, and in early diagnosis of a disease or the B-1) a Sugar chain-capturing bead; like. B-2) guanidine hydrochloride: B-3) water; BEST MODE(S) FORCARRYING OUT THE B-4) triethylamine; INVENTION B-5) 10% acetic anhydride/methanol: 65 B-6) hydrochloric acid; Hereinafter, the present invention will be described. It will B-7) methanol: be understood as a matter of course that throughout the US 8,877,454 B2 19 20 present specification, unless otherwise particularly stated, a a Sugar having a carboxyl group (for example, aldonic acid in singular expression encompasses the concept of plurality as which the C-1 position has been oxidized to carboxylic acid well. Therefore, it will be understood as a matter of course (for example, D-gluconic acid obtained from oxidized D-glu that, unless otherwise particularly stated, a singular article or cose), uronic acid in which the terminal C atom has been adjective (for example, “a”, “an”, “the or the like in English) 5 converted to carboxylic acid (D-glucuronic acid obtained encompasses the concept of plurality as well. Furthermore, it from oxidized D-); a Sugar having an amino group or will be understood as a matter of course that, unless otherwise a derivative of amino group (for example, acetylated amino particularly stated, the terms used in the present specification group) (for example, N-acetyl-D-glucosamine, N-acetyl-D- are used in the meaning as conventionally used in the related galactosamine, or the like); a Sugar having both an amino art. Therefore, unless defined otherwise, all of the jargons and 10 group and a carboxyl group (for example, N-acetyl technical terms used in the present specification have the (sialic acid), N-acetylmuraminic acid, and same meanings as generally understood by those skilled in the like); a deoxidized Sugar (for example, 2-deoxy-D-ri the art to which the present invention is pertained. In the case bose); a Sulfated Sugar containing a sulfuric acid group; a of contradiction, the present specification (including the defi phosphated Sugar containing a phosphoric acid group; and the nitions) dominates. 15 like. As used herein, when a compound is called as monosac charide, the derivatives mentioned above are also included. DEFINITIONS OF TERMS AND EXPLANATION Alternatively, in regard to forming a hemiacetal struc OF FUNDAMENTAL TECHNOLOGY ture, glycosides forming an acetal structure by reacting with alcohol, also fall in the scope of . (Sugar Chain) The term “interaction” as used herein means that, upon The term "Sugar chain” as used herein means a compound mentioning about two objects, the two objects exert force formed by one or more unit Sugars (monosacchrides and/or onto each other. Examples of such interaction include, but are derivatives thereof) linked together. When two or more unit not limited to, covalent bonding, hydrogen bonding, Van der Sugars are linked, the respective unit Sugars are linked by Waals force, ionic interaction, nonionic interaction, hydro dehydration condensation based on glycoside bond. 25 phobic interaction, electrostatic interaction, and the like. Examples of Such a Sugar chain include Preferably, the interaction is hydrogen bonding, hydrophobic present in the living body (glucose, galactose, , interaction, or the like. The term “covalent bonding as used , , N-acetylglucosamine, N-acetylgalac herein is used in the meaning as conventionally used in the tosamine, sialic acid, and complexes and derivatives thereof), related art, and means a chemical bonding formed by a pair of as well as a wide variety of Sugar chains that have been 30 electrons that are shared by two atoms. The term “hydrogen degraded or derived from complex biomolecules such as bonding as used herein is used in the meaning as conven degraded polysaccharides, glycoproteins, proteoglycans, tionally used in the related art, and means a bonding gener and glycolipids, but examples are not ated when the only extranuclear electron of a hydrogen atom limited to those. As used herein, the term Sugar chain can be is attracted to an atom having high electronegativity, so that used interchangeably with “.” “sugar,” or “car 35 the nucleus of the hydrogenatom is exposed, and this nucleus bohydrate.” Unless otherwise particularly stated, the “sugar attracts in turn another atom having high electronegativity. chain” as used herein may include both a Sugar chain and a For example, the hydrogen bonding is generated between a Sugar chain-containing Substance. hydrogen atom and an atom having high electronegativity The term "sugar chain-containing Substance' as used (such as fluorine, oxygen or nitrogen). herein means a Substance containing a Sugar chain and a 40 The term “capturing of a Sugar chain or a Sugar chain Substance other than a Sugar chain. Such a Sugar chain-con containing Substance' as used herein means that a Sugar chain taining Substance are abundant in the living body, and or a Sugar chain-containing Substance is captured using the examples thereof include polysaccharides present in the liv Sugar chain-capturing bead used in the present invention. The ing body, as well as a variety of Substances such as Sugar Sugar chain-capturing site or Sugar chain-capturing group of chains degraded or derived from complex biomolecules Such 45 the bead according to the present invention preferably as degraded polysaccharides, glycoproteins, proteoglycans, includes an aminooxy group, an N-alkylaminooxy group, a and glycolipids, but are not limited to hydrazide group, an azide group, a thiosemicarbazide group, those. a cysteine residue, and derivatives thereof. The term "monosaccharide' as used herein means polyhy The term "sugar chain-capturing carrier as used herein droxyaldehyde or polyhydroxyketone and derivatives 50 means a carrier for capturing a Sugar chain. The term "sugar thereof, which are not hydrolyzed into molecules simpler chain-capturing bead' as used herein means a bead for cap than these, and include at least one hydroxyl group and at least turing a Sugar chain. Such a carrier or bead includes a part that one aldehyde group or ketone group. are captures a Sugar chain and a part that serves as a carrier or a conventionally represented by the formula: CHO, with bead. The part that captures a Sugar chain can beformed using out being limited thereto, and also include fucose (deoxyhex 55 a Substance which specifically interacts with the Sugar chain ose), N-acetylglucosamine and the like. Here, compounds of of the present invention. For the carrier, a Support can be used, the above formula, in which n=2,3,4, 5, 6, 7, 8, 9 and 10, are and the Substance which specifically interacts with the Sugar respectively called , , tetraose, , , chain of the present invention may function as a carrier perse. , , and decose. In general, these com Such a carrier includes a carrier having “X” in the formula (I) pounds correspond to aldehydes or ketones of chain-type 60 described above. For the bead, any products can be used, and polyhydric alcohols, and the former are called as , magnetic beads are preferred. Examples of the magnetic while the latter being called as . beads include those marketed by Dynabead, but are not lim When particularly mentioned herein, the term "derivative ited thereto. of monosaccharide” means a Substance generated as a result The term "captured Sugar chain sample as used herein of Substitution of one or more hydroxyl groups on an unsub 65 means a Sugar chain Sample captured by a Sugar chain-cap stituted monosaccharide, with another substituent. Deriva turing carrier or a Sugar chain-capturing bead such as tives of such a monosaccharide include, but are not limited to, described above (for example, beads bound by a Sugar chain). US 8,877,454 B2 21 22 Specifically, such a carrier includes a carrier having “X”. by at least one group selected from the group consisting of “X Y” and “X Y—Z’’ bound to a support, and a carrier O S S—S—, N(R) C(=O) , resulting from polymerization of compounds represented by —C(=O) N(R') , and phenylene which may be substi formula (I) and formula (II). In general. Such a carrier tuted; or an alkenylene which may be substituted and may be includes, for example, a carrier having a group shown below. 5 interrupted by at least one group selected from the group consisting of —O—, S—, S–S , – N(R)- C (Sugar Chain-Capturing Functional Group)-(Spacer)-(Po (=O) . C(=O) N(R') , and phenylene which may be lymerizable Functional Group) substituted (wherein R and R are each independently a Here, the Sugar chain-capturing functional group can also hydrogen atom or an alkyl); and Z is a group represented by be described as, for example, a functional group capable of the formula: reacting with the aldehyde group of the Sugar chain in a fluid, 10 and has a structure as illustrated in FIG. 2. R in FIG.2 means the substituent group defined above, but preferably, a sub Chem 2 stituent group which is not likely to adversely affect the R4 polymerization reaction and the interaction with the Sugar chain, is preferred. More specifically, a Substance that can 15 N Z2 specifically interact with the Sugar chain of the present inven ". SC=C-C=C-Z, tion is a compound which can be represented by the following Zl formula: Zl X Y. Z. Formula (I) Z2 Wherein, X is a group represented by the formula: r YC=C-C=C-Z, R4 f Z4 Chem 1 2 25

visZl s Zl 74 Z2 30 N1 NN 2

R4 R5 s f Z3 2 35

vs.--Zl s Zl Z3

2

vs.--R4 (wherein Z' is an oxygenatom or a sulfur atom; Z and Z are each independently an alkylene which may be substituted 45 and may be interrupted by phenylene, or an alkenylene which may be substituted and may be interrupted by phenylene; Z' is an oxygen atom or a sulfur atom; R and Rare each independently a hydrogenatom oran alkyl). In the compound of formula (I) of the present invention. X' is preferably a C1-C10 alkylene or a C2-C10 alkenylene, and the chain 50 length of Y is preferably a chain length equivalent to C1-C25 alkyl. Preferred examples of Y include —(CHCH O), CHCH (wherein, n=1 to 8 is preferred, and particularly, n=1 to 6 is preferred), and Z and Z are preferably each independently a C1-C10 alkylene or a C2-C10 alkenylene. 55 Furthermore, specific examples of the phenylene which may be substituted include the following:

Chem3) 60 ON (wherein X is an alkylene which may be substituted oran O alkenylene which may be substituted;X is an oxygenatom or a sulfur atom; X is an oxygen atom or a sulfur atom; X is / \ { methylene or ethylene; R is a hydrogenatom or an alkyl; R - O O and Rare each independently a hydrogen atom or an alkyl); 65 OMe Y (the length ofY is equivalent to CO to C25) is a single bond; an alkylene which may be substituted and may be interrupted US 8,877,454 B2 23 24 According to a preferred embodiment, a polymer obtain -continued able by polymerizing the compound of the formula (I) is used. NH2 Thereby, when various films are formed from a substance that is capable of specifically interacting with the Sugar chain of the present invention, the strength and stability of the film 5 itself are enhanced, and furthermore, there is obtained an effect that the film can be fixed to a support such as a substrate. Upon fixing a film to a Support, it is preferable to use a technique of polymerizing a monolayer film that is obtainable by physically adsorbing the Z moiety of the compound rep 10 resented by the formula (I) onto the support. Thereby, a Sup port having a film fixed thereto can be directly used as the Sugar chain-capturing carrier of the present invention. Fur thermore, the polymerization may be thermopolymerization or may be photopolymerization; however, preferably, an 15 advantage that radical polymerization between diacetylene groups or between vinyl groups in the Z moiety can be made to Smoothly proceed, and polymerization can be carried out by a relatively simple operation, is taken into consideration, (wherein, X" is an alkylene which may be substituted oran and photopolymerization by irradiation with ultraviolet (UV) alkenylene which may be substituted;X is an oxygenatom or radiation near 254 nm, which is a characteristic absorption a sulfur atom; X is an oxygen atom or a sulfur atom; X is wavelength of the diacetylene group or vinyl group, is methylene or ethylene; R' is a hydrogenatom or an alkyl; R employed. Furthermore, a substance in which “X” in the and Rare each independently a hydrogen atom or an alkyl); formula (I) and a Support are directly bound, and a Substance 25 Y (the length ofY is equivalent to CO to C25) is a single bond; in which X Y” and a support are directly bound are also an alkylene which may be substituted and may be interrupted employed. As the substituent group for the “alkylene which by at least one group selected from the group consisting of may be substituted” and the “alkenylene which may be sub O—, S—, S—S—, N(R) C(=O) , stituted” in X', absence of substituent is preferred. As the —C(=O) N(R') , and phenylene which may be substi substituent group for the “alkylene which may be substituted 30 tuted; or an alkenylene which may be substituted and may be and the “alkenylene which may be substituted” in Y, absence interrupted by at least one group selected from the group of substituent is preferred. As the substituent group for the consisting of —O—, S—, S–S , – N(R)- C “alkylene which may be substituted and the “alkenylene (=O) , —C(=O) N(R)—, and phenylene which may be which may be substituted” in Z and Z, absence of substitu 35 substituted (wherein, R and R are each independently a ent is preferred. hydrogen atom or an alkyl); and Z is a group represented by According to another preferred embodiment, the Substance the formula: that is capable of specifically interacting with the Sugar chain of the present invention is a copolymer that is obtained by polymerizing a compound represented by the formula (I): 40 Chem 5) R4 X Y. Z. (I): wherein, X is a group represented by the formula: ". N Z2YC=C-C=C-Z, 45 Zl Chem 4 Zl r Z2YC=C-C=C-Z, 50 R4 f 74 2

