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Apparatus for Auto-Pretreating Sugar Chain

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Apparatus for Auto-Pretreating Sugar Chain (19) & (11) EP 2 207 030 A1 (12) EUROPEAN PATENT APPLICATION published in accordance with Art. 153(4) EPC (43) Date of publication: (51) Int Cl.: 14.07.2010 Bulletin 2010/28 G01N 27/62 (2006.01) G01N 30/06 (2006.01) G01N 30/72 (2006.01) G01N 30/88 (2006.01) (2006.01) (21) Application number: 08836267.8 G01N 35/04 (22) Date of filing: 03.10.2008 (86) International application number: PCT/JP2008/068111 (87) International publication number: WO 2009/044900 (09.04.2009 Gazette 2009/15) (84) Designated Contracting States: • MIURA, Yoshiaki AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Sapporo-shi HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT Hokkaido 001-0021 (JP) RO SE SI SK TR • YAMAZAKI, Hiroshi Designated Extension States: Hachioji-shi AL BA MK RS Tokyo 192-0031 (JP) • HORIUCHI, Michio (30) Priority: 05.10.2007 JP 2007262771 Hachioji-shi 05.10.2007 JP 2007262680 Tokyo 192-0031 (JP) • MOTOKI, Hiroaki (71) Applicants: Hachioji-shi • Hokkaido University Tokyo 192-0031 (JP) Kita-ku • KURODA, Toshiharu Sapporo-shi Hachioji-shi Hokkaido 060-0808 (JP) Tokyo 192-0031 (JP) • SYSTEM INSTRUMENTS CO., LTD. •KITA,Yoko Hachioji-shi, Tokyo 192-0031 (JP) Amagasaki-shi • Shionogi&Co., Ltd. Hyogo 660-0813 (JP) Osaka-shi, Osaka 5410045 (JP) • NAKANO, Mika Amagasaki-shi (72) Inventors: Hyogo 660-0813 (JP) • NISHIMURA, Shinichiro Sapporo-shi (74) Representative: Guder, André Hokkaido 001-0021 (JP) Uexküll & Stollberg • SHINOHARA, Yasuro Patentanwälte Sapporo-shi Beselerstraße 4 Hokkaido 001-0021 (JP) 22607 Hamburg (DE) (54) APPARATUS FOR AUTO-PRETREATING SUGAR CHAIN (57) To provide an autoanalyzer for analyzing a sug- for the mass spectrometry having the captured sugar ar chain contained in a biological sample, in particular, chain dotted thereon which comprises the step of provid- serum. Namely, it is intended to provide a method of an- ing the tagged sugar chain sample solution obtained in alyzing a sugar chain in a sample, which comprises the the step B) on a collection plate; and, if required, the step following steps: A) the sugar chain-releasing step of re- of conducting an operation in a solid phase support-en- leasing the sugar chain in the sample; B) the detection closed plate to form the plate for mass spectrometry; and sample-preparing step of preparing the released sugar D) the step of analyzing the sugar chain to be assayed. chain for detection; and, in the case of conducting mass spectrometry using a plate, C) the step of forming a plate EP 2 207 030 A1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 207 030 A1 2 EP 2 207 030 A1 Description TECHNICAL FIELD 5 [0001] The present invention relates to an apparatus for sugar chain analysis. The present invention can be applied to the diagnosis of a disease, or the like. More particularly, the present invention relates to an automatic sugar chain pretreatment apparatus. BACKGROUND ART 10 [0002] Pretreatment methods for quantitatively analyzing a sugar chain (Patent Document 1; Non-Patent Document 1). There has been reported a method of detecting a sugar chain rapidly with high sensitivity in order to rapidly analyze the sugar chain, by selectively capturing only a sugar chain released from a glycoprotein, immobilizing the sugar chain on beads, and washing the bead (Non-Patent Document 2). However, an apparatus or pretreatment apparatus that 15 automatically performs a sugar chain analysis using a large amount of a sample such as a blood serum sample, is not known yet. [0003] In the conventional art, purification of a sugar chain contained in a biologically derived mixture of a body fluid such as blood (serum) and a cell/tissue extract, is carried out according to the following steps. [0004] A. A glycoprotein in a sample is reductively alkylated in the presence of a soluble product. 20 [0005] B. A proteolytic enzyme is added thereto to digest the protein moiety with the enzyme, and then the system is heated to deactivate the enzyme. [0006] C. An enzymatic treatment or a chemical treatment which releases a sugar chain from a peptide is carried out to release the sugar chain. [0007] D. Polymer beads displaying a functional group capable of capturing a sugar chain are contacted with a sample 25 which has been finished with the treatment of item C., and thereby the released sugar chain is captured by the functional group of polymer beads. [0008] E. The sample is washed and filtered to remove those impurities that are not captured by the functional group of the polymer beads. [0009] F. The carboxylic acid of a sialic acid residue of the sugar chain captured by the functional group of the polymer 30 beads, is protected by methyl esterifying the functional group. [0010] G. The sugar chain captured by the functional group of the polymer beads is subjected to a reduction of the hydrazone bond to stabilize the binding thereof with the polymer beads. [0011] H. The sugar chain is released from the polymer beads through a hydrazone-oxime exchange reaction, by cleaving the disulfide bond included in sugar chain capturing molecules on beads, or by dispensing an aminooxy- 35 containing compound. In the case of the latter, the treatment of item G. is not carried out. [0012] I. The released sugar chain is recovered in a filtrate. [0013] J. A MALDI matrix is added to the sugar chain-containing filtrate, and then the mixture is added dropwise on a MALDI plate. 