Rapid and simple solid-phase esterification of sialic acid residues for quantitative glycomics by mass spectrometry
Rapid and simple solid-phase esterification of sialic acid residues for quantitative glycomics by mass spectrometry
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DOI：
10.1002/chem.200601872
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发表时间：
2007-01-01
影响因子：
4.3
通讯作者：
Nishimurara, Shin-ichiro
中科院分区：
文献类型：
--
作者：
Miura, Yoshiaki;Shinohara, Yasuro;Nishimurara, Shin-ichiro
建立了一种快速、定量地测定各种唾液化低聚糖羧基固相甲基酯化反应的方法。该方法采用三氮烯衍生物，3-甲基-1-对甲苯三氮烯，在多孔板中对固定在一般固体载体（如Affi-Gel Hz和金胶体纳米颗粒）上的低聚糖进行简易衍生化。优化了通过化学结扎从粗样品混合物中分离出的唾液化/非唾液化低聚糖的固相处理工作流程。从胰蛋白酶和/或PNGase f -消化的糖蛋白混合物中，通过化学选择固定纯化、酯化和回收，当与“糖块技术”（SA）结合使用时，在滤板的同一孔中，在3小时内完成。Nishimura， K. Niikura， M. Kurogochi, T Matsushita， M. Fumoto， H. Hinou， R. Kamitani， H. Nakagawa， K. Deguchi， N. Miura， K. Monde， H. Kondo，高通量蛋白糖组学：化学选择性糖blotting和MALDI-TOF/TOF质谱的联合使用化学，2005,17,93-98；Angew。化学。Int。编辑。2005,44,9196)。回收的材料可直接用于随后的质谱技术表征，如MALDI-TOF，用于中性和唾液化低聚糖的大规模糖谱分析。含唾液酸的糖聚糖的纳米珠/金衍生化使得MALDI-TOF质谱在反射和正离子模式下快速定量分析糖型。除了简单和快速之外，该方法还消除了氯仿和二氯甲烷等不利的卤化溶剂或乙醚和己烷等挥发性溶剂的使用，从而为自动化机器人适应提供了实用和绿色的化学方法。
A rapid and quantitative method for solid-phase methyl esterification of carboxy groups of various sialylated oligosaccharides has been established. The method employed a triazene derivative, 3-methyl-1-p-tolyltriazene, for facile derivatization of oligosaccharides immobilized onto general solid supports such as Affi-Gel Hz and gold colloidal nanoparticles in a multi-well plate. The workflow protocol was optimized for the solid-phase processing of captured sialylated/unsialylated oligosaccharides separated from crude sample mixtures by chemical ligation. From tryptic and/or PNGase F-digest mixtures of glycoproteins, purification by chemoselective immobilization, esterification and recovery were achieved in the same well of the filter plate within three hours when used in conjunction with "glycoblotting technology" (SA. Nishimura, K. Niikura, M. Kurogochi, T Matsushita, M. Fumoto, H. Hinou, R. Kamitani, H. Nakagawa, K. Deguchi, N. Miura, K. Monde, H. Kondo, High-throughput protein glycomics: Combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry: Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 9196). The recovered materials were directly applicable to subsequent characterization by mass spectrometric techniques such as MALDI-TOF for large-scale glycomics of both neutral and sialylated oligosaccharides. On-bead/on-gold nanoparticle derivatization of glycans containing sialic acids allowed rapid and quantitative glycoform profiling by MALDI-TOF MS with reflector and positive ion mode. In addition to its simplicity and speed, the method eliminates the use of unfavorable halogenated solvents such as chloroform and dichloromethane or volatile solvents such as diethyl ether and hexane, resulting in a practical and green chemical method for automated robotic adaptation.