55 visZl R5 s Zl 74 vs. Z2 121 R4 R5 s 4 Z3

65 US 8,877,454 B2 25 26 -continued 8 is preferred, and particularly, n=1 to 6 is preferred). Z and Zare preferably each independently a C1-C10 alkylene or a C2-C10 alkenylene, A is preferably a C1-C5 alkylene, and particularly preferably a C2 alkylene, and A' and Aare each independently a C1-C10 alkylene. Furthermore, specific examples of the phenylene which may be substituted include the following: (wherein, Z' is an oxygenatom or a sulfur atom; Z and Z are each independently an alkylene which may be substituted and may be interrupted by phenylene, oran alkenylene which 10 Chem 9 may be substituted and may be interrupted by phenylene; Z' ON is an oxygen atom or a sulfur atom; R and R are each O independently a hydrogen atom or an alkyl); and a compound represented by the formula (II): A-A (II): 15 / \ { wherein, A' is a group represented by H(OCH2CH)O - O O (wherein, n is an integer from 1 to 5) or the formula: OMe The polymerization may be thermopolymerization, or may Chem 6 be photopolymerization; however, for the same reason as R6 described above, photopolymerization by irradiation with ultraviolet (UV) radiation near 254 nm, which is a character istic absorption wavelength of the diacetylene group or vinyl R---0--0 group, is preferred. 25 (Pretreatment Technology for Sugar Chain Analysis) (wherein A is an alkylene; and R is uniformly an alkyl); As used herein, the term “release' of a Sugar chain means and that the Sugar chain is released from a complex containing the Sugar chain, which is found in biological materials and the A is a group represented by the formula: like. 30 The “sample as used herein means any sample intended Chem 7) for an analysis of Sugar chain. Preferably, the sample is a O sample derived from a biological material (for example, a body fluid, a cell extract, or a tissue extract), more preferably A, a body fluid, a cell extract, a tissue extractor the like, and even more preferably a body fluid containing blood components v-so (for example, serum and plasma). The sample is still more O preferably blood, and most preferably blood serum. 5 The “plate for reaction” as used herein means any plate on - A41 A which a chemical reaction of a sample containing a Sugar 40 chain is carried out. The plate is constituted of a material that O does not reactor is difficult to react with biological materials, 5 Such as plastic. The plate may be coated or may not be coated. -A41 A As the plate, a generally used experimental plate. Such as a multi-well plate, can be used. 45 The term "solubilizing agent” as used herein means an (wherein A' is uniformly an alkylene; and A is uniformly agent that can solubilize a protein. Examples of the solubi a group represented by the formula: lizing agent that can be used include 1-propanesulfonic acid, 2-hydroxy-3-lauramide (PHL), 1-propanesulfonic acid, Chem 8 2-hydroxy-3-myristamide (PHM), 2-hydroxy-3-sulfopropyl 50 laurate (HSD), or an equivalent thereof. Preferably, 1-pro A7 panesulfonic acid, 2-hydroxy-3-lauramide (PHL)/ammo nium bicarbonate can be used. As used herein, the “reaction conditions” for the solubiliz CEC-CEC-A, al ing agent mean any reaction conditions that cause a solubili R7 55 Zation reaction of the solubilizing agent, including, for A7 example, conditions at 25°C. to 42°C., and preferably at 37° C., for 10 minutes. The term “reducing agent as used herein means any Sub a -- stance that adds hydrogen to an element or a compound. As 60 used herein, the reducing agent is used to cleave an—S-S- (A is an alkylene; A is an oxygenatom or a sulfur atom; bond in a protein. The reducing agent may be dithiothreitol R’ is a hydrogen atom or an alkyl). In regard to the copoly (DTT), TCEP (Tris(2-carboxyethyl)phosphine hydrochlo mer of the compounds of the formulas (I) and (II) of the ride solution, 0.5 M) or an equivalent thereof. Preferably, present invention, X" is preferably a C1-C10 alkylene or a dithiothreitol (DTT) is used. C2-C10 alkenylene, and the chain length of Y is preferably a 65 As used herein, the term “reaction condition' for the reduc chain length equivalent to C1-25 alkyl. Preferred examples of ing agent means any reaction conditions under which the Y include —(CHCH O), CHCH (wherein, n=1 to reduction reaction of the reducing agent proceeds. As used US 8,877,454 B2 27 28 herein, for example, conditions at 50° C. to 80° C. can be or the like. Therefore, any of those causing these factors can used, and preferably, the reaction can be carried out at 60° C. be exemplified as protein denaturing agents. As used herein, for 30 minutes. Since the temperature is high, the temperature Such an agent includes, for example, guanidine hydrochlo may be returned to room temperature in order to carry out the ride, urea, sodium dodecyl sulfate or an equivalent. Prefer Subsequent reaction. ably, guanidine hydrochloride can be used. The term “ -SH protective agent as used herein means As used herein, the term “reaction condition for the pro any agent that protects the -SH group after an —S-S- tein denaturing agent means any conditions under which bond in a protein is cleaved. As used herein, for example, an denaturation of proteins and peptides proceed. As used agent that is capable of acetylation may be exemplified, and herein, for example, the conditions may be at room tempera the agent may be, for example, iodoacetamide (IAA) or an 10 equivalent thereof. ture from 10 seconds to 10 minutes, but the time may be even As used herein, the term “reaction condition' for the —SH longer than that. protective agent means any conditions under which the pro The “residual washing liquid” as used herein means a tection reaction of the -SH protective agent proceeds, residual liquid occurring after washing with a washing liquid including, for example, conditions under which the reaction 15 Such as water. Since the residual washing liquid can pose an is carried out at 20 to 37° C. in the dark, and preferably at impediment to the Subsequent reactions, it is usually prefer room temperature in the dark, for one hour. able to discard the residual washing liquid. The term “protein digestive enzyme” as used herein means The term "salt releasing agent of the Sugar chain-captur any enzyme that can degrade any polymer of amino acids ing group on beads as used herein means any agent that linked by peptide bond, Such as a protein, a polypeptide or a releases a salt (for example, in the case of hydrazide, the salt peptide. As used herein, for example, trypsin, chymotrypsin conjugated to hydrazide) formed with the Sugar chain-cap or an equivalent can be used, and preferably trypsin is used. turing group (including, for example, an aminooxy group, an As used herein, the term “reaction condition for the pro N-alkylaminooxy group, a hydrazide group, an azide group, a tein digestive enzyme means any condition under which the thiosemicarbazide group, a cysteine residue, and derivatives protein degradation reaction by the protein digestive enzyme 25 thereof) on the Sugar chain-capturing bead (for example, proceeds, including, for example, the conditions for carrying beads or magnetic beads). In the case of hydrazide, the salt out the reaction at 25 to 42°C., and preferably at 37°C., for releasing agent is triethylamine or an equivalent, and in the 60 minutes. case of aminooxy group, the salt releasing agent is triethy The term “deactivation of the protein digestive enzyme as lamine oran equivalent. In other cases, the salt releasing agent used herein means that the enzymatic activity of the protein 30 can be detached by protecting the group using a technology digestive enzyme is lost. Usually, since a protein digestive that is known in the related art. enzyme is also a protein, “deactivation” can be achieved by The term "discarding (. . . ) by suction” as used herein denaturing the protein itself at high temperature, but is not means that a liquid is removed by Suction using a film to limited to this. which liquid is absorbed under reduced pressure or negative The term "sugar chain releasing enzyme’ as used herein 35 pressure (for example, a commercially available multi-well means any enzyme that can release a Sugar chain from a Sugar filter plate). chain complex. As used herein, for example, PNGaseF. Endo The term “protective agent as used herein means an agent H or an equivalent, and preferably PNGaseF, can be used. that protects excess functional groups of the bead. According As used herein, the term “reaction conditions” for the sugar to the present invention, for example, acetic anhydride, Suc chain releasing enzyme mean any conditions under which the 40 cinic anhydride, other acid anhydrides or equivalents can be release of a Sugar chain by the Sugar chain releasing enzyme used, and preferably acetic anhydride can be used. In this proceed. As used herein, such a condition includes, for case, protection using an acetyl group (acetylation) is carried example, the conditions for carrying out the reaction at 25°C. Out. to 42°C., and preferably at 37°C., for 60 minutes to 12 hours. As used herein, the term “reaction condition' of the pro The term "a Sugar chain-capturing bead as used herein 45 tective agent mean any conditions under which excess func means any beads having a chemically reactive functional tional groups of the bead can be protected. As used herein, group for the Sugar chain capturing as described in the present conditions using 10% acetic anhydride/methanol at room specification. Such beads are exemplified in, for example, temperature for 30 minutes, preferably at 37° C. can be Patent Document 1. Among commercially available products, employed. BlotCilyco (Sumitomo Bakelite), AffiGel HZ (BioRad), Car 50 As used herein, the 'acid' is used to remove and free the boLink Coupling Resin (PIERCE), Surface-activated Dyna salt from the carboxyl group of Sialic acid in the captured beads (registered trademark) (Invitrogen) and the like can be sugar chain. Since it is expected that the yield of the methyl used. esterification reaction would be lowered in the absence of As used herein, the phrase “conditions for binding to the acid treatment, it is preferable to perform an acid treatment. Sugar chain-capturing bead” mean any conditions under 55 Here, in addition to hydrochloric acid, inorganic acids at pH2 which binding of a Sugar chain-capturing group (including, to 3 such as dilute sulfuric acid can be used. for example, an aminooxy group, an N-alkylaminooxy group, The term “organic reaction solvent as used herein means a hydrazide group, an azide group, a thiosemicarbazide any organic solvent used in a reaction such as an alkyl esteri group, a cysteine residue, and derivatives thereof) present on fication reaction. For example, dioxane, as well as acetonitrile a Sugar chain-capturing bead (for example, beads or magnetic 60 and tetrahydrofuran can be used. The preference for either beads) with the Sugar proceeds. For example, such a condition dioxane or acetonitrile may vary with the bead. includes, for example, a reaction at 25 to 80°C., and prefer The term “hydrophilic organic solvent as used herein ably at 80° C. means any organic solvent that is used intermediately when a The term “protein denaturing agent’ as used herein means hydrophobic organic solvent is exchanged with water (that is, changing properties such as solubility by altering the molecu 65 mediates between a water-based system and an organic Sol lar structure of the protein and the peptide. For example, Vent). For example, a lower alcohol Such as methanol or proteins denature by heating, alkali treatment, acid treatment ethanol, acetonitrile, acetone or the like can be used. US 8,877,454 B2 29 30 The term “removal of "moisture and solvent as used an SH group, an amino group, carboxylic acid), a tag having herein means any process capable of removing moisture and a functional group in a hydrophobic structure, or a tag having solvent. Alternatively, the removal also includes exclusion of a metalion ligand. a water-based solvent after a use of the water-based solvent, The term "chromophore capable of absorbing ultraviolet by purging the Solvent environment of the bead with an 5 and visible rays' as used herein means any chromophore that organic solvent. For example, bottom wiping with a filter absorbs ultraviolet and visible rays, including, for example, paper, a blotting paper, gauze, a towel, a hand towel, a tissue an aromatic compound Such as p-nitrobenzyloxylamine. paper, a cotton sheet or the like, may be exemplified. The The term "tag having a structure emitting fluorescence’ as means used in the “removal of “moisture and solvent' (also used herein means any tag that has a structure emitting fluo 10 rescence, including, for example, anthraniloyl hydrazine. referred to as “solvent absorbent sheet in the present speci The term “affinity tag having a molecule capable of inter fication) is a material which is used to absorb the solvent that acting with another molecule' as used herein means any tag has adhered to the low side of the plate. Such as water, aceto having a molecule that can interact with another molecule, nitrile (ACN) or methanol, after the plate has been subjected including, for example, His tag, biotin, biotin hydrazide and to Suctioning under reduced pressure. The means is not lim 15 the like. ited to a specific material Such as cotton, as long as it is made The term "tag having a functional group capable of spe ofa material capable of absorbing moisture and solvent. If not cifically reacting with a functional group’ as used herein wiped out, the solvent penetrates back onto the filter, and the means any tag having a functional group that specifically bead on the filter happen to contain more moisture and sol reacts with a functional group, and for example, a photoreac vent. Thus, it is preferable to prevent this. tive molecule (for crosslinking), an azide group, anSH group, The term “bottom wiping at ... as used herein means that an amino group, carboxylic acid, N-aminooxyglycine, a means for absorbing moisture and solvent (for example, a including, for example, a compound having the compound Solvent absorbent sheet Such as cotton sheet) is spread out at structure in the molecule. the bottom, and the plate which has been filtered by suction is The term 'tag having a functional group in a hydrophobic placed thereon and pressed from the top, so as to remove the 25 structure' as used herein means any tag having a functional filtration solvent adhering to the bottom of the plate, by group in a hydrophobic structure, and for example, a absorbing the filtration solvent into the absorbent sheet. hydrazide compound having a hydrophobic aromatic or The term “alkyl esterifying agent’ as used herein means hydrophobic aliphatic chain; including, for example, Girard any agent that can achieve alkyl esterification. For example, Reagent P. 30 The term "tag having a metal ion ligand’ as used herein in the case of methylation, the reaction is called methyl esteri means any tag having a metal ion ligand, including, for fication. When the carboxylic acid of sialic acid is methy example, a histidine tag or a tag that can adsorb to a metal lated, there is obtained an effect of making the detection of ion-carrying resin. sugar chain easier. This technology is described in WO 2007/ The term "tagged Sugar chain sample solution” as used 0.99856, the disclosure of which is incorporated to the present 35 herein means any sample that has been prepared to contain a specification by reference as necessary. As used herein, for tagged Sugar chain. This solution can be directly subjected to example, methyl-p-tolyl-triazene (MTT), ethyl-p-tolyl-triaz analysis by HPLC, or liquid chromatography-mass spectrom ene (ETT), butyl-p-tolyl-triazene (BTT), or an equivalent can etry (LC-MS) or the like, but can also be fixed on a plate for be used. MALDI-TOF mass spectrometry and subjected to MALDI As used herein, the term “reaction condition’ for the alkyl 40 TOF mass spectrometry. esterifying agent means any conditions under which an alkyl The term “detection sample” as used herein means a esterification reaction using an alkyl esterifying agent pro sample for detection in mass spectrometry or the like. ceeds. As used herein, for example, conditions using 100 mM The term “plate for mass spectrometry” as used herein MTT/dioxane at 60°C. for 60 minutes can be employed. means any plate used for mass spectrometry. The plate is The term "tagging as used herein means that a material for 45 constituted of a material that does not react or is difficult to mass spectrometry, such as a Sugar chain, is bound to an react with biological materials, such as plastic or metal. The additional molecule such as a label in order to prepare for plate may be coated, or may not be coated. For example, a detection. Such tagging is carried out using a chromophore sample-dotted plate that depends on the MALDI apparatus capable of absorbing ultraviolet and visible rays, a tag having can be used. a structure emitting fluorescence, an affinity tag having a 50 The term “plate for recovery” as used herein means any molecule capable of interacting with other molecules (His plate used for the recovery of sample. The plate is constituted tag, biotin, or the like), a tag having a functional group of a material that does not react or is difficult to react with capable of specifically reacting with a functional group (a biological materials, such as plastic. The plate may be coated, photoreactive molecule (for crosslinking), an azide group, an or may not be coated. As the plate, a generally used experi SH group, an amino group, carboxylic acid), a tag having a 55 mental plate such as a multi-well plate can be used. functional group in a hydrophobic structure, or a tag having a The term “multi-well as used herein means a large number metalion ligand. Tagging may be carried out in the additional of wells, and a multi-well plate means a plate having a large presence of acetic acid, acetonitrile, an acetate buffer Solu number of wells. For example, a multi-well plate having 96 tion, or an equivalent. wells, a multi-well plate having 384 wells, a multi-well plate The term “reagent' for tagging as used herein means any 60 having 1536 wells, and the like can be mentioned as reagent capable of tagging, including, for example, a chro examples. mophore capable of absorbing ultraviolet and visible rays, a The term “resin or membrane suitable for solid phase tag having a structure emitting fluorescence, an affinity tag extraction” as used herein means any resin or membrane used having a molecule capable of interacting with other mol for Solid phase extraction. In the present specification, such a ecules (His tag, biotin, or the like), a tag having a functional 65 resin or membrane includes, for example, C18 silica gel, Solid group capable of specifically reacting with a functional group phase carrier for normal phase, amino-functionalized silica (a photoreactive molecule (for crosslinking), an azide group, resin and the like. US 8,877,454 B2 31 32 The term "solid phase' as used herein means a solid state. m/Z on the horizontal axis and the relative intensity of ions on The term “concentration at which the sugar chain adsorbs the vertical axis, is a mass spectrum. The ions that give infor to a solid phase' as used herein means any concentration at mation on the molecular weight are generally referred to as which adsorption of a sample (for example, Sugar chain) to a molecular weight-related ions (there are available M result substrate or the like in the solid state is accelerated. ing from a loss of one electron from a neutral molecule M. M. The term "plate for mixing as used herein means any plate resulting from addition of one electron: M-H resulting used for mixing two or more reagents or samples. The plate is from addition of a proton; M-H resulting from a loss of a constituted of a material that does not react or is difficult to proton; M-H resulting from a loss of a hydride; M+Na" react with biological materials. Such as plastic. The plate may resulting from addition of an alkali metal (such as Na); and be coated, or may not be coated. As the plate, a generally used 10 the like). Depending on the sample or the ionization method experimental plate Such as a multi-well plate can be used. (especially, in the EI method), the molecular weight-related The term "solid phase carrier-enclosed plate' or “SPE ions may never appear; however, in that case, the molecular plate” as used herein means a column type plate for multiple weight-related ions can be confirmed by using a mild ioniza sample treatment, which encloses a carrier for Solid phase tion method. Those ions appearing on the lower mass side extraction. The plate is constituted of a plate material that 15 than molecular ions are called fragment ions, and these frag does not reactor is difficult to react with biological materials, ment ions are degradation products of molecular ions and Such as plastic; microbeads made of silica or styrene having a give information on the structure of the sample molecule. The functional group Such as aminopropyl or octadecyl, which are ion with the highest ion intensity in the spectrum is called a enclosed as a carrier for Solid phase extraction; and a filter for base peak, and this is used to normalize the spectrum by retaining the carrier for Solid phase extraction. The plate may taking the relative intensity as 100%. be coated, or may not be coated. As the plate, a generally used The "ionization' according to the present invention is car experimental plate such as a multi-well plate can be used. One ried out by appropriately selecting one among the following 7 of example products includes “MassPREPHILIC uElution methods. Plate' manufactured by Waters Corp. 1) Electron Ionization Method (EI Method) The phrase “conditioning to a solvent having a polarity 25 The electron ionization method is a method of ionizing by appropriate for the phase' as used herein means that a certain contacting hot electrons to a gasified sample, and is the most material is made compatible with a solvent having an oppo popularized ionization method. Since ionization is conducted site polarity (if water, a hydrophobic material; if a hydropho after gasifying the sample, it is necessary to gasify a liquid or bic organic solvent, a hydrophilic material). For the acclima Solid sample in advance. Because heat is applied for the tion, it is preferable to immerse the material in the solvent for 30 process of gasification, measurement of a heat-unstable Sub a long time, or to exchange the solvent several times. stance or a sparingly volatile Substance is impossible. How The term "second plate for recovery as used herein means ever, in the case of a substance which can attain volatility and a plate which is intended for conducting recovery again after thermal stability by derivatization such as methylation, sily an operation in which a plate for recovery is used, and then lation or acylation, measurement can be made. Since ioniza purification operation is carried out with the Solid phase car 35 tion is usually carried out with energy of 70 eV, fragmentions rier-enclosed plate. are generated by excess energy, along with the generation of The term “matrix for mass spectrometry” as used herein molecular ions (the ionization energy for a general organic means any matrix for preparing a sample for mass spectrom compound is about a dozen eV). Structural analysis of the etry. The matrix will be described in detail in other part of the compound is made possible from the information on these present specification. 40 fragment ions. However, because it is difficult to obtain The term “analysis of mass of a Sugar chain” as used herein molecular ions, the molecular weight information is often means any technique for analyzing the mass of a Sugar chain. unobtainable. In this case, it is necessary to drop the ioniza The methods described in the present specification, such as tion energy to about 20 eV, or to select a milder ionization high performance liquid chromatography (HPLC), an LC method (CI, DEI, DCI, FAB or ESI). ESI-TOFMS method or a MALDI-TOF method, and any 45 2) Chemical Ionization Method (CI Method) techniques known in the related art can be used. It is a method of ionizing a gasified sample by sending the The term “process of removing excess' reagent as used sample into a previously ionized reaction gas (reagent gas). herein means any process of removing the reagent used in a Since this method achieves ionization using an ion-molecule reaction. For example, for the purpose of removal. Such a reaction, the ionization energy is close to the ionization process includes, for example, washing several times with a 50 energy of organic compounds, and thus the amount of frag solvent only, or the like. ment ions is very Small, while ions having the molecular The term "pretreatment sample for analyzing a Sugar chain weight information ((M+H)", (M--NH)", (M-H), and the in a sample' as used herein means a sample used for analyZ like) appear as base ions. As the reaction gas, generally meth ing a Sugar chain in a sample. Usually, in mass spectrometry ane, isobutane or ammonia is used. or the like, it is impossible to carry out analysis with a fresh 55 3) Desorption Electron Ionization Method (DEI Method) sample as received. Thus, it is necessary to carry out a pre This is a method of conducting instantaneous heating near treatment Such as removing impurities or tagging, and a an electron beam, and thereby gasifying and ionizing the sample that has been Subjected to Such pretreatment is called sample before the sample undergoes thermal degradation. as Such in the present specification. Measurement of heat-unstable Substance or a sparingly Vola (Method for Mass Spectrometry) 60 tile Substance is made possible. Furthermore, since molecular The term “mass spectrometry” as used herein means that ions appear with stronger intensity as compared to the EI particles such as atoms, molecules or clusters are converted method as a conventional method of direction introduction, it into gaseous ions (that is, ionized) and are allowed to move is easier to obtain the molecular weight information. The about in a vacuum, and those ions are separated and detected measurement method involves attaching a sample solution at using an electromagnetic force in accordance with the mass 65 the tip of a point filament (platinum wire having a diameter of charge ratio (m/z). A spectrum obtained based on the ions 100 um), inserting the point filament into an ion Source, and separated and detected in accordance with m/z, representing then rapidly heating the filament to gasify the sample. US 8,877,454 B2 33 34 4) Desorption Chemical Ionization Method (DCI Method) diation without using a low molecular weight matrix reagent, When the operation of DEI is carried out, with the ion contrastingly to the “MALDI-TOF MS. source being put in the CI state, this operation is the DCI. The term “identical entity” as used herein means that an 5) Fast Atom Bombardment Method (FAB Method) entity that is expressed in plurality for convenience in the This is a method of thoroughly mixing the sample and present specification, is the same individual itself. matrix molecules, applying the mixture on a target, and bom The “metal as used herein means a metal element or a barding fast neutral atoms such as Xe thereon to ionize the metal composite containing a metal capable of binding, at sample. Unlike the EI and CI, it is not needed to gasify the least partially, to a Sulfur atom-containing Substance (for sample, and therefore, the method is adequate for the mea example, thiol, disulfide or the like). Representative types of Surement of a heat-unstable Substance or a sparingly volatile 10 the metal capable of binding to a sulfur atom-containing Substance. However, since intense matrix-derived back Substance include gold, silver, cadmium, selenium and the ground peaks appear in the lower mass region, measurement like. The metal may also be a magnetic Substance. of a low molecular weight sample may be difficult in some The term “metal microparticles' as used herein mean par cases. In that case, measurement by FRIT-FAB is effective. 15 ticles of metal defined as having an average particle diameter 6) FRIT-Fast Atom Bombardment Method (FRIT-FAB size of 10 nm to 1 Lim, and the microparticles may be of Method) monodisperse type or polydisperse type. The bead may be This method is also called as Continuous-flow FAB, and is constituted of such metal microparticles. When the metal a method of making the sample dissolved in a matrix solution microparticles constitute a metal composite, the Surface of the to flow continuously, and bombarding the eluent outlet with particles which serve as the core of the complex, takes a fast neutral atoms to ionize the sample. structure covered by a layer of a metal to which the sulfur 7) Electrospray Ionization Method (ESI Method) atom-containing Substance can bind. Representative metal This is an atmospheric pressure ionization method that makes use of the phenomenon in which when high Voltage is microparticles used in the present invention include micro applied on a capillary, the sample solution spontaneously particles of elemental gold or silver metal, or microparticles 25 of a metal composite formed from magnetic particles of iron, undergoes spraying and ionization. Just like FAB, there is no nickel, cobalt, iron oxide (Fe-O) or the like having the Sur need to gasify the sample, and therefore, the method is face covered with gold or silver. The “metal microparticles' adequate for the measurement of a heat-unstable Substance or of the present invention form a colloid in a solution. In addi a sparingly volatile Substance. This makes it possible to mea tion to the examples of the metal microparticles listed above, Sure a peptide or protein having a large molecular weight, 30 Quantum Dots (trade name) (Quantum Dot Corporation, even for the quadrupole type with a small mass range. In the Hayward, Calif., USA; hereinafter, referred to as “QDs) can conventional ESI measurement, because fragment ions hav also be used. Examples of the type of metal used in the QDs ing the structural information are not obtained, in-source CID include ZnSe, CdS, CdSe, CdTe and the like. A typical occurs as a Voltage slightly higher than usual is applied to example is nanoparticles having CdSe in the core, and ZnS Capillary/Skimmer-1, and thus measurement of fragment 35 Surrounding the core. An —SH compound can be presented at ions having the structural information is made possible. the QDs surface through the bond between Zn, which is The term “MALDI-TOF MS’’ as used herein is an abbre emerging from the Surface, and —SH (this bond is of the same viation for Matrix Assisted Laser Desorption Ionization type as Au-S). In regard to the production of gold micropar Time-of-Flight (Mass Spectrometer). MALDI is a technique ticles and applications thereof, descriptions are found in discovered by Tanaka et al., and developed by Hillenkamp et 40 JACS 125, 7790-7791 (2003); Angew Chem Int Ed 40(12), al. (Karas M., Hillenkamp, F., Anal. Chem. 1988, 60,2299 2257-2261 (2001); and Chem EurJ 9, 1909-1921 (2003). 