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Research paper : Development of basic tools for glycoscience and their application to cancer diagnosis (H. Narimatsu)−207−Synthesiology - English edition Vol.5 No.3 (2012) kinds of glycans can be yielded in one tube by this method. As the molecular mass of produced glycans is preliminarily known, the desired mass can be measured in an aliquot of the final mixture by mass spectrometer. We named this method Mass-tagged synthesis.[34][35]Elemental technology 5: Large-scale identification technology of glycoproteins by LC/MSBased on the newly developed LC/MS analysis method that can simultaneously identify more than 1,000 proteins in a peptide mixture sample at the same time, we established a large scale identification method for elucidation of binding sites of glycoproteins and glycan structures of glycopeptides isolated from protein digestion samples by affinity chromatography. As the peptide moiety of glycopeptides was not fragmented by MS/MS analysis with collision-induced dissociation due to the presence of large glycans, glycopeptides were not suitable for direct identification. Therefore, glycans were released from glycopeptides by glycopeptidases, and the obtained deglycosylated peptides were subjected to the large-scale identification. In this reaction, Asn in the glycosylation site was replaced by Asp, and the mass was increased by 1 Da, which indicated the glycosylation site. During this reaction, as non-glycopeptides receiving de-Asn reaction were also present in the mixture and thus the de-glycosylated peptides could not be distinguished, stable-isotope labeled water (H218O) was added to the solvent for enzymes to incorporate the labeled oxygen. As a result, the glycosylation site was labeled, and thus the highly accurate glycoprotein identification method was actualized (IGOT method). Based on the integration of LC/MS and IGOT method, Kaji et al. of RCMG are actively pursuing the high-throughput mass identification of glycoproteins. The current high speed mass analysis system enables a series of identification processes for 500-1,000 kinds of glycoproteins in 1 mg of a tissue-originated protein sample in about 10 days.[36][37] Elementary technology 6: MSn-based identification of glycan structuresIn the tandem MSn method, mass of the target glycan (mass of MS1) is measured first. Then the glycan is dissociated by weak collision of a rare gas such as argon or helium (Collision Induced Dissociation: CID), and the mass of each derived fragment is measured (MS2). Each of these fragments is isolated and again fragmented by CID to obtain MS3. Theoretically, MSn can be measured as long as a sufficient amount of the sample is available. In the actual measurements, even a slight difference of a glycan structure can be distinguished by MS4 based on the dissociation pattern obtained by CID (Fig.4). Therefore, we obtained the data up to MS4 of as many standard glycans as possible, and compiled them into a Fig. 4 Application of the tandem MSn-based structure identification on isomeric glycans400 600 800 1000 1200 1400 1600 1800 2000 Mass/Charge PA = pyridylamino Mass/Charge 200 300 400 500 600 700 800 900 1000 1100 1200 1300 MS/MS GlcNAc1-2Man1-6GlcNAc1-4Man1-3 Gal1-4GlcNAc1 2 MS/MS Man1-4GlcNAc1-4GlcNAc-PA Fuc1 6 1-4GlcNAc1-2Man1-6GlcNAc1-4Man1-3 GalGlcNAc1 2 Man1-4GlcNAc1-4GlcNAc-PA Fuc1 6 MS3 ( 1280)m/zMS3 ( 1280)m/z

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