Characterization of IgG N-Glycans Employing a Microfluidic Chip that Integrates Glycan Cleavage, Sample Purification, LC Separation, and MS Detection
Characterization of IgG N-Glycans Employing a Microfluidic Chip that Integrates Glycan Cleavage, Sample Purification, LC Separation, and MS Detection
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DOI：
10.1021/ac901326u
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发表时间：
2009-11-01
影响因子：
7.4
通讯作者：
Killeen, Kevin
中科院分区：
文献类型：
--
作者：
Bynum, Maggie A.;Yin, Hongfeng;Killeen, Kevin
开发了一种新型的带有芯片上酶反应器的聚合物微流控装置，用于重组糖蛋白的表征。用PNGase F修饰的固体载体材料填充的酶反应器芯片与微流控多糖清理芯片和商用高效液相芯片相结合，进行糖蛋白脱糖基化、蛋白质去除、多糖捕获、多聚糖LC分离和纳米电喷雾进入飞行时间质谱仪(TOF-MS)系统。利用该集成芯片，样品制备和分析的时间从几个小时减少到不到10分钟。曾经繁琐和耗时的多糖分析工作流程现在被集成到高效液相芯片设备中。仅用100毫微克的单抗就可以进行糖链图谱分析。此外，一个单一的芯片被证明保持了活性，并对来自重组抗体的250多个重复的糖链图谱进行了等价的执行。
A novel polymeric microfluidic device with an on-chip enzyme reactor has been developed for the characterization of recombinant glycoproteins. The enzyme reactor chip packed with PNGase F-modified solid support material was combined with a microfluidic glycan cleanup chip and a commercially available HPLC-chip to perform glycoprotein deglycosylation, protein removal, glycan capture, glycan LC separation, and nanoelectrospray into a time-of-flight mass spectrometry (TOF-MS) system. With this integrated chip, the combined sample preparation and sample analysis time was reduced from multiple hours to less than 10 min. A once tedious and time-consuming glycan analysis workflow is now integrated into an HPLC-chip device. Glycan profiling analysis has been achieved with as little as 100 ng of monoclonal antibody. Furthermore, a single chip was shown to retain activity and perform equivalently for over 250 replicate glycan profiles from a recombinant antibody.