2301). In this method, the sample and a matrix solution are (Application of Apparatus of Present Invention) mixed at a molar ratio of (10° to 5x10): 1, and then the A proteome analysis combining an analysis method for mixed solution is dried to Solid on a target to bring it to a crystalline state. Large energy can be applied on the matrix by Sugar chain, which is based on the mass spectrometric method 45 realized by the present invention, and a two-dimensional elec pulse laser irradiation, and sample-derived ions such as trophoresis method, also makes research aimed for rapid (M+H) and (M--Na)", and matrix-derived ions are desorbed. identification of a protein marker engaged in a disease, or Analysis can be still made even if the sample is contaminated high-throughput search for a target protein which serves as a with a trace amount of phosphate buffer solution, Tris buffer target of genomic drug discovery, to be actively carried out. solution, guanidine or the like. MALDI-TOF (MS) allows 50 Sugar chain modification is a very important and funda measurement of mass using MALDI, based on the time of mental biosynthesis process in the protein dynamic structure/ flight. When ions are accelerated at a constant accelerating function controlling mechanism, together with phosphoryla Voltage V, and when the mass of the ion is designated as m, the tion, acylation, prenylation reactions and the like. Such Velocity of the ion as V, the charge number of the ion as Z, the various modification reactions for protein are broadly called elementary electric charge as e, and the time of flight of the 55 "post-translational modification of genetic information.” ion as t, the m/z of the ion can be represented by: which is a phenomenon deeply involved in many diseases including cancer and immunity. Therefore, Sugar chain modi fication is useful in the development of new diagnosis Support In the measurement of MALDI-TOF as such, KOMPACT technologies or research and development of medicines, and MALDIII/III by Shimadzu/Kratos, or the like can be used. At 60 even in the field of clinical practice. the time of measurement, reference can be made to the pam When the present invention is used, it is also possible to phletproduced by the manufacturer. The irradiation energy of carry out high throughput quantitative glycomics as well as an the laser irradiation used upon the measurement of MALDI analysis of peptide sequence information of the carrier pro TOF is referred to as “dissociation energy” in the present tein of those Sugar chains, and a study of making a search for specification. 65 a new disease biomarker, which adopts, as a basic strategy, The term “LDI-TOF MS’’ as used herein means a method “glycoform-focused reverse genomics' which further traces of desorbing and ionizing intended molecules by laser irra back to identification of genomic data. US 8,877,454 B2 35 36 By using the present invention, glycoform-focused reverse tural analysis according to various mass spectrometric meth genomics (GFRG) can be carried out, which is a method of ods such as MALDI-LIFT-TOFFTOF or nanoLC/ESI catching a glycopeptide fragment while considering the Sugar TOFMS, can be completed within half a day. Furthermore, chain as a tag, thereby proceeding a proteomic analysis as when a selective oxidation reaction (functional group conver well as obtainment of genomic data all at one time, and thus sion) in galactose with non-reducing termini or in sialic acid implementing identification of glycoproteins, profiling of residue is carried out, followed by an oxime formation reac heterogeneity of Sugar chains, and the like. "High throughput tion at this position, glycopeptide fragments can be blotted. quantitative glycomics' is possible, in which Sugar chains Thus, a scheme as shown in FIG. 3 can be applied to the obtainable from various types of glycoproteins contained in structural analysis of a glycopeptide. biological samples of blood serum, cells, tissues and organs 10 Fragment ions of a Sugar chain can be detected before by N-glycanase digestion or the like, are selectively or com being destroyed. prehensively captured and labeled, and all of Sugar chain Mass spectrometric methods are being used already widely structures and their quantitative distribution state are ana in various aspects as very effective analytic methods for the lyzed by high performance liquid chromatography (HPLC), proteome research. This is because the methods are highly an LC-ESI-TOFMS method or the like. Partially digested 15 advantageous in that all of the peptide fragments produced by glycopeptide fragments obtainable from the same sample by enzymatic digestion or the like can be easily ionized without a peptidase treatment, are captured for each of the respective depending on their structures, and that selective fragmenta intended Sugar chain structural groups, the respective peptide tion from their stable primary ion peaks is also simple, so that sequences are determined by MALDI-TOFMS and MALDI when MS/MS spectrum patterns are collated using the above TOF/TOFMS analyses, and the like, and the results are com described MASCOT algorithm analysis or the like, candidate bined with the results of the above-described high throughput proteins including those peptide sequences can be easily con quantitative glycomics analysis. Thereby, "Glycoform-fo jectured. cused proteomics' that confirms the individual glycopeptide Furthermore, MALDI-TOFMS (for example, REFLEXIII structures can be carried out. In this case, for example, if a and BIFLEXIII by Bruker Daltonics, Ltd., or the like) can be protein containing the peptide structure can be picked up by 25 used as a simple and general-purpose method of monitoring using the MASCOT algorithm (Matrix Science, Ltd., the total the reactions for enzymatic or chemical Sugar chain modifi image of the glycoprotein containing genetic information can cation performed on peptides or proteins. In these appara be instantaneously analyzed and identified. The GFRG tuses, optimization of reaction conditions, evaluation of the method which traces back in the reverse direction and ana reaction yield, and the like can be carried out by simply lyzes the structures of a carrier protein, and even a gene, from 30 identifying the molecular weight of a reaction product, a Sugar chain tag, is one of high throughput search methods mainly from the information on monovalent molecular ion for new disease markers, and is expected to be a promising peaks only. Ultraflex, which is a higher-end model than the Strategy. two apparatuses, can detect and analyze unstable fragment In the GFRG method, two types of trace amount sugar ions with high sensitivity at high speed by the LIFT-TOF/ chain structural analysis technologies such as (1) high 35 TOF method. It is thought that similar measurement in the throughput quantitative structural analysis of Sugar chains TOF/TOF mode can also be made with MALDI type mass and (2) structural analysis of glycopeptides, are used. analyzing apparatuses of other companies, but the basic prin There, a glycoblotting method can be used. ciple is such that, as shown in FIG. 4, in TOF-1, an acceler A Sugar chain released from a complex glycoconjugates ating Voltage is selectively applied to precursor ions Subse Such as a glycoprotein, a proteoglycan or a glycolipid, nec 40 quently to laser irradiation, thereby generating fragmentions. essarily has a hemiacetal group equivalent to an aldehyde Then, each of these fragment ions is rapidly Subjected to group at a reduced terminus in the molecule. Thus, perfect instantaneous accelerating Voltage again at the stage where chemical differentiation from other biomolecules such as the fragmentions have reached a region called TOF-2, and the amino acids and peptides which constitute proteins, nucle fragmentions move directly to the detector through a reflector otides which constitute DNA/RNA, and lipids, is realized by 45 at a certain Voltage so that the respective masses are identi a specific and selective nucleophilic addition reaction to this fied. The most important point herein is that all of the frag hemiacetal group. Various Substances (irrespective of high ment ions which have been accelerated in the TOF-2 region molecular weight or low molecular weight) containing a and made to move at respectively different speeds, begin to be functional group that reacts comprehensively with the detected from after several microseconds and are succes reduced termini of all Sugar chains (an aminooxy group, a 50 sively detected during a period up to several hundred micro hydrazide group, or the like) are used to perform Sugar chain seconds thereafter. For comparison, the measurement in the capturing, probe labeling using a high sensitizing reagent or PSD mode is presented on the same time axis, but in this case, the like, and structural analysis of the Substances according to since all the fragment ions come toward the detector all at a the mass spectrometric methods, continuously at high speed time by uniform linear motion, a process of Sorting and then (Patent Document 1; FIG. 3). 55 detecting each of the fragment ions by appropriately chang By using the present technique, the process for quantitative ing the Voltage at the reflector in the middle, is required. structural analysis of an asparagine-binding type Sugar chain Consequently, all of the fragment ions take about 1 millisec released from various glycoproteins included in a serum ond after ionization to reach the detector. Upon detecting and sample (about 50 microliters) can be simplified and shortened structurally analyzing those unstable fragmentions observed to a large extent. In the conventional methods only of com 60 in the Sugar chain rapidly with high sensitivity, the “time bining various chromatographic operations, even those pro differential attack of this several hundred microseconds is cesses of Sugar chain purification from a test sample contain important. As such, when various methods are attempted for ing a large amount of foreign materials or pretreatment of a use, the automatic analysis method of the present invention is fluorescence label only require cumbersome operation taking applicable. about 2 to 3 weeks (N. Tomiya, et al. Anal. Biochem. 171, 65 By using the LIFT-TOF/TOF method, it has been made (1988)), but according to this new method, a series of pro possible to observe with good reproducibility the molecular cesses from Sugar chain capturing to comprehensive struc ion peaks or fragment ion peaks of various mucin type gly US 8,877,454 B2 37 38 copeptides including these structures, without cleaving the from the terminus. On the other hand, three types of com O-glycoside bond with serine or threonine (faster than cleav pounds which have Sugar chains of the same structure and ing). Since the TOF/TOF mass spectra of relatively large differ in the aglycone moiety (glycopeptide, glycoamino acid Sugar chains including Sialic acid or fucose can also be and fluorescent-labeled Sugar chain) were ionized, this time obtained with good reproducibility through a simple opera using CHCA as the matrix. When the molecular ions tion, it is now possible to identify structural isomers or to (M+Na') produced thereby, which respectively have defi analyze the composition ratio of isomers in an unknown nitely different mass numbers, are fragmentized, it is also sample, through a combination with the TOF/TOFMS spectra possible to perform structural analysis of the Sugar chain of a so-called known compound (spectrum matching moiety through spectrum matching. This matrix-dependent method). In regard to the spectrum matching of Sugar chains, 10 for example, a method for prediction of unique MS/MS pat selective fragmentation (MDSF) method is a method effec terns by GlycosidiOTM, which is loaded in the GlycoSuite DB tive for selective fragmentation of glycopeptides in which developed by Proteome Systems, Inc., has been already glycoside bond and amide bond co-exist in a same molecule. For the GFRG method, there is a need to receive the results reported. In this method, the spectrum pattern of actually of high throughput quantitative glycomics and to perform a observed fragment ions and the calculated database of the 15 fragment ions obtained by a theoretical simulation based on systematic analysis for each of the target Sugar chain struc the characteristics of the disintegration behavior of the Sugar tures, including the root peptide structure (glycoform focused chain, are automatically collated, and from the homologous proteomics). Therefore, it is preferable to use, in connection ness, a candidate structure is speculated. By using an MS/MS with MALDI-TOFMS, a selective MS spectrum in the database of more various and complicated Sugar chain-re MDSF methodor ESI (SSI) ion trap MS method, and to use a lated compounds, spectrum matching with high accuracy can technology intended for more selective and accurate frag be achieved. mentation Such as electron capture dissociation (ECD) The sonic spray ionization (SSI) method, which is excel method. lent in the ionization of unstable molecules of natural Sub (Constitution of Analyzing Apparatus) stances, metabolic intermediates and the like, can be said to 25 As an example of the apparatus of the present invention, the be one of optimal methods for non-destructive directioniza constitution proposed in FIG.9 can be used. tion of sugar chain. In Hitachi M-8000 3DQ type mass ana The following processes can be carried out at the respective lyzing apparatus (manufactured by Hitachi High-Technolo position numerals in FIG. 9. gies Corp.) loaded with this SSI method, stable molecularion Position Numeral peaks are detected with high sensitivity even for various oli 30 Reaction, Treatment Carried Out at the Position gosaccharide samples including neutral Sugar chains as well (1) A glycoprotein is treated with a proteolytic enzyme and as sialic acid when measurement is made in the negative ion mode, and even the four kinds of structural isomers of Sialic a Sugar chain releasing enzyme to thereby release a Sugar acid produced as a result of the difference in the bonding chain. mode can be perfectly discriminated by MS" spectrum match 35 (2) to (5) The sugar chain is captured using BLOTGLY ing obtainable from CID (collision induced dissociation) of COABC beads, and the reduced terminus of the sugar chain is helium gas. Since the MS, MS and MS spectra of these labeled. isomers are available, and the position of branch in MS has Impurities that are not captured by the bead are removed by been identified, when the MS spectrum is compared, dis filtration. crimination of the C2.6 bond and the C2.3 bond, which link 40 The carboxylic acid of the sialic acid residue is protected sialic acid with galactose residue, can be simply carried out. by methyl esterification. However, when conducting an analysis of Sugar peptides The hydrazone bond is reduced to thereby stabilize the which closely resemble in the molecular weight and structure, bond with the bead. a throughput enhancement combined with an excellent sepa The S–S bond is cleaved to release the sugar chain from ration method such as nanoLC technology is being expected. 45 the bead. Matrix Dependent Selective Fragmentation (MDSF) The sugar chain is filtered. Method (6) to (7) A MALDI matrix is added to the sugar chain 2,5-Dihydroxybenzoic acid (DHB) and C-cyano-4-hy containing filtrate? Sugar chain effluent, and then the mixture droxycinnamic acid (CHCA) are two typical matrices fre is dropped on a MALDI plate. quently used in the MALDI method. When TOF/TOFMS 50 (8) to (10) When having a small sugar content, or when measurement is made on molecular ions which have been performing HPLC analysis, this operation is carried out. produced each independently using these 2 kinds of matrices, A liquid prepared by mixing ACN with the filtrate contain even though a completely same accelerating Voltage is ing the sugar chain, is flowed over an SPE plate which applied to TOF-2 for the molecular ions having the same has been washed with ACN, and the sugar chain is mass, completely different fragment ion patterns are 55 adsorbed to the SPE plate. observed. This is conceived to be due to the difference in the The SPE plate is washed with 95% ACN, and excess distribution mode of charges in the activated molecular ions. reagents are removed. While in the fragmentation from the molecular ions produced 10% ACN is flowed to effuse the sugar chain from the SPE with DHB, most of the disintegration patterns are of exotype plate. in which monosaccharides are dissociated one by one from 60 Another exemplary embodiment is shown in FIG.5 to FIG. the end, on the contrary, in the case of the production with 6. In these diagrams, the positions in the apparatus as shown CHCA, disintegration of endo type in which relatively large in FIG. 1 are presented with numerals. FIG. 7 shows an Sugar chain fragmentions are conspicuously observed domi example of dispensing. FIG. 8 shows a bird’s-eye view of this nates. When molecular ions produced with DHB, having a embodiment. value of m/z 1862.79 (M+H") are selected and fragmentation 65 The washing valves A to H in FIG. 8 are as follows. is carried out, satisfactory fragmentation occurs at the peptide A more preferred embodiment is shown in FIG. 1. area, in addition to the exotype disintegration of sugar chains In this FIG. 1, the following process is carried out. US 8,877,454 B2 39 40 A method for analyzing a Sugar chain in a sample, the ytryptophanyl arginine methyl ester/water, benzylhy method comprising the following steps: droxylamine hydrochloride/water, or anthraniloyl A) a Sugar chain releasing step of releasing a Sugar chain in hydrazine/water, and a sample, the step comprising the following steps: B-13) a step of adding water to the tagged captured Sugar A-1) a step of providing blood serum as a sample on a filter 5 chain sample to produce a tagged Sugar chain sample plate; Solution; A-2) a step of adding 1-propanesulfonic acid, 2-hydroxy C) a step of producing a plate for mass spectrometry having 3-lauramide (PHL) or 1-propanesulfonic acid, 2-hy the tagged captured Sugar chain sample dotted thereon, the droxy-3-myristamide (PHM)/ammonium bicarbonate, step comprising: and allowing the mixture to react for 10 minutes at 37° 10 C-1) a step of disposing the tagged Sugar chain sample C.; solution obtained in the step (B) on a plate for recovery: A-3) a step of adding dithiothreitol (DTT) to the sample, C-2) a step of disposing the tagged Sugar chain sample allowing the mixture to react for 30 minutes at 60° C. solution from the plate for recovery and acetonitrile on a and then cooling the reaction mixture to room tempera 15 plate for mixing, so as to achieve a final concentration of ture; acetonitrile of 80 to 90%; A-4) a step of adding iodoacetamide (IAA), and allowing C-3) a step of providing a solid phase carrier-enclosed plate the mixture to react for 1 hour at room temperature in the which is in normal phase mode; dark; C-4) a step of washing the Solid phase carrier-enclosed A-5) adding trypsin to the sample, and allowing the mix plate sequentially with water and acetonitrile, and dis ture to react for 60 minutes at 37° C.; carding water and acetonitrile by Suction; A-6) heating the sample to 90° C. for 5 minutes, and then C-5) a step of adding the tagged Sugar chain sample solu cooling the sample to room temperature; and tion to the Solid phase carrier-enclosed plate, discarding A-7) adding PNGaseF, and allowing the mixture to react the liquid, and adding 5% acetonitrile thereto; for 12 hours at 37° C.; 25 C-6) a step of recovering the bead by suction from the solid B) a detection sample preparing step of preparing the phase carrier-enclosed plate to the second plate for released Sugar chain for use in detection, the step comprising recovery; and the following steps: C-7) a step of adding 2,5-dihydroxybenzoic acid in 30% B-1) a step of contacting the captured Sugar chain sample acetonitrile, to the tagged Sugar chain sample solution, 30 and mixing and dotting the mixture; and prepared in the step (A) with beads for capturing Sugar D) a step of performing mass spectrometry by MALDI chain, to thereby allow binding at 37°C., and thus pro TOF MS. ducing a captured Sugar chain sample: The numerals in FIG. 1 are as described in the section B-2) a step of adding guanidine hydrochloride to the cap EXPLANATIONS FOR LETTERS AND NUMERALS. tured Sugar chain sample to thereby place the captured 35 The following are deployed for the reagent rack 4°C., Sugar chain sample under a reaction condition, and then reagent rack, and washing valve. discarding the reaction liquid by Suction; B-3) a step of Washing the captured Sugar chain sample TABLE 1 with water, and then discarding the water by Suction; mL B-4) a step of Washing the captured Sugar chain sample 40 with triethylamine, and then discarding the triethy Reagentrack 4°C. lamine by Suction; Trypsin O.48 B-5) a step of adding acetic anhydride to the captured Sugar PNGaseF O.48 chain sample to thereby place the captured Sugar chain Reagentrack sample under the reaction conditions, and then discard 45 ing the acetic anhydride by Suction; PHL 1.92 DTT O.48 B-6) a step of adding hydrochloric acid to the captured IAA O.96 Sugar chain sample, and discarding the hydrochloric 10% Ac2O 9.6 acid by Suction; MTT 9.6 B-7) a step of adding methanol to the captured Sugar chain 50 20 mM reagent 1.92 sample, discarding the methanol by Suction, and then 5% ACN 9.6 adding dioxane to the captured Sugar chain sample: Washing valve B-8) a step of wiping the bottom with a cotton sheet; 2% AcOFH in ACN 34.56 B-9) a step of adding methyl-p-tolyl-triazene (MTT) to the 2M guanidine hydrochloride 38.4 55 Water 86.4 captured Sugar chain sample, and allowing the mixture 190 TEA 38.4 to react for 60 minutes at 80° C.; 10 mMHC 38.4 B-10) a step of adding dioxane to the captured Sugar chain MeOH 38.4 sample, and discarding the dioxane by Suction; Dioxane 57.6 B-11) a step of Washing the captured Sugar chain sample 95% ACN 72.96 with methanol, discarding the methanol by Suction, 60 washing the sample with a NaCl Solution, discarding the It should be understood that the process described above is NaCl Solution by Suction, washing the sample sequen Subject to modification according to necessity. tially with water, and then discarding the water by suc tion; DETAILED DESCRIPTION B-12) a step of adding acetic acid and acetonitrile to the 65 captured Sugar chain sample, and tagging the Sugar Hereinafter, a preferred embodiment of the present inven chain in the captured Sugar chain sample using aminoOX tion will be described. The embodiment provided in the fol US 8,877,454 B2 41 42 lowing is provided for the purpose of better understanding of A-4) The SH protective agent is iodoacetamide (IAA) or the present invention, and it should be understood that the an equivalent, and scope of the present invention is not intended to be limited to the reaction condition is 20 to 37° C. (typically, room the following description. Therefore, it is obvious that a per temperature) in the dark. The reaction time is typically 0.5 to son ordinarily skilled in the art can make reference to the 2 hours, and for example, one hour. descriptions in the present specification, and to appropriately A-5) The proteolytic enzyme is typically trypsin, chymot modify the invention within the scope of the present inven rypsin, or an equivalent, and preferably trypsin, and tion. the reaction condition is 25 to 42°C., and typically 37°C. (Sugar Chain Analysis Method) The reaction time is typically 30 to 120 minutes, and for According to one aspect, the present invention provides a 10 example, about 60 minutes. A-6) The conditions for the deactivation include heating to method for analyzing a Sugar chain in a sample. This method typically 65° C. or higher, and preferably 80 to 100° C. (for includes A) a Sugar chain releasing step of releasing a Sugar example, 90° C.). Here, the heating time is typically 1 to 10 chain in a sample; B) a detection sample preparing step of minutes (for example, 5 minutes), and the system is option preparing the released Sugar chain for use in detection; when 15 ally cooled to room temperature. performing mass spectrometry using a plate, the following A-7) The Sugar chain releasing enzyme is peptide-N-gly step C) a step of producing a plate for mass spectrometry cosidase F. peptide-N4-(acetyl-3-glucosaminyl)-asparagine having the captured Sugar chain sample dotted thereon; and a amidase (PNGaseF), Endo H, or an equivalent, and it is pref step of performing an analysis of the Sugar chain to be deter erable not to have evaporation of the solvent during the step. mined. A countermeasure can be taken by a heating treatment at the In the A) Sugar chain releasing step of releasing a Sugar ceiling. chain in a sample, the following processes may be carried out. The reaction conditions for the Sugar chain releasing A Sugar chain releasing step, comprising enzyme are 25°C. to 42°C. (for example, 37° C.), and the A-1) a step of providing the sample on a plate for reaction; reaction time is typically 2 to 24 hours, and for example, 12 A-2) a step of adding a solubilizing agent to the sample to 25 hours. thereby place the sample under a reaction condition; In the B) detection sample preparing step of preparing the A-3) a step of adding a reducing agent to the sample to released Sugar chain for use in detection, the following pro thereby place the sample under a reaction condition; cesses may be carried out: A-4) a step of adding an —SH protecting agent to the B-1) a step of contacting the sample prepared in the step sample to thereby place the sample under a reaction 30 (A) with a Sugar chain-capturing bead to thereby place condition; the sample under the conditions allowing the released A-5) a step of adding a proteolytic enzyme to the sample to Sugar chain in the sample to bind to the bead, and thus thereby place the sample under a reaction condition; producing a captured Sugar chain Sample; A-6) a step of deactivating the proteolytic enzyme; and B-2) a step of adding a protein denaturing agent to the A-7) a step of adding a Sugar chain releasing enzyme to the 35 captured Sugar chain sample to thereby place the cap sample to thereby release the Sugar chain. tured Sugar chain sample under a reaction condition; In sub-processes of the step (A), it is preferable to use the B-3) a step of Washing the captured Sugar chain sample, conditions shown below, but the invention is not intended to and then discarding the residual washing liquid by Suc be limited thereto. For the following conditions, any one may tion; be employed, or a plurality of them may be employed. 40 B-4) a step of adding a salt releasing agent for the Sugar A-1) The step of providing the sample on a plate for reac chain capturing agent on beads to the captured Sugar tion may be manually carried out, or may be automated. chain sample, and then discarding the salt releasing Representative examples of the sample include a body fluid, agent by Suction; a cell extract and a tissue extract, and a more representative B-5) a step of adding a protective agent to the captured example is blood serum. Typically, the sample is provided on 45 Sugar chain sample to thereby place the captured Sugar a filter plate. chain under a reaction condition; In regard to A-2), the Solubilizing agent is 1-propane B-6) a step of adding an acid to the captured Sugar chain sulfonic acid, 2-hydroxy-3-lauramide (PHL), 1-propane sample, and discarding the acid by Suction; sulfonic acid, 2-hydroxy-3-myristamide (PHM), 2-hydroxy B-7) a step of adding an organic reaction solvent to the 3-sulfopropyl laurate (HSD), or an equivalent thereto, and 50 captured Sugar chain Sample: these can be represented by the formula: R—OCOO– B-8) a step of removing the solvent and the moisture in the CHCH(OH)CH, SO ONa (wherein, R represents an bead; alkyl group). For example, it is preferable to use 1-propane B-9) a step of adding an alkyl esterifying agent to the sulfonic acid, 2-hydroxy-3-lauramide (PHL), or 1-propane captured Sugar chain sample to thereby place the cap sulfonic acid, 2-hydroxy-3-myristamide (PHM)/ammonium 55 tured Sugarchain sample under a reaction condition, and bicarbonate. As such a solubilizing agent, those listed in WO alkylating the carboxylic acid of Sialic acid; 2008/001888 (PCT/JP2007/063100) can be used. B-10) a step of adding the organic reaction solvent to the The reaction conditions described above are typically 25° captured Sugar chain sample, and discarding the organic C. to 42°C., and preferably 37° C., and the reaction time is reaction solvent by Suction; typically 5 to 60 minutes, and preferably about 10 minutes. 