Li Yan, Yan Bo, Xu Xiuqing, Deng Chunhui, Yang Pengyuan, Shen Xizhong, Zhang Xiangmin
Department of Chemistry, Fudan University, Shanghai 200433, China.
Rapid Commun Mass Spectrom. 2007;21(14):2263-8. doi: 10.1002/rcm.3073.
Peptide mapping analysis, utilizing an easily replaceable and regenerable on-column enzymatic microreactor with metal-ion-chelated adsorption of enzyme on magnetic silica microspheres, combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was developed. Firstly, magnetic microspheres of small size and strong magnetism were prepared through solvothermal reaction. Thereafter, by introducing tetraethyl orthosilicate (TEOS), magnetic silica (MS) microspheres were formed. Trypsin could then be immobilized onto the MS microspheres based on the Lewis acid-base interaction through the divalent cation chelators such as iminodiacetic acid (IDA), which was chemically bound to the microspheres through the introduction of glycidoxypropyltrimethoxysilane (GLYMO). The trypsin-immobilized MS microspheres were then locally packed into the capillary by the application of a strong magnetic field using a magnet. The performance of the method was exemplified with digestion of bovine serum albumin for 5 min at 50 degrees C and the result was comparable to the 12 h in-solution digestion. The ability of regeneration of the prepared on-column microreactor and good reproducibility of microreactor before and after regeneration were also demonstrated.
开发了一种肽图谱分析方法，该方法利用一种易于更换和再生的柱上酶微反应器，通过金属离子螯合将酶吸附在磁性二氧化硅微球上，并结合基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）。首先，通过溶剂热反应制备了小尺寸且强磁性的磁性微球。此后，通过引入正硅酸乙酯（TEOS），形成了磁性二氧化硅（MS）微球。然后，胰蛋白酶可以基于路易斯酸碱相互作用，通过二价阳离子螯合剂如亚氨基二乙酸（IDA）固定在MS微球上，IDA通过引入环氧丙氧基丙基三甲氧基硅烷（GLYMO）与微球化学结合。然后使用磁铁通过施加强磁场将固定有胰蛋白酶的MS微球局部填充到毛细管中。该方法的性能通过在50℃下对牛血清白蛋白进行5分钟的消化得到例证，结果与12小时的溶液内消化相当。还证明了所制备的柱上微反应器的再生能力以及再生前后微反应器的良好重现性。