40 Patent Document 1: WO 2004/058687 Non-patent Literature 1: Mol. Cell. Proteomics. 2007;6:1437-45. Quantitative glycomics of human whole serum glycoproteins based on the standardized protocol for liberating N- glycans. Kita Y, Miura Y, Furukawa J, Nakano M, Shinohara Y, Ohno M, Takimoto A, Nishimura S. Non-patent Literature 2: Chem. Euro. J. 2007; 13:4797-804. Rapid and simple solid- phase esterification of sialic acid 45 residues for quantitative glycomics by mass spectrometry. Miura Y, Shinohara Y, Furukawa J. Nagahori N, Nishimura S. DISCLOSURE OF THE INVENTION 50 PROBLEM TO BE SOLVED BY THE INVENTION [0014] However, conventionally, such processes have been carried out mostly by hand, and thus these processes have required extensive time and labor, while it has been difficult to carry out purification with high accuracy. [0015] The present invention was made under such circumstances, and is intended to provide an automatic sugar 55 chain pretreatment apparatus which allows purification to be carried out at high speed with high accuracy by automating the treatment processes. [0016] That is, it is an object of the present invention to provide an auto-analyzing apparatus for analyzing a sugar chain contained in a blood serum sample. It is an object to make it possible by the auto-analyzing apparatus to auto- 3 EP 2 207 030 A1 matically analyze a large amount of sample simultaneously. In order to release a sugar chain quantitatively from a glycoprotein, cumbersome pretreatments such as a use of a solubilizing agent, reductive alkylation and digestion by trypsin, are required. Furthermore, a sugar chain that has been quantitatively released can be subjected to a rapid analysis of the sugar chain by performing chemoselective capturing and thereby easily removing troublesome foreign 5 matters. However, it is difficult to treat multiple test samples all at a time. Thus, it is intended to optimize the various processes described above and to establish a system adaptable to automatic analysis. MEANS FOR SOLVING PROBLEM 10 [0017] The inventors of the present invention made a thorough investigation, and as a result, they found an apparatus that can automatically prepare a sample for mass spectrometry when a blood serum sample is applied. Thereafter, by performing mass spectrometry, an automatic analysis of sugar chain was made possible. The inventors have been able to develop an apparatus which is capable of carrying out all processes including from pretreatment to capturing of a sugar chain, modification of sialic acid, and to release of the sugar chain. 15 [0018] The present invention provides the following. (1) A method for analyzing a sugar chain in a sample, the method comprising the following steps: A) a sugar chain releasing step of releasing a sugar chain in a sample, the step comprising the following steps: 20 A-1) a step of providing the sample on a plate for reaction; A-2) a step of addinga solubilizing agent to the sample to therebyplace thesample under a reaction condition; A-3) a step of adding a reducing agent to the sample to thereby place the sample under a reaction condition; A-4) a step of adding an -SH protecting agent to the sample to thereby place the sample under a reaction 25 condition; A-5) a step of adding a proteolytic enzyme to the sample to thereby place the sample under a reaction condition; A-6) a step of deactivating the proteolytic enzyme; and A-7) a step of adding a sugar chain releasing enzyme to the sample to thereby release the sugar chain; 30 B) a detection sample preparing step of preparing the released sugar chain for use in detection, the step comprising the following steps: B-1) a step of contacting the sample prepared in the step (A) with a sugar chain- capturing bead to thereby 35 place the sample under the conditions allowing the released sugar chain in the sample to bind to the bead, and thus producing a captured sugar chain sample; B-2) a step of adding a protein denaturing agent to the captured sugar chain sample to thereby place the captured sugar chain sample under a reaction condition; B-3) a step of washing the captured sugar chain sample, and then discarding the residual washing liquid 40 by
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