60 B-11) a step of washing the captured Sugar chain sample, A-3) The reducing agent is dithiothreitol (DTT), TCEP and Subsequently discarding the residual washing liquid (Tris(2-carboxyethyl)phosphine hydrochloride solution 0.5 by Suction; M), or an equivalent, B-12) a step of releasing a Sugar chain sample from the the reaction condition is usually room temperature to 80° captured Sugar chain sample, wherein when an analysis C., preferably 50° C. to 80°C., and typically 60° C., and the 65 requiring tagging is conducted, the Sugar chain in the reaction time is typically 30 minutes to 2 hours, and prefer captured Sugar chain sample is tagged with a labeling ably about one hour. reagent and is released from the bead; and US 8,877,454 B2 43 44 B-13) a step of dissolving the released Sugar chain sample thereto. The NaCl solution can be used at any concentration, to produce a Sugar chain sample solution. but for example, a solution at 10 to 100 mM may be used, and In the sub-processes in the step (B), it is preferable to use a solution at 20 mM for example, may be used. processes characterized by one or a plurality of the conditions B-12) Tagging is carried out. Typically, the tagging is car as shown below, but the invention is not intended to be limited ried out using a chromophore capable of absorbing ultraviolet thereto. and visible rays, a tag having a structure emitting fluores In regard to B-1), the bead is a bead or magnetic bead cence, an affinity tag having a molecule capable of interacting having a Sugar chain-capturing group containing an ami with another molecule (His tag, biotin, or the like), a tag nooxy group, an N-alkylaminooxy group, a hydrazide group, having a functional group capable of specifically reacting an azide group, a thiosemicarbazide group, a cysteine residue, 10 with a functional group (a photoreactive molecule (for or a derivative thereof, and crosslinking), an azide group, an SH group, an amino group. the conditions in which the Sugar chain in the sample binds carboxylic acid, or the like), a tag having a functional group in to the bead are typically 25 to 80°C., and more typically 40 to a hydrophobic structure, or a tag having a metal ion ligand, 80°C., and for example, 80°C. is used. and the tagging is conducted by adding acetic acid, acetoni B-2) The denaturing agent is guanidine hydrochloride, 15 trile, an acetate buffer solution or an equivalent. It is prefer urea, sodium dodecyl sulfate or an equivalent, and able to subject the sample to MALDI-TOF MS. Even for mass the reaction conditions involve adding at room tempera spectrometry, another ionization method can be used. For ture, and maintaining the temperature to allow the bead to example, multi-stage mass spectrometry Such as MS/MS can sufficiently swell (from 10 seconds to 5 minutes). be carried out by ESI-MS (electrospray ionization mass spec B-3) The washing is performed using water, and then the trometry), and the respective mass spectrometric methods water is discarded by Suction. (LC-MS/MS, or the like). In addition to that, recovering the B-4) The Sugar chain capturing agent on the bead is an Sugar chain using a fluorescent dye tag and performing LC aminooxy group, an N-alkylaminooxy group, a hydrazide ESI-MS (/MS) is contemplated. For example, acetic acid and group, an azide group, a thiosemicarbazide group, a cysteine acetonitrile are added to the captured Sugar chain sample, and residue or a derivative thereof, and the salt releasing agent is 25 the Sugar chain in the captured Sugar chain sample can be triethylamine or an equivalent thereto in the case of tagged using aminooxytryptophanyl-arginine methyl ester/ hydrazide, and is triethylamine in the case of an N-alkylami water, O-benzylhydroxylamine hydrochloride/water, or nooxy group. Thereafter, the triethylamine is optionally dis anthraniloyl hydrazine/water. The Sugar chain can also be carded by Suction. recovered using a biotin tag and be subjected to an interaction B-5) The protective agent is acetic anhydride, Succinic 30 analysis based on SPR such as BIACORE, or to the produc anhydride, another acid anhydride or an equivalent, and tion of a microarray plate. When the Sugar chain is recovered the reaction conditions include (for example, 10%) acetic using a fluorescent dye tag, it is not that MALDI-TOF analy anhydride/methanol at 15 to 37°C. (for example, room tem sis cannot be performed, and the analysis can be carried out by perature) for 10 minutes to 2 hours (for example, 30 minutes). selecting appropriate conditions. Thereafter, the acetic anhydride is optionally discarded by 35 B-13) The dissolution of the tagged captured Sugar chain Suction. sample is typically carried out using water, an aqueous solu B-6) The acid is hydrochloric acid, another inorganic salt tion or an equivalent. acid, or an equivalent acid at pH 2 to 3, and the acid is When performing mass spectrometry using a plate, the optionally discarded by Suction. following process C can be carried out, and C) the step of B-7) Prior to the purging with the organic reaction solvent, 40 producing a plate for mass spectrometry having the captured a step of purging with a hydrophilic organic solvent is Sugar chain sample dotted thereon, can have at least one included. This hydrophilic organic Solvent is typically a lower feature among those described below: alcohol Such as methanol or ethanol, acetonitrile or acetone, C-1) a step of disposing the tagged Sugar chain sample and the organic reaction solvent is dioxane, acetonitrile, tet solution obtained in the step (B) on a plate for recovery; and, rahydrofuran or an equivalent. The methanol or the like is 45 the step optionally comprises the steps (C-2) to (C-6): optionally discarded by Suction, and then dioxane is added to C-2) a step of disposing the tagged Sugar chain sample the captured Sugar chain sample. Solution from the plate for recovery and the organic B-8) The step of removing the solvent and moisture in the Solvent, on a plate for mixing so as to obtain a concen bead typically includes wiping the bottom with a cotton sheet, tration at which the Sugar chain adsorbs to a solid phase; a paper, a blotting paper, gauze, a towel, a hand towel, a tissue 50 C-3) a step of providing a solid phase carrier-enclosed paper or the like. plate; B-9) The alkyl esterifying agent is typically methyl-p- C-4) a step of activating the Solid phase carrier-enclosed tolyl-triazene (MTT), ethyl-p-tolyltriazene (ETT), butyl-p- plate according to the phase of the Solid phase carrier tolyl-triazene (BTT), or an equivalent, and methyl-p-tolyl enclosed plate, and washing the Solid phase carrier-en triazene (MTT) is preferred. 55 closed plate; The reaction conditions include 100 mMMTT/dioxane at C-5) a step of adding the tagged Sugar chain sample solu 60 to 80° C. for 30 to 120 minutes (for example, 60 minutes). tion to the Solid phase carrier-enclosed plate, and con B-10) The organic reaction solvent is typically dioxane, ditioning the tagged Sugar chain sample solution to a acetonitrile, tetrahydrofuran or an equivalent, and dioxane is Solvent having a polarity appropriate for the phase of the preferred. The dioxane or the like is optionally discarded by 60 Solid phase carrier-enclosed plate; Suction. C-6) a step of recovering the tagged Sugar chain sample B-11) The washing is performed using at least one selected Solution by Suction from the Solid phase carrier-enclosed from the group consisting of methanol, a NaCl Solution and plate to a second plate for recovery; and water, and thereafter, the methanol, NaCl solution and water when subjecting the tagged Sugar chain sample solution to are optionally discarded by Suction. An amount Sufficient for 65 MALDI-TOF MS, the following step (C-7) is carried out: washing may be used, and for example, an amount of 100 ul C-7) a step of dotting the tagged Sugar chain sample solu to 1 ml, for example, 200 ul, may be used but is not limited tion on a matrix for mass spectrometry. US 8,877,454 B2 45 46 In the sup-processes for the step (C), it is preferable to use extraction Such as ion exchange. Therefore, although the type processes characterized by one or a plurality of conditions as of the SPE plate and the solvent for activation and washing shown below, but the invention is not intended to be limited depend on the properties of the reagent, it is preferable to thereto. carry out a similar procedure. If the excess reagent does not C-1) The disposing on the plate for recovery is conducted cause any problem (for example, if the reagent emits fluores under the conditions of removing the reagent for tagging. cence for the first time by binding to the Sugar chain), the C-2) The concentration at which the Sugar chain adsorbs to sample can be subjected to analysis while being retained in an the solid phase is typically 80 to 90% in an organic solvent, autosampler as received. In the case of biotin tag as well, it is and preferably, the tagged Sugar chain sample solution from preferable to remove any free (not bound to the sugar chain) the plate for recovery and acetonitrile are disposed on a plate 10 reagent. It is preferable to implement the SPE plate operation. for mixing, so as to obtain a final concentration of acetonitrile of 80 to 90%. The respective features of the above preferred embodiment C-3) The solid phase carrier-enclosed plate is typically of are such that one may be included, or two or more may be multi-well type and includes a surface of a resin or membrane combined. suitable for solid phase extraction. 15 (Sugar Chain Releasing Method for Releasing Sugar Chain C-4) When the solid phase carrier-enclosed plate is in in Sample) normal phase mode, washing is conducted sequentially with The present invention provides a Sugar chain releasing water and acetonitrile, and when the Solid phase carrier-en method for releasing a Sugar chain in a sample. This method closed plate is in reverse phase mode, washing is conducted comprises the following steps: sequentially with a lower alcohol Such as methanol and water. A-1) a step of providing the sample on a plate for reaction; C-5) The solvent having an opposite polarity is a hydro A-2) a step of adding a solubilizing agent to the sample to phobic organic solvent in the case of the normal phase mode, thereby place the sample under a reaction condition; and is a hydrophilic solvent in the case of the reverse phase A-3) a step of adding a reducing agent to the sample to mode. Here, for example, the tagged Sugar chain sample thereby place the sample under a reaction condition; Solution is added to the Solid phase carrier-enclosed plate, the 25 A-4) a step of adding an —SH protecting agent to the liquid is discarded, and typically, the plate is washed with sample to thereby place the sample under a reaction condi acetonitrile. Furthermore 1 to 20% (for example, 5%) aceto tion; nitrile can be added thereto. A-5) a step of adding a proteolytic enzyme to the sample to C-6) The second plate for recovery is of multi-well type thereby place the sample under a reaction condition; and includes a surface of a resin or membrane Suitable for 30 A-6) a step of deactivating the proteolytic enzyme; and Solid phase extraction. A-7) a step of adding a Sugar chain releasing enzyme to the C-7) the matrix for mass spectrometry is typically 2.5- sample to thereby release the Sugar chain. dihydroxybenzoic acidoran equivalent, and the dotting of the According to a preferred embodiment, a method further tagged Sugar chain sample solution on the matrix for mass having at least any one of the following features for the spectrometry is conducted in mixture or in sequence, and is 35 above-described steps is provided: diluted as necessary. Furthermore, preferably, 2,5-dihydroxy A) in the Sugar chain releasing step of releasing a Sugar benzoic acid in 20 to 40% (for example, 30%) acetonitrile can chain in a sample to prepare a Sugar chain sample for analysis: be added to the tagged Sugar chain sample solution, and the A-1) the sample is a body fluid, a cell extract or a tissue mixture can be dotted. eXtract, In the D) step of conducting an analysis of the Sugar chain 40 A-2) the solubilizing agent is 1-propanesulfonic acid, to be determined, any mass spectrometric method, typically 2-hydroxy-3-lauramide (PHL), 1-propanesulfonic acid, MADLI-TOF can be used. Upon conducting MALDI-TOF in 2-hydroxy-3-myristamide (PHM), 2-hydroxy-3-sulfo the step (D), it is preferable to carry out the overall step (C). propyl laurate (HSD) or an equivalent thereto, or The mass spectrometry of the Sugar chain to be determined is the reaction condition is at 25°C. to 42°C.; carried out by high performance liquid chromatography 45 A-3) the reducing agent is dithiothreitol (DTT), TCEP (HPLC), liquid chromatography-electrospray ionization (Tris(2-carboxyethyl)phosphine hydrochloride solu mass spectrometry (LC-ESIMS), Matrix Assisted laser Des tion, 0.5 M), or an equivalent thereto, or orption Ionization-Time-of-Flight (MALDI-TOF), or an the reaction condition is at room temperature to 80°C.; equivalent. When using a coloring reagent or biotin in the A-4) the -SH protecting agent is iodoacetamide (IAA) or tagging, it is preferable to carry out a step of removing any 50 an equivalent thereto, or excess coloring reagent. When the bead are magnetic beads, the reaction condition is at 20 to 37° C. in the dark; the magnetic beads are preferably beads having a modifiable A-5) the proteolytic enzyme is trypsin, chymotrypsin oran functional group (for example, a carboxyl group, an amino equivalent thereto, or group, an epoxy group, a tosyl group, or Streptavidin-bound the reaction condition is at 25 to 42°C.; magnetic beads). 55 A-6) the conditions for deactivating include heating to 65° If there is no problem in the Subsequent analysis such as C. or higher; MALDI-TOF MS while the sample has been recovered to the A-7) the Sugar chain releasing enzyme is peptide-N-gly plate for recovery 1, the process using the SPE plate can be cosidase F. peptide-N4-(acetyl-3-glucosaminyl)-aspar omitted. When MALDI-TOF MS is carried out under that agine amidase (PNGaseF), Endo H or an equivalent situation, the sample is transferred from the plate for recovery 60 thereto, or 1 to an MP2 plate for mixing and is mixed with the matrix, and the reaction conditions for the Sugar chain releasing then the mixture is dotted on the MALDI plate. When using enzyme are at 25° C. to 42° C. the sample directly with HPLC, LC-ESIMS or the like, the According to a more preferred embodiment, a method fur step can be omitted. ther having at least one among the following steps is pro When using a coloring reagent, if it is necessary to remove 65 vided: any excess coloring reagent, it is preferable to carry out A-1) a step of providing blood serum as a sample on a filter reverse phase or normal phase extraction, or Solid phase plate; US 8,877,454 B2 47 48 A-2) a step of adding 1-propanesulfonic acid, 2-hydroxy B-13) a step of dissolving the released Sugar chain sample 3-lauramide (PHL) or 1-propanesulfonic acid, 2-hydroxy-3- to produce a Sugar chain sample solution. myristamide (PHM)/ammonium bicarbonate, and allowing According to a preferred embodiment, a method further the mixture to react for 5 to 60 minutes at 25 to 42°C.; having at least any one of the following features for the A-3) a step of adding dithiothreitol (DTT) to the sample, above-described steps is provided: allowing the mixture to react for 10 to 60 minutes at 50 to 80° In regard to B-1), C., and then cooling the reaction mixture to room tempera the bead is a bead or magnetic bead having bound thereto a ture; Sugar chain capturing group which includes an aminooxy A-4) a step of adding iodoacetamide (IAA), and allowing group, an N-alkylaminooxy group, a hydrazide group, an the mixture to react for 0.5 to 2 hours at room temperature in 10 the dark; azide group, a thiosemicarbazide group, a cysteine residue or A-5) a step of adding trypsin to the sample, and allowing a derivative thereof, or the mixture to react for 30 to 120 minutes at 25 to 42°C.; the conditions in which the released Sugar chain in the A-6) a step of heating the sample to 80 to 100° C. for 1 to sample binds to the bead are at 25 to 80° C.; 10 minutes, and then cooling the sample to room temperature; 15 B-2) the denaturing agent is guanidine hydrochloride, urea, and Sodium dodecyl Sulfate or an equivalent, or A-7) a step of adding PNGaseF, and allowing the mixture the reaction conditions involve adding at room tempera to react for 6 to 24 hours at 25 to 42°C. ture, and maintaining the temperature to allow the bead to According to a certain embodiment, the method is used for sufficiently swell (from 10 seconds to 5 minutes); the preparation of a pretreatment sample for analyzing a Sugar B-3) the washing is performed using water; chain in a sample. B-4) the Sugar chain capturing agent on the bead is an In the above preferred embodiments, the respective fea aminooxy group, an N-alkylaminooxy group, a hydrazide tures may be included singly or in combination or two or group, an azide group, a thiosemicarbazide group, a cysteine O. residue or a derivative thereof, and the salt releasing agent is (Detection Sample Preparing Method for Preparing 25 triethylamine or an equivalent in the case of hydrazide, and is Released Sugar Chain for Use in Detection) triethylamine or an equivalent in the case of an N-alkylami The present invention also provides a detection sample noOXy group; preparing method for preparing the released Sugar chain for B-5) the protective agent is acetic anhydride, Succinic use in detection. This method includes the following steps: anhydride or another acid anhydride, or an equivalent, or B-1) a step of contacting a sample with a Sugar chain 30 capturing bead to thereby place the sample under the condi the reaction conditions use acetic anhydride/methanol at tions allowing the released sugar chain in the sample to bind 15 to 37° C.; to the bead, and thus producing a captured Sugar chain B-6) the acid is hydrochloric acid or another inorganic salt sample: acid, or an equivalent acid at pH 2 to 3: B-2) a step of adding a protein denaturing agent to the 35 B-7) the step includes a step of replacing with a hydrophilic captured Sugar chain Sample to thereby place the captured organic solvent before replacing with the organic reaction Sugar chain sample under a reaction condition; Solvent, and the hydrophilic organic solvent is a lower alcohol B-3) a step of Washing the captured Sugar chain sample, Such as methanol or ethanol, acetonitrile, or acetone, while and then discarding the residual washing liquid by Suction; the organic reaction solvent is dioxane, acetonitrile, tetrahy B-4) a step of adding a salt releasing agent for the Sugar 40 drofuran or an equivalent; chain capturing agent on beads to the captured Sugar chain B-8) the step of removing the solvent and the moisture in sample, and then discarding the salt releasing agent by Suc the bead includes wiping of the bottom with a filter paper, a tion; blotting paper, agauze, a towel, a hand towel, a tissue paperor B-5) a step of adding a protective agent to the captured a cotton sheet; Sugar chain sample to thereby place the captured Sugar chain 45 B-9) the alkyl esterifying agent is methyl-p-tolyltriazene under a reaction condition; (MTT), ethyl-p-tolyltriazene (ETT), butyl-p-tolyltriazene B-6) a step of adding an acid to the captured Sugar chain (BTT) or an equivalent thereto, or sample, and discarding the acid by Suction; the reaction conditions use 100 mMMTT/dioxane at 20 to B-7) a step of adding an organic reaction solvent to the 80° C. for 30 minutes to 5 hours: captured Sugar chain Sample: 50 B-10) the organic reaction solvent is dioxane, acetonitrile, B-8) a step of removing the solvent and the moisture in the tetrahydrofuran or an equivalent thereto; bead; B-11) the washing is performed using at least one selected B-9) a step of adding an alkyl esterifying agent to the from the group consisting of methanol, a NaCl Solution and captured Sugar chain Sample to thereby place the captured water; Sugar chain sample under a reaction condition, and alkylating 55 B-12) tagging is carried out, such that the tagging is per the carboxylic acid of sialic acid; formed using a chromophore capable of absorbing ultraviolet B-10) a step of adding the organic reaction solvent to the and visible rays, a tag having a structure emitting fluores captured Sugar chain sample, and discarding the organic reac cence, an affinity tag having a molecule capable of interacting tion solvent by Suction; with another molecule, a tag having a functional group B-11) a step of washing the captured Sugar chain sample, 60 capable of specifically reacting with a functional group, a tag and Subsequently discarding the residual washing liquid by having a functional group in a hydrophobic structure, or a tag Suction; having a metal ion ligand, and the tagging is conducted by B-12) a step of releasing a Sugar chain sample from the adding acetic acid, acetonitrile, an acetate buffer or an equiva captured Sugar chain sample, wherein when an analysis lent; or requiring tagging is conducted, the Sugar chain in the cap 65 B-13) the dissolving of the tagged captured Sugar chain tured Sugar chain sample is tagged with a labeling reagent and sample is performed using water, an aqueous solution or an is released from the bead; and equivalent. US 8,877,454 B2 49 50 According to a more preferred embodiment, a method fur on a plate for mixing so as to obtain a concentration in the ther having at least one among the following steps is pro organic solvent at which the Sugar chain adsorbs to a solid vided: phase; B-1) a step of contacting a captured Sugar chain sample C-3) a step of providing a solid phase carrier-enclosed with beads for capturing Sugar chain, to thereby allow binding 5 plate; at 40 to 80° C., and thus producing a captured Sugar chain C-4) a step of activating the Solid phase carrier-enclosed sample: plate according to the phase of the Solid phase carrier-en B-2) a step of adding guanidine hydrochloride to the cap closed plate, and washing the Solid phase carrier-enclosed tured Sugar chain sample to thereby place the captured Sugar plate; chain sample under a reaction condition, and then discarding 10 C-5) a step of adding the tagged Sugar chain sample solu the reaction liquid by Suction; tion to the Solid phase carrier-enclosed plate, and condition B-3) a step of Washing the captured Sugar chain sample ing the tagged Sugar chain sample solution to a solvent having with water, and then discarding the water by Suction; a polarity appropriate for the phase of the Solid phase carrier B-4) a step of Washing the captured Sugar chain sample enclosed plate; with triethylamine, and then discarding the triethylamine by 15 C-6) a step of recovering the tagged Sugar chain sample Suction; Solution by Suction from the Solid phase carrier-enclosed B-5) a step of adding acetic anhydride to the captured Sugar plate to a second plate for recovery; and chain sample to thereby place the captured Sugar chain when subjecting the tagged Sugar chain sample solution to sample under the reaction conditions of using 10% acetic MALDI-TOF MS, the method includes the following step anhydride/methanol at room temperature for 10 minutes to 2 (C-7): hours, and then discarding the acetic anhydride by Suction; C-7) a step of mixing the tagged Sugar chain sample solu B-6) a step of adding hydrochloric acid to the captured tion with a matrix for mass spectrometry, and dotting the Sugar chain sample, and discarding the hydrochloric acid by mixture on a plate for determination. Suction; According to a preferred embodiment, a method further B-7) a step of adding methanol to the captured Sugar chain 25 having at least any one of the following features for the sample, discarding the methanol by Suction, and then adding above-described steps is provided: dioxane to the captured Sugar chain sample; C-1) the disposing on the plate for recovery is conducted B-8) a step of wiping the bottom with a cotton sheet; under the conditions of removing the reagent for tagging; B-9) a step of adding methyl-p-tolyltriazene (MTT) to the C-2) the concentration at which the Sugar chain adsorbs to captured Sugar chain sample, and allowing the mixture to 30 the solid phase is 80 to 90% in an organic solvent; react for 30 to 120 (60) minutes at 60° C.: C-3) the solid phase carrier-enclosed plate is of multi-well B-10) a step of adding dioxane to the captured sugar chain type and includes a surface of a resin or membrane suitable sample, and discarding the dioxane by Suction; for Solid phase extraction; B-11) a step of Washing the captured Sugar chain sample C-4) when the solid phase carrier-enclosed plate is in nor sequentially with methanol, a NaCl Solution and water, and 35 mal phase mode, washing is conducted sequentially with then discarding the water by Suction; water and acetonitrile, and when the Solid phase carrier-en B-12) a step of adding acetic acid and acetonitrile to the closed plate is in reverse phase mode, washing is conducted captured Sugar chain sample, and tagging the Sugar chain in sequentially with a lower alcohol Such as methanol and water; the captured Sugar chain sample using aminooxytryptopha C-5) the solvent having an opposite polarity is a hydropho nyl-arginine methyl ester/water, O-benzylhydroxylamine 40 bic organic Solvent in the case of the normal phase mode, and hydrochloride/water, or anthraniloyl hydrazine/water; and is a hydrophilic solvent in the case of the reverse phase mode: B-13) a step of adding water to the tagged captured Sugar C-6) the second plate for recovery is of multi-well type and chain sample to produce a tagged Sugar chain sample solu includes a surface of a resin or membrane suitable for solid tion. phase extraction; and According to another preferred embodiment, the Sugar 45 C-7) the matrix for mass spectrometry is 2,5-dihydroxy chain-capturing bead are magnetic beads, and separation is benzoic acid or an equivalent, and the dotting of the tagged carried out using a magnetic field instead of discarding by Sugar chain sample solution on the matrix for mass spectrom Suction as described above. etry is conducted in mixture or in sequence, and is diluted as According to a certain embodiment, the method is used for necessary. the preparation of a pretreatment sample for analyzing a Sugar 50 According to a more preferred embodiment, a method for chain in a sample. producing a plate for mass spectrometry, further having at In the above preferred embodiments, the respective fea least one among the following steps, is provided: tures may be included singly or in combination or two or C-1) a step of disposing a tagged Sugar chain sample solu O. tion on a plate for recovery; (Method for Producing Plate for Mass Spectrometry Hav 55 C-2) a step of disposing the tagged Sugar chain sample ing Captured Sugar Chain Sample Dotted Thereon, for Mass solution from the plate for recovery and acetonitrile on a plate Spectrometry Using Plate) for mixing, so as to achieve a final concentration of acetoni According to another aspect, the present invention pro trile of 80 to 90%; vides a method for producing a plate for mass spectrometry C-3) a step of providing a solid phase carrier-enclosed plate having the captured Sugar chain sample dotted thereon, for 60 which is in normal phase mode; mass spectrometry using a plate. This method comprises the C-4) a step of washing the Solid phase carrier-enclosed following step(s): plate sequentially with water and acetonitrile, and discarding C-1) a step of disposing a tagged Sugar chain sample solu water and acetonitrile by Suction; tion on a plate for recovery; and, optionally comprises the C-5) a step of adding the tagged Sugar chain sample solu steps (C-2) to (C-6): 65 tion to the Solid phase carrier-enclosed plate, discarding the C-2) a step of disposing the tagged Sugar chain sample liquid, washing the plate with acetonitrile, and adding 1 to Solution from the plate for recovery and the organic solvent, 20% acetonitrile thereto; US 8,877,454 B2 51 52 C-6) a step of recovering the bead by suction from the solid chain and a Sugar chain-capturing bead, in which the Sugar phase carrier-enclosed plate to the second plate for recovery; chain-capturing bead bound with the Sugar chain released and from the sample are dotted on at least one hole of the plate, C-7) a step of adding 2,5-dihydroxybenzoic acid in 20 to and the Sugar chain has been tagged using a tagging reagent 40% acetonitrile, to the tagged Sugar chain sample solution, Such as aminooxytryptophanyl-arginine methyl ester/water, and mixing and dotting the mixture. O-benzylhydroxylamine hydrochloride/water oranthraniloyl According to another preferred embodiment, the Sugar hydrazine/water. chain-capturing bead are magnetic beads, and separation is In regard to the respective features shown above, the carried out using a magnetic field instead of discarding by respective features for the above preferred embodiments Suction as described above. 10 described in the present specification may be included singly, According to a certain embodiment, the method is used for or two or more of the features may be combined. the preparation of a pretreatment sample for analyzing a Sugar (Plate for Analyzing Sugar Chain in Sample) chain in a sample. The present invention also provides a plate for analyzing a In the above preferred embodiments, the respective fea Sugar chain in a sample, the plate having at least one com tures may be included singly or in combination or two or 15 partment for analysis, and at least one of the compartment O. having a Sugar chain-capturing bead previously dispensed (Kit for Production of Plate for Reaction Comprising therein. As the plate, for example, a multi-well plate Such as Sample-Derived Sugar Chain and a Sugar Chain-Capturing a 96-well plate, or a filter plate is used. In regard to 121 Bead) (BlotGlyco H) in FIG. 1 and Start2 (BlotGlyco ABC) in FIG. The present invention provides a kit for production of a 8, instead of manually dispensing a Sugar chain-capturing plate for reaction comprising a sample-derived Sugar chain bead by hand, a plate having such a Sugar chain-capturing and a Sugar chain-capturing bead, which kit is provided with bead previously dispensed, can be used. Analysis can be a means for realizing any of the operations of the method of carried out more conveniently with Such a plate, by combin the present invention for analyzing a Sugar chain in a sample. ing the plate with an auto-analyzing apparatus for Sugar An example of Such a kit, includes a kit comprising the 25 chain. following: In regard to the respective features shown above, the A-1) the sample: respective features for the above preferred embodiments A-2) 1-propanesulfonic acid, 2-hydroxy-3-lauramide described in the present specification may be included singly, (PHL) or 1-propanesulfonic acid, 2-hydroxy-3-myristamide or two or more of the features may be combined. (PHM)/ammonium bicarbonate; 30 (Exemplary Specific Embodiment of Automatic Sugar A-3) dithiothreitol (DTT): Chain Pretreatment Apparatus) A-4) iodoacetamide (IAA); Hereinafter, the best embodiment for implementing the A-5) trypsin; automatic Sugar chain pretreatment apparatus according to A-6) aheating unit; the present invention will be described with reference to the A-7) PNGaseF: 35 drawings. B-1) a Sugar chain-capturing bead; FIG. 15 is a plane view showing the automatic Sugar chain B-2) guanidine hydrochloride: pretreatment apparatus according to an embodiment of the B-3) water; present invention, with some parts being omitted from the B-4) triethylamine; diagram: FIG. 16 is a frontal view of the dispensing head; B-5) 10% acetic anhydride/methanol: 40 FIG. 17 is a cross-sectional view of FIG. 2, dissected along B-6) hydrochloric acid; the line A-A, FIG. 18 is a central vertical cross-sectional view B-7) methanol: of the first constant-temperature bath; FIG. 19 is an exploded B-8) cotton sheet; perspective view showing the first low-pressure recovering B-9) methyl-p-tolyltriazene (MTT); device and the filter plate moving mechanism: FIG. 20 is a B-10) dioxane: 45 plane view showing the filter plate moving mechanism, with B-11) methanol, a NaCl solution and water; Some parts being omitted from the diagram: FIG. 21 is a B-12) acetic acid and acetonitrile, and aminooxytryp Vertical cross-sectional view showing the vertical moving tophanyl-arginine methyl ester/water, O-benzylhydroxy unit in the filter plate moving mechanism, with some parts lamine hydrochloride/water or anthraniloyl hydrazine/water; being omitted from the diagram: FIG.22 is a perspective view B-13) water; 50 of the filterplate moving mechanism and the second constant C-1) a plate for recovery: temperature bath; FIG. 23 is a central vertical cross-sectional C-2) acetonitrile and a plate for mixing; view of the second constant-temperature bath; FIG. 24 and C-3) a solid phase carrier-enclosed plate; FIG. 25 are flow diagrams of the treatment operation. C-4) water and acetonitrile; The present invention allows the Sugar chain purification C-5) acetonitrile and 1 to 20% acetonitrile; 55 treatment to be carried out at high speed with high accuracy C-6) a second plate for recovery; and by automating the treatment, and provides, inside a casing C-7) 2,5-dihydroxybenzoic acid in 20 to 40% acetonitrile. base 1, a dispensing head 2 to move in longitudinal and In regard to the respective features shown above, the transverse directions by means of a moving mechanism 11. respective features for the above preferred embodiments Dispensing needles 10, 10, 10, ... are retained in the dispens described in the present specification may be included singly, 60 ing head 2 so as to move up and down. A first and second or two or more of the features may be combined. constant-temperature baths 12 and 29 are provided, which are (Plate for Analysis Comprising Sample-Derived Sugar equipped with a means to conduct heating and cooling of a Chain and a Sugar Chain-Capturing Bead) receiving stand, with the upper part of the receiving stand The present invention also provides a plate produced in being covered with a lid. A reagent rack 22, and microplates connection with the method for analyzing a Sugar chain in a 65 for mixing 23 and 24 are provided. In front of the second sample of the present invention. Such a plate can also be said constant-temperature bath 29, a first low-pressure recovering to be a plate for analysis comprising a sample-derived Sugar device 26, a first suction discarding device 27, and a bottom US 8,877,454 B2 53 54 wiper 28 are disposed in a row. In front of a target plate apparatus is made to operate each of the devices according to receiving stand 59, a second low-pressure recovering device the operation protocol inputted to the control device 60. 