Tempels F W Alexander, Underberg Willy J M, Somsen Govert W, de Jong Gerhardus J
Division of Biomedical Analysis, Department of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Electrophoresis. 2007 May;28(9):1319-26. doi: 10.1002/elps.200600403.
An on-line SPE-CE-MS system has been developed for the analysis of peptides. Analytes are preconcentrated using a C(18) microcolumn (5 x 0.5 mm id), and then introduced into the CE system via a valve interface. The CE system with a Polybrene-poly(vinylsulfonate) bilayer coated capillary is combined with an ion-trap mass spectrometer via ESI using a coaxial sheath-liquid sprayer. The on-line coupling of the SPE and CE step by the valve interface is advantageous because it allows an independent functioning of the system parts. Optimization of the SPE-CE system was performed using UV detection. Subsequently, the SPE-CE system has been coupled to the ion-trap mass spectrometer. Test solutions with enkephalin peptides (50 ng/mL) were used for evaluation of system performance. Repeatability of effective mobility and peak area ratio of the two enkephalins were within 1.2% and 9% RSD, respectively. The analysis of 1:1 v/v diluted cerebrospinal fluid samples spiked with enkephalin peptides showed detection limits (S/N = 3) in the range of 1.5-3 ng/mL (around 5 nM), which were similar to those obtained for enkephalin test solutions. Moreover, the potential of the on-line SPE-CE-MS system was demonstrated by the analysis of a cytochrome C digest. Some hydrophilic peptides did not show sufficient retention on the SPE column, and were lost during preconcentration. Nonetheless, positive identification of the protein was achieved, indicating the feasibility of the system for proteomics.
已开发出一种用于肽分析的在线固相萃取-毛细管电泳-质谱联用系统。使用C(18)微柱（内径5×0.5 mm）对分析物进行预浓缩，然后通过阀接口将其引入毛细管电泳系统。带有聚凝胺-聚（乙烯磺酸盐）双层涂层毛细管的毛细管电泳系统通过同轴鞘液喷雾器经电喷雾电离与离子阱质谱仪联用。通过阀接口在线连接固相萃取和毛细管电泳步骤具有优势，因为它允许系统各部分独立运行。使用紫外检测对固相萃取-毛细管电泳系统进行了优化。随后，将固相萃取-毛细管电泳系统与离子阱质谱仪联用。使用含脑啡肽肽（50 ng/mL）的测试溶液评估系统性能。两种脑啡肽的有效迁移率和峰面积比的重复性分别在相对标准偏差1.2%和9%以内。对添加了脑啡肽肽的1:1 v/v稀释脑脊液样品的分析显示，检测限（信噪比 = 3）在1.5 - 3 ng/mL（约5 nM）范围内，这与脑啡肽测试溶液获得的结果相似。此外，通过对细胞色素C消化产物的分析证明了在线固相萃取-毛细管电泳-质谱联用系统的潜力。一些亲水性肽在固相萃取柱上没有足够的保留，在预浓缩过程中损失了。尽管如此，仍实现了蛋白质的阳性鉴定，表明该系统在蛋白质组学方面的可行性。
