Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599, United States.
Anal Chem. 2013 Apr 16;85(8):4100-6. doi: 10.1021/ac400205a. Epub 2013 Mar 26.
A hybrid multidimensional separation system was made by coupling capillary liquid chromatography (LC) to a microfluidic device. The microfluidic device integrated flow splitting, capillary electrophoresis (CE), electroosmotic pumping, and electrospray ionization (ESI) emitter functional elements. The system was used with a time-of-flight mass spectrometer for comprehensive online LC-CE-MS of proteolytic digests. Analysis of a complex mixture of peptides yielded a peak capacity of approximately 1400 in 50 min. Three replicate runs demonstrated mean reproducibility for LC retention and CE migration times of 0.32% and 0.75% relative standard deviation (RSD), respectively. The same LC-CE-MS method was also used to characterize the N-linked glycosylation of a monoclonal antibody. Glycopeptides from two different N-linked glycosylation sites were separated from all other tryptic peptides and identified using MS data. The relative amounts of each glycoform and total site occupancy were quantified using LC-CE-MS data.
通过将毛细管液相色谱 (LC) 与微流控装置耦合，构建了一种混合多维分离系统。该微流控装置集成了分流、毛细管电泳 (CE)、电动泵送和电喷雾电离 (ESI) 发射器功能元件。该系统与飞行时间质谱仪联用，实现了对蛋白酶解物的全面在线 LC-CE-MS 分析。对复杂的肽混合物进行分析，在 50 分钟内得到了约 1400 的峰容量。三次重复运行表明，LC 保留时间和 CE 迁移时间的平均重现性分别为 0.32%和 0.75%的相对标准偏差 (RSD)。同样的 LC-CE-MS 方法也用于表征单克隆抗体的 N 连接糖基化。来自两个不同 N 连接糖基化位点的糖肽与所有其他胰蛋白酶肽分离，并使用 MS 数据进行鉴定。使用 LC-CE-MS 数据定量每种糖型和总位点占有率的相对量。