56 and a second suction discarding device 57 are disposed. Furthermore, in regard to the above constitution, a bottom The operation process inputted into the control device 60 wiper 28 which is provided with a planar wiping material on disposed in the casing base 1, operates the respective units the Surface, and is intended to wipe out the liquid adhering to mentioned above. the lower surface of the bottom of the filter plate, may be The automatic Sugar chain pretreatment apparatus accord installed between the first suction discarding device 27 and ing to the present embodiment is equipped with a casing base the second constant-temperature bath 29, as necessary. 1 provided with a cover capable of freely opening and closing Furthermore, the dispensing head moving mechanism 11 (not depicted); a dispensing head moving mechanism 11 that 10 in the constitution described above may employ a well known is installed inside the casing base 1 and moves a below moving mechanism making use of a motor or a rail. described dispensing head in longitudinal and transverse The dispensing head 2 is a dispensing head constituted to directions; a dispensing head 2 that raises and lowers a plu have a supporting frame 3; an elevator stand 5that slides in the rality of dispensing needles arranged in a row, all at the same 15 Vertical direction along a guide rod installed on the Supporting time by means of an elevating mechanism; a first constant frame 3 in parallel with the dispensing needles; a driving temperature bath 12 installed above the installation space 1 a motor 6 fixed on the Supporting frame 3; an elevator stand for the casing base 1, which is equipped with a unit that moving mechanism 8 that has a ball screw 7 connected to the conducts heating and cooling of a receiving stand 13 that rotating axis of the driving motor 6 and moves the elevator holds a microplate, and is provided with a lid 21 having an stand 5 in the vertical direction; and dispensing needles 10, inner lid 19 to cover the upper part of the receiving stand 13: 10, 10, ... retained in a dispensing needle holder 9 installed a reagentrack 22 and a plurality of microplates for mixing 23 on the elevator stand 5. and 24, which are installed above the installation space for the According to the present embodiment, a cylinder 2A is casing base 1; a first low-pressure recovering device 26, being fixed vertically downward at the lower part of the supporting in a frame form, which depressurizes while having a filter 25 frame 3 in the dispensing head 2, and at the same time, a plate mounted on the upper opening, and receives the liquid pressurizing plate 2B is adhered at the tip of the piston rod of that has passed through the filter of the filter plate, into a the cylinder 2A. If constituted as such, when Suctioning under microplate 25 installed inside the recovering device; a first reduced pressure is carried out for the first and second low Suction discarding device 27, being in a frame form, which pressure recovering devices 26 and 56, and the first and sec depressurizes and Suctions while having a filter plate 30 ond suction discarding devices 27 and 57, airtightness can be mounted on the upper opening, and discards the liquid that increased by suppressing the filter plate or the SPE plate from has passed through the filter; a second constant-temperature above, and thus the efficiency is enhanced. bath 29, which is equipped with a unit that conducts heating The first constant-temperature bath 12 is a first constant and cooling of a receiving stand 30 that holds a filterplate, and temperature bath having a main body part 12A that is con is provided with an automatically opening and closing lid 38 35 structed by including a cartridge heater 14 built in at the to cover the upper part of the receiving stand 30, all of these internal center of the receiving stand 13 formed from an devices being disposed and arranged in a row along the lon aluminum block, and also by disposing a Peltier element 15 gitudinal direction and above the installation space 1a for the and a heat sink 16 at the lower part of the receiving stand 13. casing base 1; a filterplate moving mechanism 39 that retains wherein the upper part of the receiving stand 13 in the main a filter plate and moves the filter plate to each of the first 40 body part 12A is covered by a lid 21 which has a silicone sheet low-pressure recovering device 26, the first Suction discard pasted on the inside and has an inner lid 19 with a built-in ing device 27 and the second constant-temperature bath 29 in cartridge heater 18 at the internal center, the inner lid being sequence; a second low-pressure recovering device 56, being elastically supported via a spring 20. Furthermore, for this in a frame form, which depressurizes while having an SPE first constant-temperature bath 12, while opening and closing plate mounted on the upper opening, and receives the liquid 45 of the lid is operated manually in the present embodiment, the that has passed through the solid phase body of the SPE plate, opening and closing may also be operated automatically. into a microplate 55 installed inside the recovering device; a Furthermore, the silicone sheet 17 is intended to prevent second Suction discarding device 57, being in a frame form, evaluation of the sample inside when an aluminum sheet 61a which depressurizes and suctions while having an SPE plate of the microplate 61 has been perforated by dispensing mounted on the upper opening, and discards the liquid that 50 needles 10, 10, 10, . . . . has passed through the Solid phase body; a target plate receiv The filter plate moving mechanism 39 is a moving mecha ing stand 59, which holds a target plate 58that is dotted on the nism having pulleys 41 and 42 installed at both ends in the surface with the sample that has been finished with the final length direction of a Supporting plate 40 that is installed to treatment step, all of these devices 56, 57 and 59 being dis stand along the longitudinal direction of the casing base 1, posed and arranged in a row along the longitudinal direction 55 with one of the pulleys 41 and 42 being made to be rotary and above the installation space 1a for the casing base 1; an driven by a step motor 43, wherein a horizontally moving SPE plate moving mechanism 39 that retains an SPE plate and plate 45 is connected to a belt 44 hung between the two moves the SPE plate between the second low-pressure recov pulleys 41 and 42 and revolved, a vertical moving unit 46 is ering device and the second Suction discarding device; a installed on the horizontally moving plate 45, and the vertical control device 60 that is installed in the casing base 1 and has 60 moving unit 46 is ascended and descended by a vertically been inputted with the operation protocol; a microplate 61 moving rod 51 that supports a receiving frame 48 intended to having a plurality of wells arranged in a matrix array, which is receive an inner frame 47 at the upper end, and slides in the covered with a sheet while biological samples have been vertical direction along a guide 50 installed vertically inside injected into each well; a filter plate 62 having a plurality of the supporting frame 49, and by a ball screw 53 connected to filters arranged in a matrix array; and an SPE plate (not 65 the rotating axis 52a of a motor 52 fixed inside the supporting depicted) for sample in a trace amount, having a plurality of frame 49, and wherein the mechanism is constituted to have Solid phase bodies arranged in a matrix array, wherein the an elevating rod 51 connected to the vertically moving rod 54. US 8,877,454 B2 55 56 Furthermore, gaskets 47a and 48a for maintaining airtight 13. The lid is opened, and the constant-temperature bath is ness are installed between the inner frame 47 and the receiv cooled at room temperature. ing frame 48. (The process up to this point corresponds approximately to The SPE plate moving mechanism 39 has the same consti the step (A-5).) tution as that of the filterplate moving mechanism, and there 14. Peptide-N-glycosidase F and peptide-N4-(acetyl-3- fore, explanation will be omitted. glucosaminyl)-asparagine amidase (PNGaseF) (2 U) are dis The second constant-temperature bath 29 is a second con pensed in an amount of 5ul to each well of the microplate 61. stant-temperature bath having a main body part 29A that is 15. The lid is closed, and the first constant-temperature constructed by including a cartridge heater 31 built in at the bath is heated at 37° C. for 12 hours. internal center of the receiving stand 30 formed from an 10 (The process up to this point corresponds approximately to aluminum block, and also an air circulation path 32 installed the step (A-6).) across from the internal center to the Surface, and by dispos 16. 1 Macetic acid (AcOH) is dispensed in an amount of ing a Peltier element 33 and a heat sink34 at the lower part of 200 ul to each well of the filter plate 62 containing polymer the receiving stand 30, wherein an air circulation path 32 beads, which is set up in the suction discarding device 27. which is in communication with the above air circulation path 15 17. The acetic acid is discarded by suction. 32 is provided between the upper part of the receiving stand (The process up to this point corresponds approximately to 30 in the main body part 29A and the filter plate located the step (B-1).) thereon, and a duct 36 and a fan 37 are further provided to let 18.50% ACN is dispensed in an amount of 300 ul. the air discharged out of the filter plate flow again into the 19. Acetonitrile is discarded by suction. receiving stand 30 to thereby circulate, while the upper part of 20. ACN is dispensed in an amount of 300 ul. the receiving stand 30 in the main body part 29A is covered by 21. ACN is discarded by suction. a lid 38. Furthermore, in the second constant-temperature (The process up to this point corresponds approximately to bath 29, the lid 38 is made to open and close automatically. the step (B-2.3).) Next, the treatment operation of the automatic Sugar chain 22. The filter plate 62 containing polymer beads is moved pretreatment apparatus related to the embodiments described 25 to the position of the bottom wiper 38, and bottom wiping is above will be explained according to the flow diagrams performed. The filter plate is moved to the second constant shown in FIG. 24 and FIG. 25. temperature bath 29 (the constant temperature bath opens 23. The second constant-temperature bath 29 is opened, 1. The cover of the casing base 1 is opened, the first con 30 and the enzymatically treated sample is dispensed in an stant-temperature bath 12 is opened, and the microplate 61, amount of 20 ul to the filter plate 62 containing polymer covered with an aluminum sheet while having a biological beads, which is placed on the receiving stand 30. sample injected in each well, is placed on the receiving stand (The process up to this point corresponds approximately to 13. the step (B-4).) (The process up to this point corresponds approximately to 35 24. 20 AcOH/ACN is dispensed in an amount of 200 ul. the step (A-1).) 25. The lid is closed, and the second constant-temperature 2. The dispensing head 2 is moved to suction 15ul of 0.33 bath is heated at 80° C. for 60 minutes. Mammonium bicarbonate from a container of the reagent 26.6 M guanidine hydrochloride is dispensed in an amount rack 22, and is moved to perform dispensing to each well of of 300 ul. the microplate 61. 40 27. The second constant-temperature bath is cooled at 3. In the same manner as in 2., 30 ul of a 0.4% solubilizing room temperature for 10 minutes, with the lid open. agent is dispensed. 28. The filter plate 62 is moved to the suction discarding 4. The first constant-temperature bath 12 is closed and is device 27. heated at 37° C. for 10 minutes. 29. Guanidine hydrochloride is discarded by suction. (The process up to this point corresponds approximately to 45 (The process up to this point corresponds approximately to the step (A-2).) the step (B-5).) 5. The lid is opened, and 5ul (CV (Coefficient of Variation) 30.6 M guanidine hydrochloride is dispensed in an amount 5%) of 120 mM dithiothreitol (DTT) is dispensed. of 300 ul. 6. The lid is closed, and the first constant-temperature bath 31. Guanidine hydrochloride is discarded by suction. is heated at 60° C. for 30 minutes. 50 32. Water is dispensed in an amount of 300 ul. 7. The lid is opened, and the first constant-temperature bath 33. Water is discarded by suction. is cooled at room temperature. 34. 10 M hydrochloric acid is dispensed in an amount of (The process up to this point corresponds approximately to 300 ul. the step (A-3).) 35. Hydrochloric acid is discarded by suction. 8. 123 mMiodoacetamide (IAA) is dispensed in an amount 55 (The process up to this point corresponds approximately to of 10 ul (CV 5%). the step (B-6).) 9. The first constant-temperature bath is cooled at room 36. MeOH is dispensed in an amount of 300 ul. temperature for 60 minutes in the dark. 37. MeOH is discarded by suction. (The process up to this point corresponds approximately to (The process up to this point corresponds approximately to the step (A-4).) 60 the step (B-7).) 10. Trypsin (400 U) is dispensed in an amount of 5 ul to 38. The filterplate 62 is moved to the position of the bottom each well of the microplate 61 which has been set up again in wiper 28, and bottom wiping is performed. the first constant-temperature bath 12, with the lid open. 39. The filter plate 62 is moved to the second constant 11. The lid is closed, and the first constant-temperature temperature bath 29. bath is heated at 37° C. for 60 minutes. 65 (The process up to this point corresponds approximately to 12. The first constant-temperature bath is further heated at the step (B-8).) 90° C. for 5 minutes. 40. A reducing reagent is dispensed in an amount of 200 ul. US 8,877,454 B2 57 58 41. Light is shielded, and the second constant-temperature 73. End. bath is cooled at room temperature for 30 minutes. (The process up to this point corresponds approximately to 42. The filter plate 62 is moved to the suction discarding the step (B-13).) device 27. When the Sample is in a Trace Amount 43. The reducing reagent is discarded by Suction. 74. The sample in the microplate 25 is dispensed in an 44. MeOH is dispensed in an amount of 300 ul. amount of 20 Jul to the microplate for mixing 23. 45. MeOH is discarded by suction. 75. ACN is dispensed in an amount of 400 ul to the micro 46. The filterplate 62 is moved to the position of the bottom plate for mixing 23. wiper 28, and bottom wiping is performed. 76. Water is dispensed in an amount of 200 ul to the SPE 10 plate positioned in the suction discarding device 57. 47. Water is dispensed in an amount of 300 ul. 77. Water is discarded by suction. 48. Water is discarded by suction. 78. ACN is dispensed in an amount of 200 ul to the SPE 49. ACN is dispensed in an amount of 300 ul. plate. 50. ACN is discarded by suction. 79. ACN is discarded by suction. 51. Bottom wiping is performed. 15 The sample in the microplate for mixing 23 is dispensed in 52. The filter plate 62 is moved to the second constant an amount of 420 ul to the SPE plate. temperature bath 29. 81. The sample is discarded by suction. 53. 100 mM MTT in DMSO/ACN is dispensed in an 82.95% ACN is dispensed in an amount of 200 ul to the amount of 100 ul. SPE plate. 54. The lid is closed, and the second constant-temperature 83. ACN is discarded by suction. bath is heated at 60° C. for 60 minutes. 84.10% ACN is dispensed in an amount of 20 ul to the SPE (The process up to this point corresponds approximately to plate. the step (B-9).) 85. The SPE plate is moved to the low-pressure recovering 55. The filter plate 62 is moved to the suction discarding device 56. device 27. 25 86. Pressure is reduced, and the sample is withdrawn into 56. MTT is discarded by suction. the microplate 55 and recovered. 57. ACN is dispensed in an amount of 300 ul. 87. The sample in the microplate 55 is dispensed in an 58. ACN is discarded by suction. amount of 2 ul to the microplate for mixing 24. (The process up to this point corresponds approximately to 88. MALDI matrix is dispensed in an amount of 2 ul to the 30 microplate for mixing 24. the step (B-10).) 89. The sample in the microplate for mixing 24 is dotted in 59. The filterplate 62 is moved to the position of the bottom an amount of 2 ul on the target plate 58 on the target plate wiper 28, and bottom wiping is performed. receiving stand 59. 60. The filter plate 62 is moved to the suction discarding 90. End. device 27. 35 C) When performing mass spectrometry using a plate, the 61. Water is dispensed in an amount of 300 ul. process of producing a plate for mass spectrometry having the (The process up to this point corresponds approximately to captured Sugar chain Sample dotted thereon is carried out. the step (B-11).) 91. A process of disposing the tagged Sugar chain sample 62. Water is discarded by suction. solution obtained by the processes up to 73. on a plate for 63. 50 mM DTT is dispensed in an amount of 50 ul. 40 recovery; and optionally, 92. to 93. are carried out. 64. The filter plate 62 is moved to the second constant 92. The tagged Sugar chain sample solution from the plate temperature bath 29. for recovery and the organic solvent are disposed on the plate 65. The lid is closed, and the second constant-temperature for mixing, so as to obtain a concentration of the organic bath is heated at 60° C. for 5 minutes. Solvent at which the Sugar chain adsorbs to a solid phase. 66. The lid is opened, and the second constant-temperature 45 93. The solid phase carrier-enclosed plate is provided. bath is cooled at room temperature for 15 minutes. 94. The solid phase carrier-enclosed plate is activated in 67. The filter plate 62 is moved to the low-pressure recov accordance with the phase of the Solid phase carrier-enclosed ering device 26. plate, and the plate is washed. 68. Pressure is reduced, and the sample is withdrawn into 95. The tagged Sugar chain sample solution is added to the the microplate 25 and recovered. 50 Solid phase carrier-enclosed plate, and the sample solution is (The process up to this point corresponds approximately to conditioned to a solvent having a polarity appropriate for the the first half of the step (B-12).) phase of the Solid phase carrier-enclosed plate. 69. The sample is cooled to 10° C., and the process is 96. The tagged Sugar chain sample solution is recovered by completed. Suction from the Solid phase carrier-enclosed plate to the 55 second plate for recovery. Thereafter, the treatment procedure is divided depending When Subjecting the tagged Sugar chain sample solution to on the amount of the sample. MALDI-TOF MS, the following processes are carried out. When the Sample is Sufficient 97. The tagged Sugar chain Sample solution is mixed with 70. The sample in the microplate 25 is dispensed to the a matrix for mass spectrometry, and the mixture is dotted on microplate for mixing 24. 60 a plate for measurement. 71. MALDI matrix is dispensed in an amount of 2 ul to the 98. Analysis of the sugar chain to be determined is carried microplate for mixing 24. out. This analysis can be performed using any known method, 72. The sample in the microplate for mixing 24 is dotted in as described in the present specification. an amount of 2 ul on the target plate 58 on the target plate The reference documents such as Scientific articles, patents receiving stand 59. 65 and patent applications that are cited in the present invention (The process up to this point corresponds approximately to have been incorporated in their entirety by reference to the the second half of the step (B-12).) same extent as they are respectively described in detail. US 8,877,454 B2 59 60 As discussed above, the present invention has been illus was thermally deactivated, and then 2 units of PNGase F was trated using preferred embodiments of the present invention, added to the mixture. The resulting mixture was incubated but the present invention is not intended to be interpreted as overnight at 37°C. The entirety of released N-glycans (neu being limited to these embodiments. It should be understood tral glycans and Sialylglycans) in the digestion mixture was that the scope of the present invention is definitely interpreted directly used for glycoblotting using BLOTGLYCOABCTM based only on the scope of claims. Those skilled in the art will beads as follows. understand, from the specific descriptions of the preferred (The General Protocol of Glycoblotting by BLOTGLYCO embodiments of the present invention, that an equivalent ABCTM Beads) Scope of invention can be carried out based on the descrip 40 ul BLOTGLYCOABCTM beads (50% suspension) was tions of the present invention and common technical knowl 10 dispersed on the wells of a filter plate MultiScreen Solvinert edge. It is to be understood that the disclosures of the patents, (Millipore). Those beads were rinsed with 1M of aqueous patent applications and documents cited in the present speci acetic acid (AcOH), 50% aqueous acetonitrile and acetoni fication have been definitely incorporated in the present trile (ACN) using a reduced pressure manifold in order. 20 ul specification by reference, in the same manner as that the of whole serum (equivalent to 2.5 ul of serum) digested with disclosures are specifically described per se in the present 15 trypsin and PNGase F was transferred to the wells and then, specification. 200 ul of 2%. AcOH in acetonitrile was added thereto. The plate was incubated at 80°C. in a TurboVap 96 concentrator EXAMPLES (Caliper Life Sciences, Inc., Hopkinton, Mass.) without using nitrogen gas flow and dried. It took usually 30 min. The plate Hereinafter, the present invention will be described in more was rinsed three times with 300 ul of 6M guanidine hydro detail by way of Examples, but this invention is not intended chloride in 50 mM of ammonium and then, to be limited to the following examples. rinsed three times with 300 ul of water. After methyl esteri fication on beads (Miura, Y., Shinohara, Y. Furukawa, J. I. Example 1 Nagahori, N., and Nishimura, S. I. (2007) Chem. Eur. J. 13, 25 4797-4804), the beads were rinsed using 300 ul of 10 mM of BLOTGLYCOABCTM Beads: Integrated Type HCl, 50% aqueous acetonitrile and acetonitrile in order. The Glycoblotting Technology for Rapid, Large-Scale wells were incubated at 60° C. for 60 min together with 100 Clinical Glycomics ul of 100 mM 3-methyl-1-p-tolyl triazene in dimethylsulfox ide-ACN (1:1). The solution was removed by applying The following abbreviations will be used as necessary. 30 reduced pressure and then, the beads were rinsed with ACN, CDG: Congenital disorders of glycosylation water and MeOH (3x300 ul) in order. Hydrazone bonding DTT Dithiothreitol between and fluorescent probe on the bead HCC: Hepatic cell cancer was stabilized by incubation with a reducing agent (0.8 M HPLC: High performance liquid chromatography borane-pyridine) according to a process previously reported MALDI-TOF: Matrix-assisted laser desorption ionization 35 (Lohse, A., Martins, R., Jorgensen, M. R., and Hindsgaul, O. Time-of-flight (2006) Solid-phase oligosaccharide tagging (SPOT):Valida (Order of Experiment) tion on glycolipid-derived structures. Angew. Chem. Int. Ed. (Materials) Engl. 45, 4167-4172). The reduction was carried out at room The clinical study of the present Example was approved by temperature in dark for 30 minutes. After removing the mix the Ethics Committee of Hokkaido University Hospital. 40 ture, the bead were rinsed with MeOH, CAN and water Explanation was offered to and consent was obtained from all (3x300 ul) in order. On the wells, 50 ul of 50 mM DTT in 5 of serum donors. Peptide-N-glycosidase F (PNGase F) was mMammonium carbohydrate was added, the plate was incu purchased from Roche (Mannheim, Germany). 3-Methyl-1- bated at 60° C. for 5 min and further incubated at room p-tolyltriazene (MTT), 8 Mborane-pyridine complex and temperature for 15 min. The concentrated N-glycan treated as dithiothreitol (DTT) were obtained from Sigma-Aldrich, Inc. 45 described above was collected from the bead in the DTT (St. Louis, Mo.). BLOTGLYCOABCTM beads were prepared Solution. as will be described additionally. This is now commercially (Mass Spectrometry) available from Sumitomo Bakelite Co., Ltd. (Tokyo, Japan). The collected N-glycan (0.5 ul) in the DTT solution was Other reagents and solvents were obtained, unless stated oth directly arranged on Anchor Chip 400/384 (Bruker Daltonics, erwise, from Wako Pure Chemical Industries, Ltd. (Tokyo, 50 Germany), mixed with the equal Volume of matrix solution Japan). The blood serum of CDG patients was kindly sup (the 9:1 mixture of DHB and DHB sodium salt (respectively plied by Professor H. H. Freeze of Burnham Institute for 10 mg/ml in 30% ACN)) and dried under reduced pressure to Medical Research (La Jolla, Calif.). obtain analyte crystal. Its mass spectrum was obtained repre Release of N-Glycans from Human Blood Serum) sentatively summarizing 100x5 shots using UltraFlex IITOF/ Whole human blood serum was treated according to the 55 TOF (Bruker Daltonics) in reflector positive ion mode. conditions previously reported by the group of the present (Statistical Analysis) inventors (Kita, Y. Miura, Y. Furukawa, J. I., Nakano, M., In the present research, the maximum 44N-glycan peaks in Shinohara, Y., Ohno, M., Takimoto, A., and Nishimura, S. I. MALDI-TOF MS spectrum were selected using a software (2007) Mol. Cell. Proteomics 6, 1437-1445), to release reduc called FlexAnalysis version 3 (Bruker Daltonics, German). ing type N-glycans. Briefly speaking, 10ul of a serumaliquot 60 All statistical analyses were carried out using those originally was diluted 6-fold in 83 mMammonium hydrogen carbonate developed with MATLAB (Version 7.4) (The Mathworks, containing a surfactant and 10 mMDTT. After incubating the Inc.) language using Statistics Toolbox unless otherwise mixture at 60° C. for 30 minutes, one volume (10 ul) of 123 noted. mM iodoacetamide was added thereto, and the mixture was (CDG Case) incubated for one hour at room temperature in the dark. 400 65 Respective subtypes were repeatedly measured (n-6). The Units of trypsin (Sigma-Aldrich) was added to this solution, reason was that only one for respective samples was available and the mixture was incubated for one hour at 37°C. Trypsin for these Subtypes. The isotopic areas of respective glycans US 8,877,454 B2 61 62 were normalized against the known amount of internal stan- TABLE 2-continued dard. In order to identify N-glycans that may be used for discrimination between CDG-I subtype and normal control, Supplementery Scheme 1 the present inventors carried out classification using algorism is for expected value maximization. The algorism separates the NH2 mixtures of various data distributions in the repeating steps of H / maximum evaluation method (Redner, R. A., and Walker, H. i-N-i- F. (1984) Soc. Indust. Appl. Math. Rev. 26, 195-239.) Pre- to O dH, O-CH, clinical component analysis (PCA) was carried out using HN-NH Spotfire DecisionSite (version 9.0 Somerville, Mass.). The present inventors plotted initial two principal components. NH2 (HCC Case) 15 C H3 Cut-off line for calculation was regulated as 0.3% among C total area. The statistical difference of disorder condition O VOO against normal condition was calculated using student t cali- 3 bration. The difference was regarded as statistically signifi- 20 cant in case of PK0.001. In order to identify essential charac teristics for optimally classifying serum between the two conditions of disorder condition and normal condition, the present inventors applied successively progressive selection 25 algorism. The algorism selected the better combination of A N-glycan peaks in order based on the error rate of the one N H - C extract method (LOO) of k-proximal discriminator (k=3). HNH3 (Result) 30 (The Principal Illustration of Clinical Glycomics at BLOT DTT GLYCOABCTM Beads Base) The novel method described in the present specification ()-N( )-NHH can provides extremely simple and convenient approach for the concentration analysis of composite glyco-adelphus with- 35 d C out any special training (FIG. 10a). The present inventors designed N-(2-aminobenzoyl)cysteine hydrazide (ABCh) in 5 order to establish “all in one' protocol, bonded the adelphus with thiopropyl Sepharose 6B and used it in order to obtain stable hydrazide-functionalized polymer Support (namely, 40 BLOTGLYCOABCTM beads) (FIG. 10b, refer to the synthe NH2 sis of BLOTGLYCOABCTM beads and illustration after the characterization, and the following scheme 1). N C (Table 2) The synthesis route of ABCh (N-(2-aminoben- 45 HNH3 Zoyl)cysteine hydrazide) and the preparation of BLOTGLY COABCTM beads. : ( 6 TABLE 2 50 Supplementery Scheme 1

O M in-i- NH2 N H, O-CH, pH 55 2N

S \O 6 He

i -e-2 60 HOEt CH3 y CH TCA HN-CH-C WBC V O 1 65 ThiopropylSepharose6B US 8,877,454 B2 63 64 TABLE 2-continued method of the present inventors without losing sialic acid at all. Disulfide bond bonding thiopropyl Sepharose 6B with Supplementery Scheme 1 ABCh probe enabled the quantitative collection of N-glycan enriched by being treated with DTT in eluate buffer. The collected sample contains neutral and acidic glycans and can be immediately used for the following quantitative analysis, using some of HPLC and MS analysis application. Although intact neutral glycan and sialylated glycan are collected by the above-mentioned method, it is known that sialylated oligosaccharide is decomposed by their minus charge in general MS analysis. Since the stabilization ofsialic acid is essential for the quantitative MS analysis of sialic oligosaccharide, the simple O-methyl esterification of Sialic acid residue by 3-methyl-1-p-tolyltriazene (MTT) was incor porated in the protocol. In the capture of whole N-glycan by BlotClycoABC the intervention of stable hydrazone bond by BLOTGLYCO ABCTM beads, methyl esterification on the bead of sialic acid and the reduction of hydrazone bond on the bead were carried The synthesis route of ABCh (N-(2-aminobenzoyl)cysteine out in order to enable mass measurement that is high in hydrazide) and the preparation of BLOTGLYCOABCTM reliability and high in reproducibility for both of neutral gly beads. coform and acidic glycoform in positive reflector mode in The hydrazide group reacts with an aldehyde group or a MALDI-TOF. ketone group that is very rare in a general biological sample, In order to exemplify a novel method, the present inventors excepting glycans having reductive hemiacetal terminal. The provided human serum (2.5ul) for optimal protocol that used formation of the hydrazone bond between BLOTGLYCO 25 BLOTGLYCOABCTM beads. The portion of sample solution ABCTM beads and glycan is reversible and enables the dis equivalent to 25 nil serum that “can be immediately analyzed charge of partially . Otherwise, it can be was directly deposited on a target plate to be provided for reduced for converting stable C N bond (Lohse, A., Mar MALDI-TOF analysis (positive reflector mode). The present tins, R., Jorgensen, M. R., and Hindsgaul, O. (2006) Angew. inventors quantified 44 kinds of N-glycans (FIG. 11A and Chem. Int. Ed. Engl. 45, 4167-4172). The chemical property 30 Table 3). 44 Kinds of N-glycans derived from human serum includes various reagents (for example, dithiothreitol (DTT), were set as the target in the present research. The peak 33 is iodoacetamide, Surfactant, etc.). It is suitable for enriching internal standard that was added for quantification. The anno glycans from complex biological materials even in the pres tation of composition and deduced structure shown in the ence of various amines and reagents that are used in the column of abbreviated description were obtained by Pro preparation of proteomics sample. The quantitative ligation 35 teome Systems (glycosuite-proteomesystems.com/glyco of general N bond type oligosaccharide is generated by the suite/glycodb) that is GlycoSuite online database. TABLE 3

Peak Abbreviated number mz Composition description 1 495.511 (Hex) + (Man)(GlcNAc), Man5 2 520.543 (HexNAc)(Deoxyhexose) + (Man)(GlcNAc), CoreFGN1 3 636.538 (Hex). (HexNAc) + (Man)(GlcNAc), CoreG1GN1 4 577.565 (HexNAc) + (Man)(GlcNAc), NA2GO 5 657.564 (Hex) + (Man)(GlcNAc), Manó 6 723.628 (HexNAc)(Dexoxyhexose) + (Man)(GlcNAc), NA2FGO 7 739.617 (Hex). (HexNAc) + (Man)(GlcNAc), NA2G1 8 780.644 (HexNAc) + (Man)(GlcNAc), bisCO 9 819.617 (Hex) + (Man)(GlcNAc), Manf 10 841.548 (Hex). (HexNAc)(NeuAc) + (Man)(GlcNAc), A1(G1GN1) 11 885.675 (Hex). (HexNAc)(Deoxyhexose) + (Man) (GlcNAc), NA2FG1 12 901.670 (Hex)(HexNAc) + (Man)(GlcNAc), NA2 13 828.702 (HexNAc)(Deoxyhexose) + (Man) (GlcNAc), bis GO 14 942.097 (Hex). (HexNAc) + (Man)(GlcNAc), bis(G1) 15 981.670 (Hex)5(Man)(GlcNAc), Man& 16 987.707 (Hex). (HexNAc)(Deoxyhexose) (Neu Ac) + (Man)(GlcNAc), A1F(G1GN1) 17 2003.702 (Hex)2(HexNAc)(NeuAc) + (Man)(GlcMAc), A1M1(G1GN1) 18 2044.729 (Hex). (HexNAc)(NeuAc) + (Man)(GlcNAc), A1 (G1) 19 2047.728 (Hex),(HexNAc),(Deoxyhexose) + (Man). (GlcNAc), NA2F 2O 2088.755 (Hex). (HexNAc)(Deoxyhexose) + (Man) (GlcNAc), bish (G1) 21 2104.750 (Hex)2(HexNAc) + (Man)(GlcNAc), bis(G2) 22 2143.725 (Hex) + (Man)(GlcNAc), Man 23 21 65.755 (Hex). (HexNAc)(NeuAc) + (Man)3(GlcNAc), A1Mans 24 2206.781 (Hex)2(HexNAc)(NeuAc) + (Man)3(GlcNAc), A1 25 2247.808 (Hex). (HexNAc). (Neu Ac) + (Man)3(GlcNAc), NA3(G1A1) 26 2250.808 (Hex)2(HexNAc)(Deoxyhexose) + (Man) (GlcNAc), bish (G2) 27 2206.802 (Hex). (HexNAc) + (Man)(GlcNAc), NA3 28 2352.839 (Hex),(HexNAc),(Deoxyhexose) (Neu Ac) + (Man)(GlcNAc), A1F 29 2393.866 (Hex). (HexNAc)(Deoxyhexose) (NeuAc) + (Man)(GlcNAc), NA3F(G1A1) 30 2409,861 (Hex)2(HexNAc)(NeuAc) + (Man)(GlcNAc), bis(G2A1) US 8,877,454 B2 65 66 TABLE 3-continued

Peak Abbreviated number mz Composition description 31 2412.860 (Hex). (HexNAc)(Deoxyhexose) + (Man) (GlcNAc), bsF(G3) 32 2469.882 (Hex). (HexNAc) + (Man). (GlcNAc), NA4(G3)/NA3bis 33 2481.893 (Hex)(HexNAc)(NeuAcAmide) + (Man)(GlcNAc), A2 amidE 34 2511.892 (Hex)(HexNAc)(NeuAc) + (Man)(GlcNAc), A2 35 2539.924 (Hex) (HexNAc). (Deoxyhexose) (Neu Ac) + (Man). (GlcNAc), 36 2555.919 (Hex)(HexNAc)(Deoxyhexose)1 (NeuAc) + (Man)(GlcNAc), bish (G2A1) 37 2571.912 (Hex). (HexNAc)(NeuAc) + (Man)(GlcNAc), NA3 (A1) 38 2615.940 (Hex). (HexNAc). (Deoxyhexose) + (Man) (GlcNAc), NA3Fbis 39 2631.935 (Hex). (HexNAc) + (Man). (GlcNAc), NA4 40 2657.960 (Hex)(HexNAc)(Deoxyhexose) (NeuAc) + (Man) (GlcNAc), A2F 41 2717.971 (Hex). (HexNAc)(Deoxyhexose) (NeuAc) + (Man) (GlcNAc), NA3F(A1) 42 2861.030 (Hex)(HexNAc)(Deoxyhexose) (NeuAc) + (Man) (GlcNAc), bish (A2) 43 2677.025 (Hex). (HexNAc). (Neu Ac) + (Man)(GlcNAc), NA3 (A2) 44 3023.083 (Hex). (HexNAc)(Deoxyhexose) (NeuAc) + (Man) (GlcNAc), NA3F(A2) 45 3162.136 (Hex). (HexNAc)(NeuAc) + (Man)(GlcNAc), A3

These glycans can be detected in all samples and these One” GlycoBlotting protocol based on BLOTGLYCO quantities were normalized for peak number 33 (Spike, that is, ABCTM beads can be preliminarily transferred to automated A2 amide abruptly raised) as internal standard. Once N-gly platform by use in combination with standard multi-wells cans are applicable by the PNGase F treatment (Kita, Y., filter plate form under the flow chart optimized for human Miura, Y., Furukawa, J. I., Nakano, M., Shinohara, Y., Ohno, serum application (FIG. 8). The effectiveness of the auto M., Takimoto, A., and Nishimura, S.-I. (2007) Mol. Cell. mated protocol was evaluated by simultaneously performing Proteomics 6, 1437-1445.) of whole serum glycol protein, it 25 same human serum digestive articles (FIG. 14). Further, its takes only less than 4 hours to identify the whole N-glycan precise reliability was confirmed by good reproducibility profile by the idiomatic operation of BLOTGLYCOABCTM (FIG. 4). beads and MALDI-TOF MS. It should be marked that the (Table 4). CV: Fluctuation Counting solid phase protocol is the first example of “All in One' It should be noted that hydrazone bond is not reduced in the GlycoBlotting technology in a single automated work flow. 30 automated GlycoBlotting because of collecting oligosaccha The present inventors are now studying and developing a ride in reducing Sugar mode by acid processing and the mass sample processing machine Suitable for automated GlycoB difference of -2Da is generated in comparison with the mass lotting that uses BLOTGLYCOABCTM beads. In fact, “All in of Tables 3 and 4. TABLE 4

Number of 25% 75% Standard mz figure Minimum Percentile Median Percentile Maximum Average deviation CV 1493.365112 8 O.O94 O-110 O. 120 O.126 O.148 O. 119 O.O16 13.50% 1855.395142 8 O.122 O.129 O.140 O.154 O.172 O.142 O.O17 11.88% 1721.453491 8 1079 1186 1.177 1.198 1.264 1.178 O.OS2 4.37% 1737.439331 8 O.066 O.O82 O.O86 O.097 O112 O.O89 O.O15 16.39% 1778.443481 8 O.OOO O.044 O.OS4 O.OS2 O.O72 O.049 O.O22 45.08% 1839.444214 8 O.O63 O.078 O.O94 O.109 O.130 O.O94 O.O23 24.02% 1883.491821 8 2.130 2.247 2.315 2.344 2.382 2.291 O.O82 3.56% 1899.47 1436 8 O.083 O.109 O. 119 0.144 O.149 O.122 O.O23 18.48% 1924.493O42 8 O.143 O.154 O.166 O.18O O.213 O.170 O.O22 13.08% 1940.455688 8 O.O72 O.O82 O.088 O.107 O.118 O.092 O.O16 17.68% 1979.456787 8 O.OOO O.OS1 O.069 O.08O O.107 O.O63 O.O32 SO.04% 2001.482666 8 O.OOO 0.057 O.062 O.O86 O.093 O.O63 O.O29 46.72% 2042.4854O3 8 O.092 O.095 O. 111 O.121 O.167 O.114 O.O2S 21.73% 2O4S.S19043 8 1.OOO 1.OOO 1.OOO 1.OOO 1.OOO 1.OOO O.OOO O.00% 2O86.525879 8 O.244 O.253 O.268 O.28O O311 O.270 O.O22 8.21% 2188.534424 8 O.129 O.143 0.157 O. 161 O.2O7 0.157 O.O23 14.74% 2204.SS9814 8 3.122 3.453 3.521 3.782 3.966 3.600 O.277 7.70% 2.245.557617 8 O.083 O.096 O. 113 O.141 O161 O.118 O.O28 23.98% 2248.559082 8 O.109 O.128 O. 142 O.154 O.173 O.141 O.O2O 14.14% 23S0.59082 8 1.120 1.152 1.174 1231 1296 1.191 O.OS8 4.87% 2381.575439 8 O.O41 O.OS6 O.069 O.08O O.O88 O.O68 O.O16 24.19% 24O7.578369 8 O.128 O.143 O.151 O.182 O.208 O161 O.O26 16.37% 2467.575195 8 O.O41 O.OS6 O.074 O.087 O.100 O.O72 O.O21 29.09% 2SO9.64624 8 19.400 22.080 24.27O 25.980 28.860 24.110 2.995 12.42% 2S37.617188 8 O.O73 O. 106 O.143 O.181 O.30S 0.155 O.O72 46.72% 2551.622314 8 O.159 O.210 O.276 O.334 O416 O.277 O.088 31.79% 2553.6381.84 8 O403 O.458 O.480 O.SO3 OSO4 O.474 O.O34 7.27% 2569.622O7 8 O.246 O.340 O.433 0.597 O.682 O484 O.209 43.20% 2658.66O156 8 O.702 O.760 O.845 O.874 O.898 O.82O O.O71 8.62% 2858.700195 8 O.195 O.218 O.241 0.255 O.281 O.235 O.O24 10.33% 2874.6994.63 8 O.O86 O.109 O.143 O.147 O.163 O.131 O.O26 20.17% 3179,767672 8 O.115 O.146 O.169 O.181 O.197 O.163 O.O26 16.11% US 8,877,454 B2 67 68 (Detection of Change in Congenital Glycol Deficiency N-glycan profiles of 10 human serum samples (83 subjects (CDG)) and 20 normal control). In order to most suitably classify The detection of N-glycan in whole serum protein and the serums between two relative classes, disease and normal by determination of profile are very useful for the rapid identi identifying essential characteristics, the present inventors fication of abnormal protein glycosylation provoked by con applied successive forward selection algorism that selects the genital change and acquired change. The present inventors better combination of N-glycan peaks based on the error rate tested the technology of the present inventors for serum (k=3) of one extract method (LOO) of k proximal discrimi derived from a Subject Suffering from congenital deficiency nator (FIGS. 12a and 12b). When the present inventors of glycosylation (CDG) that is rare disease that is hereditary selected the proportion of whole two peaks between acquired human disorder group (Mandato, C., Brive, L., Miura, Y. 10 peaks that show significant difference (t calibration at both Davis, J. A., Di Cosmo, N., Lucariello, S. Pagliardini, S., sides and P<0.001) between disease and control, the algorism Seo, N. S., Parenti, G., Vecchione, R., Freeze, H. H., and selected the proportion (FIG. 12c) of the quantities of N-gly Vajro, P. (2006) Pediatric Res. 59,293–298.). CDG's exceed cans that discriminated HCC sample at 100% accuracy from ing 19 subgroups have beenhitherto identified (Freeze, H. H., normal control (FIG. 12a). Accordingly, it was verified that and Aebi, M. (2005) Curr. Opin. Struc. Biol. 15, 490-498.). 15 the determination of whole serum N-glycan profiles of These CDG's are generated from the biological synthesis of BLOTGLYCOABCTM beads base provides simple noninva N-glycan precursor or specified type N-glycan assembly or sive diagnostic tool for disease that has been previously dif the deficiency of either of them that are provoked by the ficult in early diagnosis or discrimination. rejection (type 1 CDG) or immature processing (type 2 CDG) (Application to Cell Glycomics) of sugar chain bonded with protein. The GlycoBlotting tech Cell and tissue in addition to serum are important biologi nology of the present inventors is based on BLOTGLYCO cal materials in clinical glycomics. The present inventors ABCTM beads and MALDI-TOF MS, but the present inven applied the purification protocol established in the above tors could detect abnormal glycosylated pattern in whole description, to cultured cells in order to study the N-glycan serum glycoprotein. Respective analyses were repeated 6 forms of whole cells. The PC-3 cell of human prostate cancer times (n-6) and peak area in MALDI-TOFMS spectrum was 25 and the PrEC cell of normal human prostate epithelial were normalized against the fixed amount of A2 amide that is cultured and their whole protein extracts were provided for internal standard. As anticipated, the present inventors BLOTGLYCOABCTM beads treatment. In similar manner as observed abnormal N-glycan profile (for example, some the N-glycan profile of serum, whole cell glycan forms were immature small size N-glycan chains) for a type 2 CDG clarified by the small amount of starting material (5-8x10 (CDG-II) subject elapsing the concrete step of glycosylation 30 cells). The N-glycan profiles unique to respective cell species deficiency (FIG. 11B, the increase of iv-vii) unclassified were successfully detected by the result and it was shown that subgroups of CDG-I and CDG-II). On the other hand, N-gly the approach of the present inventors for serum glycomics can enriched from the serum of a type 1 CDG (CDG-I) subject shown in the above-description is equally suitable for cell showed N-glycan profile that is extremely similar as a normal glycomics (FIG. 13). The optimization of cell glycomics/ analyte (FIG. 11B ii and iii). However, its result showed also 35 tissue glycomics can let general protocol applicable to char the significant decrease of total N-glycan in comparison with acteristic with further high throughput. normal donor (1011+69 uM) (575+46 uMand 692+41 uM for (The Synthesis and Characterization of BLOTGLYCO respective CDG-Ia subject and CDG 1b subject; and refer to ABCTM Beads) Table 4). This suggests clearly that deficiency in CDG-I loses General method and materials: All commercially available the whole glycan chains, to a certain degree caused by that the 40 starting materials and solvents are reagent grade and they biological synthesis of precursor is not possible. were used as they were at purchase. 2-Aminobenzoate, 1-hy The advantage of quantitative glycomics in the diagnosis droxybenzotriazole hydrate (HOBt) and L-cystine dimethyl marker of the CDG 1 subject is clear. The reason is that data esters dihydrochloride were bought from Sigma-Aldrich distribution formed by the quantities of 42 kinds of N-glycans Chemical. N-(3-dimethylaminopropyl)-N-ethylcarbodiim detected can be used for the simple differentiation of a subject 45 ide mono hydrochloride (WSC) was purchased from TCI from normal serum (FIG. 11C, a panel at left side and a panel (Japan). Thiopropyl Sepharose 6B was purchased from GE at center). It was clarified by the result that the pair of com Healthcare Biosciences. All other chemical substances are bination of the quantities of N-glycans verifies diagnostic ultra high purity grade and purchased from Wako Pure differentiation between the CDG-I subject, CDG-Ia subject Chemicals Co., Ltd. (Japan). 1H NMR spectra and 13C NMR and CDG-Ib subject (FIG.11C, a panel at right side). Further, 50 spectra were measured at 600 MHz with a Bruker DPX-600 the present inventors used the principal component analysis spectrometer using DMSO as solvent. All other chemical (PCA) of all CDG subsystems of the acquired whole N-gly reactions were carried out under nitrogen atmosphere in can profiles in order to interpret complex multi spectra data anhydrous solvent shielding light unless otherwise noted. set. The initial two principal components discriminated TLC was carried out on a Merck precoated plate (20x20 cm, clearly CDG Subsystem containing normal donor. It was con 55 layer thickness of 0.25 mm, and Silica Gel 60F254); spots firmed from these results that it can be discriminated from were visualized by spraying the solution of 90:5:5 (v/v/v) of healthy control by the profiles of N-glycan set of whole methanol:p-anis aldehydes: concentrated Sulfuric acid and serums and the specific subsystem of CDG can be discrimi heating it at 180°C. for about 30 seconds, or under ultraviolet nated from other subsystem of CDG. rays (256 nm or 365 nm) when it is applicable. The organic (Diagnostic Discrimination of Hepatocellular Carcinoma) 60 extract was dried on anhydrous MgSO and the solution was In order to verify the elasticity of the present protocol in concentrated at less than 50° C. under reduced pressure. large scale clinical glycomics, the present inventors applied N,N'-(2-aminobenzoyl)cystine dimethylesters 3:2-ami the technology of the present inventors to the serums of plural nobenzoic acid (8.22 g (60.0 mmol)) was dissolved in 60 ml number of Subjects Suffering from hepatocellular carcinoma of tetrahydrofuran in a round-bottom flask and the mixture (HCC). As recognized, the use of Glycoblotting protocol 65 was cooled in an ice bath. HOBt (9.18 g, 26.9 mmol), dim based on BLOTGLYCOABCTM beads and MALDI-TOF ethyl ester of L-cystine dihydrochloride (10.23g, 30.0 mmol) analysis enabled the speedy and quantitative determination of and triethylamine (8.37 mL) were added to the solution. WSC US 8,877,454 B2 69 70 (11.49 g, 60.0 mmol) was added to the solution, the solution and left still standing on the ice for 1 hour. This lysate was was stirred for 15 minutes on an ice bath, and the mixture was subjected to centrifugation at 15,000 rpm at 4°C. for 10 min. stirred at room temperature over night. After completion of To this Supernatant was added 4 by Volume cold acetone to the reaction, the solvents were removed by evaporation and cause precipitation of protein Substances and left overnight at the residue was dissolved in 150 ml of chloroform. The chlo 5 -20° C. Precipitates were collected by centrifugation at roform solution was successively rinsed with the saturated 12,000 rpm, 4°C. for 15 minutes; their pellets were washed aqueous solution of NaHCO and the saturated aqueous solu with 200 ul of acetonitrile and subjected to centrifugation as tion of NaCl, its organic phase was dehydrated with NaSO mentioned above. These pellets were dissolved in 50 ul of 80 and the solvents were removed by evaporation to obtain yel mMammonium hydrogen carbonate (including 0.02% 1-pro low powder (16.4g, 32.4 mmol. 108%). 10 panesulfonic acid, 2-hydroxy-3-lauric amide (PHL)), and 1H NMR (500 MHz, CDC1) 8: 3.32 (d. J=5.3 Hz, 2H, incubated at 60°C. for 10 minutes. To this solution was added CH2), 3.77 (s.3H, CH3), 5.03 (dd, J=5.3, 12.6 Hz, 1H, CH), 1 ul of 0.5M dithiothreitol (DTT) aqueous solution, incubated 3.32 (d. J=5.3 Hz, 2H, CH,), 6.62 (t, J=7.3 Hz, 1H, Ph), 6.69 at 60° C. for 30 minutes, and then alkylated with iodoaceta (d. J=8.1 Hz, 1H, Ph), 6.98 (bd, J=6.6 Hz, 1H, NH), 7.20 (t, mide by incubating at room temperature for 30 minutes under J=7.2 Hz, 1H, Ph), 7.43 (d. J=8.0 Hz, 1H, Ph). 15 light shielded condition. Subsequently, this mixture was N,N'-(2-aminobenzoyl)cystine hydrazide 5: Hydrazine treated with trypsin overnight at 37° C. and heatinactivated at monohydrate (31.6 g. 631 mmol) was added to the solution of 90° C. for 10 minutes. After cooled down to room tempera 320 mL of methyl alcohol, and the mixture was stirred at ture, this mixture was incubated overnight at 37°C. together room temperature over night to obtain precipitate. The pre with 2.5 U of PNGase F (Roche). Prepared substance (20-40 cipitate was collected by filtration and rinsed with 320 mL of ul) equivalent to cells per /2 culture dish was used in the methyl alcohol. The residue was dried under reduced pressure following Glycoblotting protocol that uses BLOTGLYCO to obtain a pure compound 5 (10.8 g. 21.3 mmol, 67.4%). ABCTM beads as described in this specification. N-(2-aminobenzoyl)cystine hydrazide 6:1 Mhydrochloric (Automation of Glycoblotting Protocol that Uses BLOT acid (14.22 mL) was added to the solution of 96 mL of GLYCOABCTM Beads) acetonitrile-water (50%, V/v) and the solution was stirred. 25 Using 8 wells of filter plate, equivalent volume (20 ul) of The pH of the solvent was adjusted at 7.0 by adding the PNGase F digestion blood serum sample was applied to aqueous solution (17.06 mL) of 1 Mammonium carbohy BLOTGLYCOABCTM beads, and Glycoblotting protocol drate, then the aqueous solution (4.74 mL) of 1 Mdithiothrei was executed simultaneously according to the flow diagram tol (DTT) and the mixture was stirred at room temperature for shown in FIG. 8. N-glycan thus collected was analyzed by 2 hours. After completion of the reaction by TLC test, the 30 MALDI-TOFMS and detected area was statistically ana Solution was extracted with ethyl acetate. The organic phase lyzed. was dehydrated with NaSO and the solvent was removed by (Discussion) evaporation to obtain white powder 6 (2.4 g., 9.44 mmol. Recent development of mass spectrometry brought new quantification). 1H NMR (500 MHz, DMSO)8: 2.36 (bs, 1H, difficult problem in Glycomics. As this difficult problem, SH), 2.84 (m, 2H, CH,), 4.26 (bs, 2H, NHNH2), 4.45 (dd. 35 development of rapid glycan enrichment technology is men J=2.1, 8.0 Hz, 1H, CH), 6.36 (bs, 2H, PhNH2), 6.52 (t, J–7.5 tioned. Easy technology for retrieval of biomarkers related to HZ, 1H, Ph), 6.69 (d. J=8.2 Hz, 1H, Ph), 7.15 (t, J=8.2 Hz, 1H, carbohydrate is important. This is because the proteomics Ph), 7.60 (d. J=7.9 Hz, 1H, Ph), 8.16 (d. J=7.9 HZ, CHNH), research targets glycation that is a posttranslational modifi and 9.21 (s, 1H, NHNH2). cation. In this specification, the present inventors report “all The preparation of BLOTGLYCOABCTM beads: 10 mL of 40 in-one” type protocol for high-throughput clinical Glycom thiopropyl Sepharose 6B resin was rinsed with the aqueous ics. With this novel technology, glycan concentration of solution of 0.1 M ammonium carbohydrate in a dispensable Glycoblotting base on BLOTGLYCOABCTM beads, stabili column. These beads were immersed in 6.7 mL of acetoni Zation on sialic acid beads, and fluorescent labeling of oli trile/water (50%, v/v) and then, 0.1 M solution of ABCh in gosaccharide are unified into one workflow on the multi-well acetonitrile/water (50%, V/v) (3.3 mL) was added thereto. 45 filter plate. Advantages of this protocol and MALDI-TOF The slurry was mixed at room temperature for 30 minutes. mass analysis were verified through distinction of serum After the reaction, the beads were adequately rinsed with N-glycan profile of specimen having congenital glycation acetonitrile/water (50%, V/v), 0.1 M acetic acid and ethanol/ disorder, serum N-glycan profile of specimen having hepato water (20%, V/v). The substance obtained was stored at 4°C. cellular cancer, and serum N-glycan profile of a healthy in 10 mL of ethanol/water (20%, V/v) until usage. 50 donor. Further, above-mentioned method enabled whole cell (Cell Glycomics by Using BLOTGLYCOABCTM Beads) Glycomics of human prostate cancer cell and normal human Cell culture: The PC-3 cell of human prostate cancer and prostate epidermal cell. These results suggest possibility of the cell (PrEC) of normal human prostate epithelial were glycan condensation/treatment for biomarker discovery. respectively obtained from Health Science Researches Bank Large-scale quantitative Glycomics is important and and Cambrex Bio Science Walkersville, Inc. (Walkersville, 55 encouraging approach. This is because a difference of glycan Md.). The PC-3 cell was kept in Ham's F-12 replenishing expression between disorder state and healthy state is 10% bovine embryo serum, 50 U/ml of penicillin and 50 expected to be favorable tool for diagnosis of the disorder or ug/mL of streptomycin. The PrEC cell was maintained prognostic judgment (Hakomori, S. (2001) Adv. Exp. Med. according to the illustration of the producer using Clonetics Biol. 491, 369-402.). At present, mass spectrometry (MS) is Prostate Epithelial Growth Media (PrEGMTM) BULLET 60 used exclusively for structure analysis of carbohydrate and KITR) kit. These cells were cultured at 37° C. and 5% CO, in study of sequence determination (Zaia, J. (2004) Mass Spec a humidifying incubator. trom. Rev. 23, 161-227.: Dell, A., and Morris, H. R. (2001) Preparation of N-glycan from cells: Cells proliferated in a Science 291, 2351-2356.). Due to remarkable improvements 10 cm dish were washed with ice bath PBS, scraped off in in MS technology (ionization, fragmentation and detection PBS containing 10 mM EDTA, and then washed with PBS. 65 method are cited) (Kurogochi, M., and Nishimura, S. (2004) Cells collected in a 1.5 ml tube were suspended in PBS (60 Anal. Chem. 76, 6097-6101.; Takegawa, Y., Deguchi, K., Ito, ul), dissolved by adding /10 by volume of 10% Triton X-100 S., Yoshioka, S., Nakagawa, H., and Nishimura, S. (2005) US 8,877,454 B2 71 72 Anal. Chem. 77, 2097-2106.: Takegawa, Y., Deguchi, K., Ito, Shinohara, Y. Furukawa, J. I., Nagahori, N., and Nishimura, S., Yoshioka, S., Nakagawa, H., and Nishimura, S. (2005) S. I. (2007) Chem. Eur. J. 13, 4797-4804; Kita, Y. Miura, Y., Anal. Chem. 77, 2097-2106.), MS is more preferable in gly Furukawa, J. I., Nakano, M., Shinohara, Y., Ohno, M., comics than other analysis methods (e.g., high pressure liquid Takimoto, A., and Nishimura, S. I. (2007) Mol. Cell. Pro chromatography (HPLC) and nuclear magnetic resonance 5 teomics 6, 1437-1445.). The optimum protocol for this needs analysis (NMR). Specifically, moderate ionization of glycan only 5ul of human serum for quantitative profiling of 30 to 40 and matrix Support laser desorption ionization-time-of-flight types of primary glycoform in 5 to 8 hours (Miura, Y., Shi (MALDI-TOF) MS for delivery of high-throughput analysis, nohara, Y. Furukawa, J.I., Nagahori, N., and Nishimura, S.I. and electrospray ionization (ESI) MS are potential candidates (2007) Chem. Eur.J. 13,4797-4804). These attempts clearly for primary technology. 10 showed such advantages that higher recovery is attained and However, PCR-like glycan amplification technology for complicated manipulations are reduced by incorporating a glycomics is not available. Because glycan biosynthesis pro Solid Supporter in easy and efficient manipulations of con cess is not template driven type, but is provided for multiple, densed glycan. The present inventors present in this specifi continuous and enzyme processes. Although partial peptide cation “all-in-one” type solution for automated high-through fragment detected in proteomics is completely Supported by 15 put N-glycan condensation treatment. This solution is very full length protein sequence database/full length DNA beneficial in “real world’ clinical glycomics. With this sys sequence database, condensation of total glycan from highly tem, multiple processes (e.g., selective capture of whole gly combined mixture (e.g., serum, cell and texture) is necessary can, methyl esterification of Sialic acid and fluorescent tag for glycomics. Therefore, one of significant bottlenecks in ging) can be unified in one workflow based on the use of the structure-function glycomics is tiresome and time-consum combination of handling of hydrazide functionalized beads ing multiple processes for glycan purification. For example, and multi-way filterplate. The present inventors consider that after once being separated from glycoproteins or glycolipids, with this de facto standard technology, large-scale clinical glycan is frequently subjected to fluorescent labeling and glycomics/large-scale clinical glycoproteomics will be made purification for detection in HPLC analysis (Neville, D.C., possible and discovery of novel disease-related biomarkers Coquard, V., Priestman, D. A., to Vruchte, D.J., Sillence, D. 25 will be accelerated. J., Dwek, R. A., Platt, F. M., and Butters, T. D. (2004) Anal. (Conclusions) Biochem. 331, 275-282.: Tomiya, N., Kurono, M., Ishihara, Much research for verification of changes in glycosylation H., Tejima, S. Endo, S., Arata, Y., and Takahashi, N. (1987) of proteins by disease state has been reported (Baldus, S. E., Anal. Biochem. 163, 489-499.). In addition, oligosaccharide Wienand, J. R. Werner, J. P. Landsberg, S., Drebber, U. containing sialic acid is further modified for stabilization and 30 Hanisch, F. G., and Dienes, H. P. (2005) Int. J. Oncol. 27, amplification of sensitivity of anionic glycan in MS measure 1289-1297.: Comunale, M. A., Lowman, M., Long, R. E., ment (Powell, A. K., and Harvey, D. J. (1996) Rapid Com Krakover, J., Philip, R., Seeholzer, S., Evans, A.A., Hann, H. mun. Mass Spectrom. 10, 1027-1032.: Sekiya, S. Wada, Y. W., Block, T. M., and Mehta, A.S. (2006).J. Proteome Res. 5, and Tanaka, K. (2005) Anal. Chem. 77,4962-4968.: Mechref, 308-315.; Dwek, M. V., Lacey, H. A., and Leathem, A. J. Y., Kang, P., and Novotny, M. V. (2006) Rapid Commun. 35 (1998) Clin. Chim. Acta 271, 191-202.; J. Proteome Res. 6, Mass Spectrom. 20, 1381-1389.). In several cases, although 1822-1832.). The results of the inventors show potential clini important information are lost, sialic acid is removed due to cal value of quantitative analysis of whole serum N-glycan simplification or limitation of detection. In recent years, DNA profile obtained by MALDI-TOFMS. In recent years, usabil sequence determination is also used for clinical N-glycan ity of MALDI-TOFMS for profiling of N-glycan in the serum profiling (Callewaert, N., Van Vlierberghe, H., Van Hecke, A., 40 is expected in both quantitative analysis and qualitative analy Laroy, W., Delanghe, J., and Contreras, R. (2004) Nat. Med. sis for promotion of clinically related detection of a tumor 10, 429-434.; Laroy, W., Contreras, R., and Callewaert, N. (Morelle, W., Flahaut, C., Michalski, J. C., Louvet, A., (2006) Nat. Protoc. 1, 397-405.). In this clinical N-glycan Mathurin, P., and Klein, A. (2006) Glycobiology 16, 281 profiling, multiple processes and derivation procedures as 293.; Kranz, C., Ng, B.G., Sun, L., Sharma, V. Eklund, E.A., mentioned above suited for capillary electrophoresis are still 45 Miura, Y. Ungar, D., Lupashin, V., Winkel, D. R., Cipollo, J. necessary. In general, the protocol for preparation of glycan F., Costello, C. E., Loh, E., Hong, W., and Freeze, H. H. derivatives varies depending on the analysis method and (2007) Hum. Mol. Genet. 16, 731-74.1.). The method by the needs expertise for handling in every process. Due to these inventors imparts very high feasibility of glycan condensa technical but significant problems in glycan condensation, it tion and derivatization for quantitative and exhaustive MS is not possible to accomplish reliable high-throughput gly 50 analysis in the clinical evaluation. By incorporating inte comics (Morelle, W., Canis, K., Chirat, F. Faid, V. and grated type Glycoblotting beads (BLOTGLYCOABCTM Michalski, J. C. (2006) Proteomics 6,3993-401). beads introduced here) and methyl esterification on the bead, Recent efforts by the inventors were directed to practical true gross N-glycomics analysis was made possible. The glycan condensation method (i.e., Glycoblotting) (Nish present protocol needed only 2.5ul of aliquot of human whole imura, S., Nikura, K., Kurogochi, M., Matsushita, T., 55 serum and performed multiple processes for Subsequent Fumoto, M., Hinou, H., Kamitani, R., Nakagawa, H., Degu derivatization Successfully on the Solid phase by single chi, K., Miura, N., Monde, K., and Kondo, H. (2005) Angew. sweeping by manipulations (including MALDI-TOFMS) for Chem. Int. Ed. Engl. 44, 91-96. Lohse, A., Martins, R., four hours. This resulted in drastic improvements in effi Jorgensen, M. R., and Hindsgaul, O. (2006) Solid-phase oli ciency and time formanipulation. Since meaningful improve gosaccharide tagging (SPOT): Validation on glycolipid-de 60 ments were verified in preparation and analysis of the serum rived structures. Angew. Chem. Int. Ed. Engl. 45,4167-4172.: glycan, the protocol proposed by the inventors could be used Nikura, K. Kamitani, R., Kurogochi, M., Uematsu, R., Shi for glycan concentration analysis from cell sample. For nohara, Y., Nakagawa, H., Deguchi, K., Monde, K. Kondo, human prostate cancer PC-3 cell being proliferated in a single H., and Nishimura, S. (2005) Chem. Eur. J. 11, 3825-3834.: 10 cm dish and for normal cell corresponding thereto, the Shimaoka, H., Kuramoto, H., Furukawa, J., Miura, Y., 65 inventors have preliminarily verified their overall cell N-gly Kurogochi, M., Kita, Y., Hinou, H., Shinohara, Y., and Nish come by means of Glycoblotting using BLOTGLYCO imura, S. (2007) Chem. Eur. J. 13, 1664-1673.; Miura, Y., ABCTM beads and MALDI-TOFMS analysis. The cell gly US 8,877,454 B2 73 74 come profiling method was essentially identical with the glycomics (83 patients and 20-healthy donors) of HCC serum profiling method except for Small modifica sample. The inventors distinguished HCC samples and the tions made prior to pretreatment of the sample (FIG. 13). The normal control with as high as 100% accuracy by the specific present results clearly showed that there was a significant N-glycan profile mentioned above. Therefore, information difference in structure of N-glycan (pattern and volumes of 5 obtained by the present study, while still requiring verifica N-glycan) between PC-3 cells and normal prostate epidermal tion using correlation with other clinical variables and medi cells (PrEC). It is considered that use of the approach which cal facts such as hepatitis B virus infection and/or Chepatitis the inventors proposed will greatly promote glycomics at cell C virus infection, quantitative profiling of the selected glyco level and tissue level. Recent finding about defect that results form has potential clinical information for a novel and effec in each CDG identified that glycan analysis is insufficient to 10 tive diagnosis biomarker for human HCC. point out correctly a defect in part of CDG-II (Wu. X., Steet, Mass spectrometry with reproducibility over entire R. A., Bohorov, O., Bakker, J., Newell, J., Krieger, M., MALDI-TOFMS analysis was realized by quantitative Spaapen, L., Kornfeld, S., and Freeze, H. H. (2004) Nat. Med. methyl esterification of sialic acid residue. This will allow 10, 518-523.; Redner, R. A., and Walker, H. F. (1984) Soc. MALDI-TOFMS to be capable of withstanding simultaneous Indust. Appl. Math. Rev. 26, 195-239.). However, although 15 and quantitative profiling for neutral N-glycan and acidic multiple tests could not be performed for each of the sub N-glycan. Even if there are such advantages which are better types, the present results clearly verified that the dataset about than other analysis methods, it was necessary to note the fact the abundance of N-glycan in patients of each Subtype was that intensity of mass signal is frequently dependent on indi definitely separated at places of expression by principal com vidual MALDI device used during analysis on N-glycan with ponent analysis. Therefore, in general screening tests (e.g., 20 broad range molecular weight. It seems that this is attribut newborn screening program), discovery of a rare case may able to the laser source mounted to each of MALDI systems become essential by quantitative glycomics for CDG. This is and is influenced by ionization efficiency of particular mol because CDG could be detected together with other disorders ecule (e.g., high molecular weight glycan having plural Sialic by a simple blood examination. The present Glycoblotting acid residues). By comparison of the present results (Table 4) technique could then become a method for realizing the above 25 and previous data (Kita, Y. Miura, Y. Furukawa, J. I. due to its reproducibility and easiness. It is interesting if this Nakano, M., Shinohara, Y., Ohno, M., Takimoto, A., and technique is applied to other disorders (e.g., congenital galac Nishimura, S.I. (2007) Mol. Cell. Proteomics 6, 1437-1445.), tosemia and chronic alcohol intake) characterized by alter it was assumed that glycan amount of triantennary added ation of glycosylation of transferrin to identify usability of the thoroughly to sialic acid is reduced from the amount in the analysis performed in this research. 30 previous data. It is now possible to identify a set of specific N-glycan (Table 5) Statistics of automation trial of human serum expression ratios best suited for pattern classification by sta N-glycan condensation/treatment (n=8) tistical approach based on N-glycan profiling by large-scale Peak area was normalized with regard to 2045 m/z. TABLE 5 Normal (IM CDG-Ia (IIM CDG-Ib (IIM Peak No mz Average stol Average stol P Average Stod P O 333.460 N.D. O.OO 135 0.13 2.9E-10 1.16 O.11 4E-10 2.70 O41 2.48 O.34 13E--OO 1.66 0.18 2.OE-04 N.D O.OO N.D. O.OO NaN N.D. O.OO NaN 3 S36.538 N.D. O.OO N.D. O.OO NaN N.D. O.OO NaN 4 577.565 O.OO O.OO O.OO O.OO NaN 0.72 0.57 .1E-02 5 657.564 2.78 O.47 1.7O O.26 S.7E-04 O48 OS4 3E-OS 6 723.623 74.56 6.86 16.52 2.11 23E-09 27.9S 2.30 2.1E-08 7 739.617 2.20 O2O O.47 O.S2 1.8E-OS 248 0.34 .1E-O 8 780.644 N.D O.OO N.D. O.OO NaN N.D. O.OO NaN 9 819.617 N.D. O.OO N.D. O.OO NaN N.D. O.OO NaN 10 841.649 2.0S 0.39 2.OO O.33 8.5E-0 149 0.74 4E-O 11 885.675 119.SS 9.29 35.08 399 1.7E-09 49.1S 3.04 7.3E-09 12 901670 3.24 O.28 1.96 O2O 33E-06 3.01 O.32 2.2E-O 13 928.702 853 1.41 3.25 O.69 9.2E-08 3.85 0.45 SE-OS 14 942.697 15 1.34 N.D. O.OO 6.2E-02 N.D. O.OO 6.2E-O2 15 981.670 O.29 O.71 N.D. O.OO 34E-O N.D. O.OO 34E-O 16 997.707 OS1 O.81 N.D. O.OO 1.5E-0 N.D. O.OO SE-O 17 2003.702 97 1.07 O.48 O.74 1.9E-02 O.27 O.66 7.8E-03 18 2O44.729 2.52 1.29 N.D. O.OO 7.2E-04 O.62 0.97 6E-O2 19 2047.728 31.61 2.76 15.38 2.12 4.6E-07 2O3O 17O 6.6E-06 2O 2088.755 11.27 1.28 3.20 O.S1 S4E-08 3.29 O.34 4.1E-08 21 2104.7SO O.23 O.56 N.D. O.OO 34E-O N.D. O.OO 34E-O 22 2143.723 38 0.71 N.D. O.OO 7.6E-04 N.D. O.OO 7.6E-04 23 2185.755 O.22 O.S.S O.83 0.67 1.2E-O 1.65 0.26 1.8E-04 24 22O6.781 87.58 3.93 38.09 3.SS S.8E-10 48.2O 2.68 1.9E-09 25 2247.808 OS 1.72 N.D. O.OO 1.7E-O N.D. O.OO 1.7E-O 26 22SO.808 S38 1.OO 3.20 O.S9 1.OE-03 182 150 7.OE-04 27 2266.802 94 O.34 O.3.6 OS6 1.6E-04 O.32 O.S4. 9.8E-OS 28 23S2.839 31.91 2.18 25.07 2.65 8.7E-04 1212 0.94 1.7E-09 29 2393.866 O.31 O.75 N.D. O.OO 34E-O N.D. O.OO 34E-O 30 2409,861 11 1.23 N.D. O.OO S1E-02 N.D. O.OO 5.1E-O2 31 2412.860 N.D. O.OO N.D. O.OO NaN O.16 O4O 34E-O 32 2469.882 3.23 O.77 1.54 OS1 1.OE--OO 2.59 1.03 2.5E-0 33 2481.893 32O.OO O.OO 32O.OO O.OO NaN 32O.OO O.OO NaN US 8,877,454 B2

TABLE 5-continued Normal (LIM CDG-Ia (LIM CDG-Ib (IIM Peak No mz Average stol Average stol P Average Stod P 34 2511892 542.12 16.82 364.28 8.78 5.6E-10 16046 13.29 3.OE-06 35 2S39.924 5.17 O.77 2.51 1.47 2.8E-O3 3.71 0.88 1.2E-O2 36 2555.919 15.77 1.62 7.08 1.11 6.8E-07 3.11 O.71 7.2E-09 37 2571.914 1993 0.96 1166 5.89 6.8E-03 14.73 1.75 7.9E-OS 38 2615.940 1.2S 1.38 O.8O O.89 S1E-O1 O.98 109 7.1E-O1 39 2631.935 O.S.S. O.86 1.35 0.67 1.OE-01 1.25 101 2.2E-O1 40 2657.950 11.74 111 16.63 1.73 1.7E-04 15.14 141 9.3E-04 41 2717.971 10.20 O3O 11.88 1.22 8.2E-03 9.40 106 1.OE-01 42 2861.030 4.21 O.60 1.55 O.88 1.6E-04 N.D. O.OO 9.3E-09 43 2877.025 O.S2 O.80 1.47 0.75 S.8E-O2 1.09 (0.56 16E-O1 44 3O23.083 O.S3 O.60 1.93 0.32 47E-04 N.D. O.OO 5.6E-O2 45 3182.136 1.67 0.35 3.66 0.43 4.9E-06 1.2O 0.67 1.6E-O1 total (M) 1013.05 68.45 576.52 44.57 693.22 41.92 N.D.: Not detected in the specimen NaN: Nonnumerio 2O These results are attributable to both differences of chemi A-2-2) adding 50ll of 1-propane Sulfonic acid, 2-hydroxy cal structure between high-sensitivity tag of peptide base 3-lauric amide (PHL) or 2-hydroxy-3-myristamide (Kita, Y. Miura, Y. Furukawa, J. I., Nakano, M., Shinohara, (PHM) 0.24%/bicarbonate of ammonium 105 Y., Ohno, M., Takimoto, A., and Nishimura, S.I. (2007) Mol. mM/dithiothreitol (DTT) 12 mM to the sample (CV 56). Cell. Proteomics 6, 1437-1445.) and the probe designated by 25 When A-2-1) is employed in the above, BLOTGLYCOABCTM beads, and of the output and wave A-3-1) adding 5 ul of 120 mM dithiothreitol (DTT) (CV length of the laser used in each measurement. To accomplish 56) to the sample. more accurate and reliable quantitative analysis in the gly When A-2-2) is employed in the above or upon completion comics of MALDI mass spectrometry base, it is likely that a 30 of above A-3-1), new type of molecular probe with higher ionization effi A-3-2) adding 12 Jul of A2 amide Sugar chain that is an ciency, which provides Such high molecular Weight glycan, internal standard (peak No. 33 in above Table 3), to should be developed. For plural serum samples and cell cause reaction at 60°C. for 30 minutes and then cooling samples, integrated type Glycoblotting technique utilizing to room temperature. BLOTGLYCOABCTM beads greatly facilitates discovery of 35 A-4) adding 10 ul of 123 mM iodoacetamide (IAA) (CV novel biomarkers for various disorders. Chemical properties 5%) to the sample to cause reaction at room temperature used for handling of enriched N-glycan in the Solid phase are and in dark place for 1 hour. well suited for the derivatization needed in the subsequent A-5) adding 5 ul of trypsin 400 U to the sample to cause analysis method and therefore, recovered N-glycan tagged reaction at 37° C. for 60 minutes. with an optimum chemical probe can be used for conven 40 A-6) heating the sample at 90° C. for 10 minutes and then tional HPLC, LC-MS and/or analysis of DNA sequencer cooling to room temperature, wherein cooling is per base. formed sufficiently to avoid deactivation of PNGaseF. A-7) adding 5ul of PNGaseF2U to cause reaction at 37°C. Example 2 for 12 hours, wherein care is taken so that solvent evapo 45 ration may not occur during operation while correspond Example of type H ing to ceiling by heating treatment. B) Step for preparation of detection sample for detection of In the present example, analysis example using type H is the Sugar chain being separated: exemplified. Essentially, the experiments performed under B-1) contacting the capture Sugar Sample prepared in step the conditions stated in Example 1. FIG. 1 exemplify the 50 A) with beads for Sugar capture and causing binding at analysis example diagrammatically. In particular, the follow 80°C. to prepare a capture sugar sample, wherein 180 ul of 2% acetic acid/acetonitrile is added, heat is kept at 80° ing steps are performed. Preparation of beads of BLOTGLY C. for 45 minutes, and washed. COHTM are carried out in accordance with Example 1 (syn B-2) adding 200 ul of 2Mguanidine hydrochloride twice to thesis of beads of BLOTGLYCOABCTM and 55 the captured Sugar chain as necessary, placing the cap characterization) using ABC and descriptions of Japanese tured Sugar chain sample under the reaction condition Patent Application No. 2006-217165 and Japanese Patent and then discharging reaction liquid by Suction. Application No. 2006-73170. B-3) washing 200 ul of the captured sugar sample twice A) A Sugar chain separation step for separating a Sugar with water as necessary and then discarding the water by chain in a sample comprising the steps of 60 Suction. A-1) providing 10 ul of serum which is the sample to PCR B-4) washing 200 ul of the captured Sugar sample twice plate; with trimethylamine/methanol and then discarding the A-2-1) adding 45ul of 1-propane Sulfonic acid, 2-hydroxy trimethylamine by suction, wherein after suction, bot 3-lauric amide (PHL) or 2-hydroxy-3-myristamide tom wiping is performed in a sheet with cotton. (PHM) 0.26%/bicarbonate of ammonium 0.11 M to the 65 B-5) adding 100 ul of 10% acetic anhydride/methanol to sample (CV 5%) to cause reaction at 37° C. for 10 the captured Sugar chain sample, placing the captured minutes; or Sugar chain sample under the reaction conditions of US 8,877,454 B2 77 78 room temperature for 30 minutes and then discarding the although prompt analysis of Sugar chains is possible while acetic anhydride by Suction, wherein methanol is added removal of troublesome foreign matters are made easy by to acetic anhydride to prepare 10% acetic anhydride/ performing chemoselective capture, this invention enabled methanol. one-time treatment of many specimen samples. Each of the B-6) adding 200 ul of 10 mM hydrochloric acid to the 5 steps above were optimized and a system capable of coping captured Sugar chain sample twice as necessary and with automatic analysis was constructed. discarding the hydrochloric acid by Suction. Analysis of multiple specimens is made possible by the B-7) adding 200 ul of methanol to the captured sugar chain device of the present invention. Searching of Sugar chain sample twice as necessary, adding 200 ul of dioxane to biomarkers is expected, and applications concerning diagno the captured Sugar chain sample twice as necessary after 10 sis and early diagnosis of diseases are considered. the methanol is discarded by Suction, and discarding by Suction. BRIEF DESCRIPTION OF DRAWINGS B-8) performing bottom wiping in a sheet with cotton. B-9) adding 100 ul 100 mM methyl-p-tolyl-triazene FIG. 1 is the flow chart for BLOTGLYCOHTM beads, (MTT)/dioxane to the captured Sugar chain sample to 15 cause reaction at 80° C. for 60 minutes. which is a representative embodiment of the Sugar chain B-10) adding 200 ul of dioxane to the captured sugar chain auto-analyzing apparatus or the pretreatment apparatus for sample and discarding the dioxane by Suction. Sugar chain auto-analysis of the present invention. Descrip B-11) (1) adding 200 ul of methanol to the captured sugar tions for the respective numerals are described in the section chain sample, discarding by Suction; (2) adding 200ul of of EXPLANATIONS OF LETTERS OR NUMERALS or in a 20 mM NaCl Solution to captured Sugar chain sample, the specification; discarding by Suction; (3) washing the captured Sugar FIG. 1A is a magnified view (upper left part) of FIG. 1; chain sample with 200 ul of water and discarding the FIG. 1B is a magnified view (lower left part) of FIG. 1; water by Suction. FIG.1C is a magnified view (upper middle part) of FIG. 1; B-12) adding 180 ul of 2% acetic acid/acetonitrile to the 25 FIG.1D is a magnified view (lower middle part) of FIG. 1; captured Sugar chain sample, tagging Sugar chain in the FIG. 1E is a magnified view (upper right part) of FIG. 1; captured sugar chain sample using 20 ul of 20-50 mM FIG.1F is a magnified view (lower right part) of FIG. 1; aminooxy tryptophanyl arginine methyl ester/water, FIG. 2 is a schematic diagram of a Sugar chain-capturing O-benzylhydroxylamine hydrochloride/water oranthra molecule; niloyl hydrazine/water and discarding by Suction. 30 FIG. 3 is a conceptual diagram of Sugar chain blotting. B-13) adding 100 ul of water to the captured sugar chain Since a Sugar chain released from a complex carbohydrate sample being tagged to produce tagged sugar chain solu Such as a glycoprotein, a proteoglycan or a glycolipid, nec tion. essarily has a hemiacetal group equivalent to an aldehyde C) Step for producing mass spectrometry plate on which group at a reduced terminus in the molecule, complete chemi the captured Sugar chain samples being tagged are deposited: 35 C-1) disposing tagged Sugar chain sample solution cal discrimination from other biological molecules Such as obtained in step B) on the collection plate. the amino acid/peptide constituting a protein, the nucleotide C-2) adding 15ul of a 50% methanol/1% glycerol aqueous constituting DNA/RNA, and lipids, is realized by a selective Solution that is liquid matrix to the plate for mixing. nucleophilic addition reaction specific to this hemiacetal C-3) adding 15 Jul of the tagged Sugar chain sample from 40 group. Various Substances (regardless of being a macromol the plate for collection to plate for mixing, mixing by ecule or a small molecule) containing a functional group pipette to cause ignition. capable of generally reacting with reduced termini of all Mass spectrometry plate is generated from this by dispens kinds of Sugar chains (an aminooxy group, a hydrazide group ing 2 ul and depositing on MALDI Plate (#209512 MTP or the like) are used to conduct Sugar chain capturing, probe Anchor ChipTM400/384T F (384 anchors, 400 um diameter: 45 labeling using a highly sensitive reagent or the like, and a #209520 MTP 384 target plate polished steel T F, Bruker structural analysis according to those mass spectrometric Daltonik GmbH). methods, continuously at high speed. The MADLI analysis, D) executing MALDI-TOF according to the ordinary MS analysis and MS/MS analysis results shown in the lower method. right corner are given as examples; The present invention has been exemplified as mentioned 50 FIG. 4 is a diagram showing the fundamental principles of above using preferred embodiments of the present invention. MALDI-TOF MS; However, it should be understood that the scope of the present FIG. 5 is an exemplary flow chart of a process making use invention is interpreted only by the claims. It should be under of BLOTGLYCOABCTM beads: stood that contents of patents, patent applications and litera FIG. 6 is an exemplary flow chart of a process making use tures cited in the specification are incorporated herein for 55 of BLOTGLYCOABCTM beads. FIG. 6 is a continuation of reference of the present specification. FIG. 5; FIG. 7 is an exemplary flow chart for the dispensing in the INDUSTRIAL APPLICABILITY process making use of BLOTGLYCOABCTM beads: FIG. 8 is an exemplary bird’s-eye view of a process making The inventors succeeded in providing an automatic analy 60 use of BLOTGLYCOABCTM beads. A flow diagram of the sis device for analyzing Sugar chains contained in the sample automated glycoblotting protocol is shown. Application to a Such as serum sample. This enabled analysis of a large quan 96-well format robot is shown. FIG. 8 is different from FIG. tity of samples simultaneously and automatically. For sepa 1 in that a reduction treatment of hydrazone bond using a 8M ration of Sugar chains quantitatively from Sugar proteins, borane-pyridine complex is further included in FIG.8. Such complicated pre-treatments such as reduction alkylation and 65 a treatment can be carried out by referring to Angew Chem Int trypsin digestion as well as solubilizing agent were auto Ed Engl. 2006 Jun. 19:45(25):4167-72; Lohse A, Martins R, mated. For Sugar chains being quantitatively isolated, Jorgensen MR, Hindsgaul O. et al. The descriptions for the US 8,877,454 B2 79 80 various numerals are described in the section of EXPLANA amount of the detected 42 types of N-glycan can be used for TIONS OF LETTERS OR NUMERALS or in the specifica simple discrimination of the patients from normal blood tion; serum. (c) Classification of CDG-I patients using the expec FIG. 8A is a magnified view (upper left part) of FIG. 8: tation maximization algorithm. The selected set of concen FIG.8B is a magnified view (lower left part) of FIG. 8: tration of N-glycan is used to discriminate between healthy FIG. 8C is a magnified view (upper middle part) of FIG. 8: patients and CDG-I patients (panels on the left and the middle FIG. 8D is a magnified view (lower middle part) of FIG. 8: columns). Similarly, CDG-Ia and CDG-Ib can be success FIG.8E is a magnified view (upper right part) of FIG. 8: fully discriminated using the same set of N-glycan. FIG.8F is a magnified view (lower right part) of FIG. 8: FIG. 11D is a diagram showing the principal component FIG.9 is a disposition chart as an example of the apparatus 10 of the present invention. A dotted-line rectangle indicates the analysis (PCA) of seven kinds in total of the blood serum location where a dispensing needle for reagent or the like is tested in the present study with regard to the cases of CDG. placed. A solid-line rectangle indicates the location where a The first principal component (PC1) and the second principal dispensing needle for reagent or the like is not placed. A component (PC2) were plotted. These two principal compo dotted-line arrow indicates the movement of a liquid with a 15 nents occupy 41% and 37%, respectively, of the distribution. dispensing needle for reagent or the like. A solid-line arrow FIG. 12A is a diagram showing the classification based on indicates the movement of a filter plate/SPE plate. A double N-glycans derived from the blood sera of hepatocyte cancer line arrow indicates the connection to a vacuum pump. A and normal control. (a) Process of feature-subset selection. In position marked by * indicates the location where cooling to this process, the most significant N-glycan ratio in each step 10% occurs; was added in Succession, and 276 models in total were tested. FIG. 10 is a diagram showing an example for a very con As a result, the minimum relative error became 0%. (b) Hit Venient and feasible approach to the concentrated analysis of map diagram of three species of N-glycan ratios selected as a complex type Sugar conjugate of the present invention (FIG. feature for the classification (Lighter colors represent higher 10a). FIG. 10b is a diagram showing a stable hydrazide ratios of the presence of glycan). (c) Manifestation in box functionalized polymer carrier (that is, BLOTGLYCO 25 plots of the selected features (The ratio of presence of N-gly ABCTM beads) prepared by designing N-(2-aminobenzoyl) can, structure of oligosaccharide and number of peaks are cysteine hydrazide (ABCh), a multifunctional molecular shown in the diagram). probe, and binding this probe to thiopropyl-Sepharose 6B. FIG.12B is a diagram showing the classification based on This is a general protocol for the integrated glycoblotting N-glycans derived from the blood sera of hepatocyte cancer technology. (a) Workflow of glycoblotting-based high 30 and normal control. (a) Process of feature-subset selection. In throughput clinical glycomics. The process includes the fol this process, the most significant N-glycan ratio in each step lowing: 1) a reduced sugar is chemoselectively captured onto was added in succession, and 276 models in total were tested. hybridized functionalized beads using the glycoblotting tech As a result, the minimum relative error became 0%. (b) Hit nology 15, 2) washing is performed to remove all of the map diagram of three species of N-glycan ratios selected as a impurities, 3) the sialic acid residue is subjected to on-bead 35 feature for the classification (Lighter colors represent higher methyl esterification, and then the hydrazone bond is ratios of the presence of glycan). (c) Manifestation in box reduced, 4) modified N-glycan is recovered by reducing the plots of the selected features (The ratio of presence of N-gly disulfide bond. (b) Glycoblotting based on BLOTGLYCO can, structure of oligosaccharide and number of peaks are ABCTM beads. The chemical structure of N-(2-aminoben shown in the diagram). Zoyl)cysteine hydrazide (ABCh), the probe, is included; ref 40 FIG. 12C is a diagram showing the classification based on erence may be made to the descriptions in the specification N-glycans derived from the blood sera of hepatocyte cancer with respect to the synthesis: and normal control. (a) Process of feature-subset selection. In FIG. 11A is a diagram showing the 44 types of N-glycan this process, the most significant N-glycan ratio in each step used as examples in the present invention. Profiling of human was added in Succession, and 276 models in total were tested. serum glycoforms. (a) MALDI-TOF MS spectrum of a rep 45 As a result, the minimum relative error became 0%. (b) Hit resentative normal human serum N-glycan. Blood serum that map diagram of three species of N-glycan ratios selected as a had been digested using trypsin and PNGase F, was subjected feature for the classification (Lighter colors represent higher directly to the protocol for concentration and derivatization of ratios of the presence of glycan). (c) Manifestation in box N-glycan, using BLOTGLYCOABCTM beads. The sialyl plots of the selected features (The ratio of presence of N-gly N-glycan treated by on-bead methyl esterification was stable 50 can, structure of oligosaccharide and number of peaks are against desialylation, and as a result, it should be noted that shown in the diagram). quantitative glycomics analysis of total N-glycans is made FIG. 13 is a total cellular glycome of human prostate can possible; cer PC-3 cells and of normal human prostate epithelial cells FIG. 11B is a diagram showing the results for a Type-II (PrEC). Cells that had been proliferated in a dish measuring CDG (CDG-II) patient who has been through the specific 55 10 cm in size were subjected to glycoblotting and MS analysis stages of the failure of glycosylation. The inventors of the using the BLOTGLYCOABCTM beads (also, see the descrip present invention observed an abnormal N-glycan profile (for tions in the specification). example, several immature Small N-glycan chains). (b) FIG. 14 is a MALDI-TOF MS spectrum of blood serum Serum N-glycan profile of a DCG patient found by MALDI samples (n=8) prepared by the automated glycoblotting pro TOF MS. i: Healthy donor, ii. CDG-Ia, iii. CDG-Ib, iv: CDG 60 tocol using BLOTGLYCOABCTM beads. IIh, v. CDG-IIx1, vi: CDG-IIx2, and vii: CDG-IIx3. The FIG. 15 is a plane view showing the automatic Sugar chain asterisk indicates the internal standard (A2 amide) in a pre pretreatment apparatus according to an embodiment of the determined amount; present invention, with some parts being omitted from the FIG.11C is a diagram showing pairs of the combinations of diagram. the amount of N-glycan, which show the diagnostic differ 65 FIG. 16 is a frontal view of the dispensing head. ences between CDG-I patients, CDG-Ia patients and CDG-Ib FIG. 17 is a cross-sectional view of FIG. 16, dissected patients. It is shown that the data distribution formed by the along the line A-A. US 8,877,454 B2 81 82 FIG. 18 is a central vertical cross-sectional view of the first 46 VERTICAL MOVING UNIT constant-temperature bath. 47 INNER FRAME FIG. 19 is an exploded perspective view showing the first 48 RECEIVING FRAME low-pressure recovering device and the filter plate moving 49 SUPPORTING FRAME mechanism. SO GUIDE FIG. 20 is a plane view showing the filter plate moving S1 VERTICALLY MOVING ROD mechanism, with some parts being omitted from the diagram. 52 MOTOR FIG. 21 is a vertical cross-sectional view showing the ver S3 BALL SCREW tical moving unit in the filter plate moving mechanism, with 54 ELEVATING ROD Some parts being omitted from the diagram. 10 SS MICROPLATE FIG. 22 is a perspective view of the filter plate moving 56 SECOND LOW-PRESSURE RECOVERING mechanism and the second constant-temperature bath. DEVICE FIG. 23 is a central vertical cross-sectional view of the 58 TARGET PLATE second constant-temperature bath. 59 TARGET PLATE RECEIVING STAND FIG. 24 is a flow diagram of the treatment operation. 15 6O CONTROL DEVICE FIG. 25 is a flow diagram of the treatment operation that 61 MICROPLATE follows the treatment operation of FIG. 24. 62 FILTER PLATE 101101 OF SERUM EXPLANATIONS OF REFERENCE NUMERALS 102 SERUM-CONTAIN I NG FILTER PLATE 103 KEPT WARMAT 3 C, FOR 10 MINUTES 1 CASING BASE 104 SERUM-CONTAINING FILTER PLATE 1. DISPOSITION SPACE OF CASING BASE 105 KEPT WARMAT 60° C. FOR 30 MINUTES 2 DISPENSING HEAD 106 SERUM-CONTAINING FILTER PLATE 2A. CYLINDER 107 COOLED TO ROOM TEMPERATURE 2B PRESSURIZING PLATE 25 108 SERUM-CONTAINING FILTER PLATE 3 SUPPORTING FRAME 109 KEPT WARMAT ROOM TEMPERATURE, FOR 1 4 GUIDE ROD HOUR IN THE DARK SELEVATOR STAND 110 SERUM-CONTAINING FILTER PLATE 6 DRIVING MOTOR 111 KEPT WARMAT 37° C. FOR 60 MINUTES 7 BALL SCREW 30 112 SERUM-CONTAINING FILTER PLATE 8ELEVATOR STAND MOVING MECHANISM 113 KEPT WARMAT 90° C. FOR 5 MINUTES 9 DISPENSING NEEDLE HOLDER 114 SERUM-CONTAINING FILTER PLATE 10 DISPENSING NEEDLES 115 COOLED TO ROOM TEMPERATURE 11 DISPENSING HEAD MOVING MECHANISM 116 SERUM-CONTAINING FILTER PLATE 12 FIRST CONSTANTTEMPERATURE BATH 35 117 KEPT WARMAT 37° C. FOR 12 HOURS 12A MAIN BODY PART 118 SERUM-CONTAINING FILTER PLATE 13 RECEIVING STAND 119 REAGENT RACK AT 4°C., 1.5 ml 14 CARTRIDGE HEATER 120 REAGENT RACK, ROOM TEMPERATURE, 15 PELTIERELEMENT LIGHT SHIELDED 16 HEAT SINK 40 121 SERUM-CONTAINING FILTER PLATE 17 SILICONE SHEET 122 KEPT WARMAT 80° C. FOR 45 MINUTES 18 CARTRIDGE HEATER 123 SERUM-CONTAINING FILTER PLATE 19 INNER LID 124 DISCARDING BY SUCTION 21 LID 125 SERUM-CONTAINING FILTER PLATE 22 REAGENTRACK 45 126 DISCARDING BY SUCTION 23, 24 MICROPLATE FOR MIXING 127 SERUM-CONTAINING FILTER PLATE 25 MICROPLATE 128 DISCARDING BY SUCTION 26 FIRST LOW-PRESSURE RECOVERING DEVICE 129 SERUM-CONTAINING FILTER PLATE 27 FIRST SUCTION DISCARDING DEVICE 130 KEPT WARMAT ROOM TEMPERATURE, FOR 30 28 BOTTOM WIPER 50 MINUTES 29 SECOND CONSTANTTEMPERATURE BATH 131 SERUM-CONTAINING FILTER PLATE 29A MAIN BODY PART 132 DISCARDING BY S UCTION 3O RECEIVING STAND 133 DISCARDING BY SUCTION 31 CARTRIDGE HEATER 134 SERUM-CONTAININGI FILTER PLATE 32 AIR CIRCULATION PATH 55 135 DISCARDING BY SUCTION 33 PELTIERELEMENT 136 SERUM-CONTAININGI FILTER PLATE 34. HEAT SINK 137 DISCARDING BY SUCTION 35AIR CIRCULATION PATH 138 BOTTOM WIPER 36 DUCT 139 SERUM-CONTAINING FILTER PLATE 37 FAN 60 140 KEPT WARMAT 80° C. FOR 60 MINUTES 38 LID 141 SERUM-CONTAINING FILTER PLATE 39 FILTER PLATE MOVING MECHANISM 142 DISCARDING BY SUCTION 4O SUPPORTING PLATE 143 SERUM-CONTAINING FILTER PLATE 41,42 PULLEY 144 DISCARDING BY SUCTION 43 MOTOR 65 145 SERUM-CONTAINING FILTER PLATE 44 BELT 146 KEPT WARMAT 80° C. FOR 45 MINUTES 45 HORIZONTALLY MOVING PLATE 147 SERUM-CONTAINING FILTER PLATE US 8,877,454 B2 83 84 148 EXTRACTION BY SUCTION 310 KEPT WARMAT ROOM TEMPERATURE, FOR 1 149 WASHING VALVE HOUR IN THE DARK 15O PLATE FOR RECOVERY 1 311 SERUM-CONTAINING PCR PLATE 151 DISPENSING20 ul 312 KEPT WARMAT 37° C. FOR 60 MINUTES 152 MP1 FOR MIXING 313 SERUM-CONTAINING PCR PLATE 153 DISPENSING 380 ul 314 KEPT WARMAT 90° C. FOR 5 MINUTES 154. SPE PLATE 315 SERUM-CONTAINING PCR PLATE 1SS DISCARDING BY SUCTION 316 COOLED TO ROOM TEMPERATURE 156 SPE PLATE 317 SERUM-CONTAINING PCR PLATE 157 DISCARDING BY SUCTION 10 318 KEPT WARMAT 37° C. FOR 12 HOURS 319 SERUM-CONTAINING PCR PLATE 158 SPE PLATE 320 REAGENTRACK, 4°C., 1.5 ml 159 DISCARDING 321 REAGENT RACK, ROOM TEMPERATURE 16O SPE PLATE 322 DISPENSING20 ul 161 DISCARDING BY SUCTION 15 323 DISCARDING BY SUCTION 162 SPE PLATE 324 DISCARDING BY SUCTION 163 RECOVERY BY SUCTION 325 DISCARDING BY SUCTION 164 PLATE FOR RECOVERY 2 326 BOTTOM WIPER 165 DISPENSING 2 ul 327 BEADS-CONTAINING FILTER PLATE 166 MP2 FOR MIXING 328 BEADS-CONTAINING FILTER PLATE 167 DISPENSING 2 ul 329 BEADS-CONTAINING FILTER PLATE 168 MALDI PLATE 33 OBEADS-CONTAINING FILTER PLATE 169 END 331 KEPT WARMAT 80° C. FOR 60 MINUTES 201 TRYPSIN400 U.5ul 332 BEADS-CONTAINING FILTER PLATE 202 PNGase F, 2 U, 5ul 25 333 KEPT WARMAT ROOM TEMPERATURE, FOR 10 203 0.26% PHL (SOLUBILIZING AGENT)/0.11 M MINUTES AMMONIUM BICARBONATE, 45ul (CV 5%) 334 DISCARDING BY SUCTION 204 120 mM DTT, 5 ul (CV 5%) 335 BEADS-CONTAINING FILTER PLATE 205 123 mM IAA, 10 ul (CV 5%) 336 DISCARDING BY SUCTION 206 10% AcO/MeOH, 100 ul 30 337 BEADS-CONTAINING FILTER PLATE 207 150 mMMTT/DIOXANE, 100 ul 338 DISCARDING BY SUCTION 208.20 mM REAGENT, 20 ul 339 BEADS-CONTAINING FILTER PLATE 2092% AcOH/ACN, 180 ul 340 DISCARDING BY SUCTION 210 2 MGUANIDINE HYDROCHLORIDE, 200 ulx2 341 BEADS-CONTAINING FILTER PLATE 211 WATER, 200 ulx2 35 342 DISCARDING BY SUCTION 212 1% TEA/MeOH, 200 ulx2 343 BOTTOM WIPER 213 10 mM HCl, 200 ulx2 344 BEADS-CONTAINING FILTER PLATE 214 MeOH, 200 ulx2 345 KEPT WARM, LIGHT SHIELDED, ROOM TEM 215 DIOXANE, 200 ulx2 PERATURE, FOR 30 MINUTES 216 DIOXANE, 200 ulx2 40 346 BEADS-CONTAINING FILTER PLATE 217 WATER, 200 ulx1 347 DISCARDING BY SUCTION 2182% AcOH/ACN, 180 ul 348 BEADS-CONTAINING FILTER PLATE 219 WATER, 100 ulx1 349 DISCARDING BY SUCTION 220.95%, ACN, 360 ul 350 BOTTOM WIPER 221 WATER, 200 ul 45 351 BEADS-CONTAINING FILTER PLATE 222 95%, ACN, 200 ul 352 DISCARDING BY SUCTION 223.95%, ACN, 200 ul 353 BEADS-CONTAINING FILTER PLATE 224.5%, ACN, 100 ul 354. DISCARDING BY SUCTION 225 DHB/30% ACN, 18 ul 355 BOTTOM WIPER In 149, 50 356 BEADS-CONTAINING FILTER PLATE A 2% ACETIC ACID (AcOH)/ACETONITRILE (ACN) 357 KEPT WARMAT 60° C. FOR 60 MINUTES BWATER 358 BEADS-CONTAINING FILTER PLATE C 2 MGUANIDINE HYDROCHLORIDE 359 DISCARDING BY SUCTION D 95% ACETONITRILE (ACN) 36O BEADS-CONTAINING FILTER PLATE EDIOXANE 55 361. DISCARDING BY SUCTION F METHANOL (MeOH) 362 BOTTOM WIPER G 10 mM HCl 363 BEADS-CONTAINING FILTER PLATE H 1% TEA/METHANOL (MeOH) 364 DISCARDING BY SUCTION 301 101 SERUM 365 BEADS-CONTAINING FILTER PLATE 302 PCR PLATE 60 366 50 nM DTT IN 50 nMAMMONIUM BICARBON 303 SERUM-CONTAINING PCR PLATE ATE, 50 ul 304 KEPT WARMAT 37° C. FOR 10 MINUTES 367 KEPT WARMAT 60° C. FOR 5 MINUTES 305 SERUM-CONTAINING PCR PLATE 368 KEPT WARMAT ROOM TEMPERATURE, FOR 15 306 KEPT WARM 60° C. FOR 30 MINUTES MINUTES 307 SERUM-CONTAINING PCR PLATE 65 369 BEADS-CONTAINING FILTER PLATE 3O8 COOLED TO ROOM TEMPERATURE 370 RECOVERY BY SUCTION 309 SERUM-CONTAINING PCR PLATE 371 PLATE FOR RECOVERY 1 US 8,877,454 B2 85 86 372 KEPT WARMAT 10° C., TO COMPLETION 517B50% ACN, 300 ulx3 373 SPE INITIATION 5181 M AcOH, 200 ul 374 DISPENSING20 ul 519 10% ACN, 20 ul 375 MP FOR MIXING 1. 520 MALDI MATRIX, 2 ul 376 DISPENSING 420 ul 521 200 ul 377 SPE PLATE 522 100 mM MTT IN DMSO/ACN, 100 ul 378 DISCARDING BY SUCTION 523 20 AcOH/ACN, 200 ul 379 SPE PLATE 524 6 MGUANIDINE HYDROCHLORIDE, 300 ul 38O DISCARDING BY SUCTION 5256 MGUANIDINE HYDROCHLORIDE, 300 ulx2 381 SPE PLATE 10 526 WATER, 300 ul 382 DISCARDING BY SUCTION 527 10 mM HYDROCHLORIC ACID, 300 ulx3 383 SPE PLATE 528 MeOH,300 ulx3 384 DISCARDING BY SUCTION 529 MeOH,300 ulx3 385 SPE PLATE 530 WATER, 300 ulx3 386 RECOVERY BY SUCTION 15 531 ACN, 300 ulx3 387 PLATE FOR RECOVERY 2 532 ACN, 300 ulx3 388 KEPT WARMAT 10° C., TO COMPLETION 533 WATER, 300 ulx3 389 DISPENSING 2 ul 534 WATER, 200 ul 390 MALDI DISPENSING INITIATED 535 ACN, 400 ul 391 MP FOR MIXING 2 536 ACN, 200 ul 392 DISPENSING 2 ul 537 95% ACN, 200 ul 393 MALDI PLATE S38 MALDI DISPENSING ONLY 394 WASHING VALVE 539 MALDI DISPENSING AFTER SPETREATMENT 395 BEADS-CONTAINING FILTER PLATE The invention claimed is: 396 DISCARDING BY SUCTION 25 1. A method for analyzing a Sugar chain in a sample, the 397 BOTTOM WIPER method comprising the following steps: In 394, A) a Sugar chain releasing step of releasing a Sugar chain in A 2% ACETIC ACID (AcOH)/ACETONITRILE (ACN), a sample, the step comprising the following steps: 20 ml A-1) a step of providing the sample on a plate for reac B 95% ACETONITRILE (ACN) IN WATER, 20 ml 30 tion; C METHANOL (MeOH), 200 ml A-2) a step of adding a solubilizing agent to the sample D WATER to thereby place the sample under a reaction condi E 95% ACETONITRILE (ACN), 330 ml tion; F50% ACETONITRILE (ACN) A-3) a step of adding a reducing agent to the sample to G 10 mM HYDROCHLORIC ACID, 100 ml 35 thereby place the sample under a reaction condition; H 6 MGUANIDINE HYDROCHLORIDE, 100 ml A-4) a step of adding an —SH protecting agent to the 4O1 REGULATION OF REDUCING REAGENT WORK sample to thereby place the sample under a reaction 402 REDUCING REAGENT condition; 403 REAGENT RACK, ROOM TEMPERATURE/ A-5) a step of adding a proteolytic enzyme to the sample LIGHT SHIELDED 40 to thereby place the sample under a reaction condi 404 DISPENSING20 ul tion; 405 DISPENSING 40 ul A-6) a step of deactivating the proteolytic enzyme; and 406 R9 (2 ml) A-7) a step of adding a Sugar chain releasing enzyme to 4O78M BORANEPYRIDINE the sample to thereby release the Sugar chain; 408 EMPTYRACK 45 B) a detection sample preparing step of preparing the 409 R11 (20 ml) released Sugar chain for use in detection, the step com 410 R10 (4 ml) prising the following steps: 411 MeOH, 140 ul B-1) a step of contacting the sample prepared in the step 412.50% AQUEOUS SOLUTION OF TRICHLOROACE (A) with a Sugar chain-capturing bead to thereby TIC ACID 50 place the sample under the conditions allowing the 501 0.33 MAMMONIUM BICARBONATE, 15 ul, CV released Sugar chain in the sample to bind to the bead, 5% and thus producing a captured Sugar chain sample; 502 0.4% SOLUBILIZINGAGENT, 30 ul, CV 5% B-2) a step of adding a protein denaturing agent to the 503 120 mM DTT, 5ul, CV5% captured Sugar chain sample to thereby place the cap 504123 mM IAA, 10 ul, CV 5% 55 tured Sugar chain sample under a reaction condition; 505 TRYPSIN, 400 U, 5ul B-3) a step of washing the captured Sugar chain sample, 506 PNGase F, 2 U, 5ul and then discarding the residual washing liquid by 508 R1 (1.5 ml) Suction; 509 R2 (3 ml) B-4) a step of adding a salt releasing agent for the Sugar 510 R12 (20 ml) 60 chain capturing agent on beads to the captured Sugar 511 R3 (0.5 ml) chain sample, and then discarding the salt releasing 512 R4 (1 ml) agent by Suction; 513 R5 (2 ml) B-5) a step of adding a protective agent to the captured 514 R6 (0.2 ml) Sugar chain sample to thereby place the captured 515 R7 (10 ml) 65 Sugar chain under a reaction condition; 516 R8 (5 ml) B-6) a step of adding an acid to the captured Sugar chain 517AACN, 300 ulx3 sample, and discarding the acid by Suction; US 8,877,454 B2 87 88 B-7) a step of adding an organic reaction solvent to the A-2) the Solubilizing agent is 1-propanesulfonic acid, captured Sugar chain sample; 2-hydroxy-3-lauramide (PHL), 1-propanesulfonic B-8) a step of removing the solvent and the moisture in acid, 2-hydroxy-3-myristamide (PHM), 2-hydroxy the bead; 3-sulfopropyl laurate (HSD) or an equivalent thereto, B-9) a step of adding a methyl esterifying agent to the and captured Sugar chain sample to thereby place the cap the reaction condition is at 25°C. to 42°C.; tured Sugar chain sample under a reaction condition, A-3) the reducing agent is dithiothreitol (DTT), TCEP and alkylating the carboxylic acid of Sialic acid; (Tris(2-carboxyethyl)phosphine hydrochloride solu B-10) a step of adding the organic reaction solvent to the tion, 0.5 M), or an equivalent thereto, and captured Sugar chain sample, and discarding the 10 the reaction condition is at room temperature to 80°C.; A-4) the -SH protecting agent is iodoacetamide (IAA) organic reaction solvent by Suction; or an equivalent thereto, and B-11) a step of washing the captured Sugar chain sample, the reaction condition is at 20 to 37° C. in the dark; and Subsequently discarding the residual washing liq A-5) the proteolytic enzyme is trypsin, chymotrypsin or uid by Suction; 15 an equivalent thereto, and B-12) a step of releasing a Sugar chain sample from the the reaction condition is at 25 to 42°C.; captured Sugar chain sample, wherein when an analy A-6) the conditions for deactivating include heating to sis requiring tagging is conducted, the Sugar chain in 65° C. or higher; the captured Sugar chain sample is tagged with a A-7) the Sugar chain releasing enzyme is peptide-N- labeling reagent and is released from the bead; and glycosidase F. peptide-N4-(acetyl-3-glucosaminyl)- B-13) a step of dissolving the released Sugar chain asparagine amidase (PNGaseF), Endo Horan equiva sample to produce a Sugar chain sample solution; lent thereto, and the reaction conditions for the Sugar C) when performing mass spectrometry using a plate, a chain releasing enzyme are at 25°C. to 42°C.; with step of producing a plate for mass spectrometry having regard to the step (B), the captured Sugar chain dotted thereon, the step com 25 B-1) the bead is a bead or magnetic bead having a Sugar prising: chain capturing group which includes an aminooxy C-1) a step of disposing the tagged Sugar chain sample group, an N-alkylaminooxy group, a hydrazide solution obtained in the step (B) on a plate for recov group, an azide group, a thiosemicarbazide group, a ery; cysteine residue or a derivative thereof bound thereto, C-2) a step of disposing the tagged Sugar chain sample 30 and Solution from the plate for recovery by adding an the conditions in which the released Sugar chain in the organic solvent to the plate and mixing so as to obtain sample binds to the bead are at 25 to 80° C.: a concentration at which the Sugar chain adsorbs to a B-2) the denaturing agent is guanidine hydrochloride, solid phase, wherein the concentration at which the urea, sodium dodecyl Sulfate or an equivalent thereto, sugar chain adsorbs to the solid phase is 80 to 90% in 35 and the organic solvent; and wherein the step optionally the reaction conditions involve adding at room tempera comprises the steps C-3) to C-6): ture, and maintaining the temperature to allow the bead C-3) a step of providing a solid phase carrier-enclosed to sufficiently swell (from 10 seconds to 5 minutes); plate; B-3) the washing is performed using water, C-4) a step of activating the Solid phase carrier-enclosed 40 B-4) the Sugar chain capturing agent on the bead is an plate according to the phase of the solid phase carrier aminooxy group, an N-alkylaminooxy group, a enclosed plate, and washing the Solid phase carrier hydrazide group, an azide group, a thiosemicarbazide enclosed plate; group, a cysteine residue or a derivative thereof, and C-5) a step of adding the tagged Sugar chain sample the salt releasing agent is triethylamine or an equiva Solution to the Solid phase carrier-enclosed plate, and 45 lent thereto in the case of hydrazide, and is triethy conditioning the tagged Sugar chain sample solution lamine or an equivalent thereto in the case of an to a solvent having apolarity appropriate for the phase N-alkylaminooxy group; of the Solid phase carrier-enclosed plate; B-5) the protective agent is acetic anhydride, Succinic C-6) a step of recovering the tagged Sugar chain sample anhydride or another acid anhydride, or an equivalent Solution by Suction from the Solid phase carrier-en 50 thereto, and closed plate to a second plate for recovery; and the reaction conditions use acetic anhydride/methanol at when subjecting the tagged Sugar chain sample solution to 15 to 37° C.; MALDI-TOFMS, comprising the following step (C-7): B-6) the acid is hydrochloric acid or another inorganic C-7) a step of mixing the tagged Sugar chain sample acid, or an equivalent acid at pH 2 to 3: Solution with a matrix for mass spectrometry, and 55 B-7) the step includes steps of adding a hydrophilic dotting the mixture on a plate for MALDI-TOF MS organic solvent and discarding the hydrophilic determination; and organic solvent before adding the organic reaction D) a step of conducting an analysis of the Sugar chain to be Solvent, and the hydrophilic organic solvent is metha determined. nol, ethanol, acetonitrile, or acetone, while the 2. The method according to claim 1, wherein the preceding 60 organic reaction solvent is dioxane, acetonitrile, tet steps are associated with at least any one of the following rahydrofuran or an equivalent thereto; conditions: B-8) the step of removing the solvent and the moisture in A) in the Sugar chain releasing step of releasing a Sugar the bead includes wiping of the bottom of the plate chain in a sample to prepare a Sugar chain sample for with a filter paper, a blotting paper, a gauze, a towel, a analysis: 65 hand towel, a tissue paper or a cotton sheet; A-1) the sample is a body fluid, a cell extract or a tissue B-9) the methyl esterifying agent is methyl-p-tolyl-tria eXtract; Zene (MTT) or an equivalent thereto, and US 8,877,454 B2 89 90 the reaction conditions use 100 mMMTT/dioxane at 20 to 2-hydroxy-3-myristamide (PHM)/ammonium bicar 80° C. for 30 minutes to 5 hours: bonate, and allowing the mixture to react for 5 to 60 B-10) the organic reaction solvent is dioxane, acetoni minutes at 25 to 42°C.; trile, tetrahydrofuran or an equivalent thereto; A-3) a step of adding dithiothreitol (DTT) to the sample, B-11) the washing is performed using at least one allowing the mixture to react for 10 to 60 minutes at selected from the group consisting of methanol, a 50 to 80°C., and then cooling the reaction mixture to NaCl solution and water; and room temperature; B-12) the tagging is carried out, such that the tagging is A-4) a step of adding iodoacetamide (IAA), and allow performed using a chromophore capable of absorbing ing the mixture to react for 0.5 to 2 hours at room ultraviolet and visible rays, a tag having a structure 10 emitting fluorescence, an affinity tag having a mol temperature in the dark; ecule capable of interacting with another molecule, a A-5) a step of adding trypsinto the sample, and allowing tag having a functional group capable of specifically the mixture to react for 30 to 120 minutes at 25 to 42° reacting with a functional group, a tag having a func C.; tional group in a hydrophobic structure, or a tag hav 15 A-6) a step of heating the sample to 80 to 100° C. for 1 ing a metalion ligand, and the tagging is conducted by to 10 minutes, and then cooling the sample to room adding acetic acid, acetonitrile, an acetate buffer oran temperature; and equivalent thereto; and A-7) a step of adding PNGaseF, and allowing the mix B-13) the dissolving of the tagged captured Sugar chain ture to react for 6 to 24 hours at 25 to 42°C.; sample is performed using water, an aqueous Solution B) a detection sample preparing step of preparing the or an equivalent thereto; released Sugar chain for use in detection, the step com C) in the step of producing a plate for mass spectrometry prising the following steps: having the captured Sugar chain sample dotted thereon; B-1) a step of contacting the captured Sugar chain C-1) the disposing on the plate for recovery is conducted sample prepared in the step (A) with beads for cap under the conditions of removing the reagent for tag 25 turing Sugar chain, to thereby allow binding at 40°C. glng or higher (for example, 80°C.), and thus producing a C-3) the solid phase carrier-enclosed plate is of multi captured Sugar chain sample: well type and includes a Surface of a resin or mem B-2) a step of adding guanidine hydrochloride to the brane suitable for solid phase extraction: captured Sugar chain sample to thereby place the cap C-4) when the solid phase carrier-enclosed plate is in 30 normal phase mode, washing is conducted sequen tured Sugar chain sample under a reaction condition, tially with water and acetonitrile, and when the solid and then discarding the reaction liquid by suction; phase carrier-enclosed plate is in reverse phase mode, B-3) a step of washing the captured Sugar chain sample washing is conducted sequentially with a lower alco with water, and then discarding the water by Suction; hol Such as methanol and water; 35 B-4) a step of washing the captured Sugar chain sample C-5) the solvent having an opposite polarity is a hydro with triethylamine, and then discarding the triethy phobic organic solvent in the case of the normal phase lamine by Suction; mode, and is a hydrophilic solvent in the case of the B-5) a step of adding acetic anhydride to the captured reverse phase mode; Sugar chain sample to thereby place the captured C-6) the second plate for recovery is of multi-well type 40 Sugar chain Sample under the reaction conditions of and includes a Surface of a resin or membrane Suitable using 10% acetic anhydride/methanol at room tem for Solid phase extraction; and perature for 10 minutes to 2 hours, and then discard C-7) the matrix for mass spectrometry is 2,5-dihydroxy ing the acetic anhydride by Suction; benzoic acid or an equivalent thereto, and the dotting B-6) a step of adding hydrochloric acid to the captured of the tagged Sugar chain sample solution on the 45 Sugar chain sample, and discarding the hydrochloric matrix for mass spectrometry is conducted in mixture acid by Suction; or in sequence, and is diluted as necessary; B-7) a step of adding methanol to the captured Sugar D) the analysis of the sugar chain to be determined is chain sample, discarding the methanol by Suction, and conducted by high performance liquid chromatography then adding dioxane to the captured Sugar chain (HPLC), liquid chromatography-electrospray ioniza 50 sample; tion mass spectrometry (LC-ESIMS), matrix assisted B-8) a step of wiping the bottom of the filter plate with a laser desorption ionization-Time-of-Flight (MALDI cotton sheet; TOF), or an equivalent thereto, while when using a col B-9) a step of adding methyl-p-tolyl-triazene (MTT) to oring reagent or biotin in the tagging, a step of removing the captured Sugar chain sample, and allowing the any excess coloring reagent is carried out as necessary, 55 mixture to react for 30 to 120 minutes (for example, and 60 min.) at 60° C. or higher; when the beads are magnetic beads, the magnetic beads are B-10) a step of adding dioxane to the captured Sugar beads having a modifiable functional group, a hydrazide chain sample, and discarding the dioxane by Suction; group or an aminooxy group. B-11) a step of washing the captured Sugar chain sample 3. The method according to claim 1, further comprising at 60 sequentially with methanol, a NaCl solution and least one step among the following steps: water, and then discarding the water by Suction; and A) a Sugar chain releasing step of releasing a Sugar chain in B-12) a step of adding acetic acid and acetonitrile to the a sample, the step comprising the following steps: captured Sugar chain sample, and tagging the Sugar A-1) a step of providing blood serum as a sample on a chain in the captured Sugar chain sample using ami filter plate: 65 nooxytryptophanyl arginine methyl ester/water, A-2) a step of adding 1-propanesulfonic acid, 2-hy O-benzylhydroxylamine hydrochloride/water, or droxy-3-lauramide (PHL) or 1-propanesulfonic acid, anthraniloyl hydrazine/water; and US 8,877,454 B2 91 92 B-13) a step of adding water to the tagged captured sugar C-5) a step of adding the tagged sugar chain sample chain sample to produce a tagged sugar chain sample solution to the solid phase carrier-enclosed plate, dis solution; carding the liquid, washing the plate with acetonitrile, C) a step of producing a plate for mass spectrometry having and adding 1 to 20% acetonitrile thereto; the tagged captured sugar chain sample dotted thereon, C-6) a step of recovering the bead by suction from the the step comprising: solid phase carrier-enclosed plate to the second plate C-1) a step of disposing the tagged sugar chain sample for recovery; and solution obtained in the step (B) on a plate for recov ery; C-7) a step of adding 2,5-dihydroxybenzoic acid in 20 to C-2) a step of disposing the tagged sugar chain sample 40% acetonitrile, to the tagged sugar chain sample Solution from the plate for recovery by adding aceto solution, and mixing and dotting the mixture; and nitrile to the plate and mixing, so as to achieve a final D) a step of performing mass spectrometry by MALDI concentration in acetonitrile of 80 to 90%; TOF MS. C-3) a step of providing a solid phase carrier-enclosed 4. The method according to claim 3, wherein the sugar plate which is in normal phase mode: chain-capturing bead is a magnetic bead, and separation is C-4) a step of washing the solid phase carrier-enclosed conducted by means of a magnetic field instead of the dis plate sequentially with water and acetonitrile, and carding by suction. discarding water and acetonitrile by suction; UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 8,877,454 B2 Page 1 of 1 APPLICATIONNO. : 12/681573 DATED : November 4, 2014 INVENTOR(S) : Shinichiro Nishimura et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

On the Title Page, item (73) the Assignee “Shionogi & Co. Ltd. should be changed to --Sumitomo Bakelite Co., Ltd.--

Signed and Sealed this Twenty-eighth Day of April, 2015 74-4-04- 2% 4 Michelle K. Lee Director of the United States Patent and Trademark Office