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Thermally-initiated free radical polymerization for reproducible production of stable linear polyacrylamide coated capillaries and their application to proteomic analysis using capillary zone electrophoresis-mass spectrometry

机译：热引发的自由基聚合可重现生产稳定的线性聚丙烯酰胺涂层毛细管并将其应用于使用毛细管区带电泳质谱的蛋白质组学分析中

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Proteomic analysis using capillary zone electrophoresis (CZE) typically is performed with linear polyacrylamide (LPA) coated capillaries. These capillaries both minimize the adsorption of peptides and proteins to the inner wall of the capillary and decrease electroosmosis, which increases the separation capacity. LPA coating protocols were first reported by Hjerten in 1985. Conventional LPA production is based on the use of TEMED to catalyze the free-radical polymerization that couples acrylamide to a capillary wall that has been pretreated with γ-methacryloxypropyltrimethoxysilane. The treated capillary is filled with a mixture of monomer, TEMED, and ammonium persulfate; free radical polymerization forms the LPA coating. Over many years, we have observed significant variation in the electroosmotic properties of commercial LPA coated capillaries both along the capillary length and between lots. We believe this variation is due to differences in the time between initiation of the reaction and the filling of the capillary. Here, we report a simple method for the generation of very stable and reproducible coatings. In this protocol, the monomer mixture and an ammonium persulfate initiator are introduced into the capillary without TEMED initiator. The mixture is stable and does not begin polymerization at room temperature. The filled capillary is then heated in a water bath to initiate polymerization in a well-controlled manner. A mixture of four standard proteins was used to evaluate the coating performance. Compared with commercialized LPA capillaries, our LPA capillaries generate much better separation performance and superior protein peak shape in CZE analysis. We also analyzed an intact antibody (MW 150k) by CZE-MS with the new LPA capillary in triplicate runs. The intact antibody generated a Gaussian-shaped electrophoresis peak with 1.2% relative standard deviation in migration time and 8.5% in base peak intensity. An automated CZE-MS system was used to generate 97 successive separations of a BSA tryptic digest over a 145-hour period. Separation efficiency averaged over 100,000 theoretical plates across this period with no systematic variation. The LPA coating protocol had excellent batch-to-batch reproducibility with relative standard deviation in migration time < 7%, and in separation window < 1%.
机译：使用毛细管区带电泳（CZE）的蛋白质组学分析通常是使用线性聚丙烯酰胺（LPA）涂覆的毛细管进行的。这些毛细管既可将肽和蛋白质对毛细管内壁的吸附降至最低，又可减少电渗，从而增加了分离能力。 LPA涂层协议由Hjerten于1985年首次报道。常规的LPA生产基于使用TEMED催化自由基聚合，该聚合将丙烯酰胺偶联到已用γ-甲基丙烯酰氧基丙基三甲氧基硅烷进行预处理的毛细管壁上。处理过的毛细管中充满了单体，TEMED和过硫酸铵的混合物；自由基聚合形成LPA涂层。多年来，我们已经观察到商用LPA涂层毛细管的电渗特性沿毛细管长度和批次之间存在显着变化。我们认为，这种变化是由于反应开始和毛细管填充之间的时间不同所致。在这里，我们报告了一种生成非常稳定且可重现的涂层的简单方法。在该方案中，将单体混合物和过硫酸铵引发剂引入毛细管中，而没有TEMED引发剂。该混合物是稳定的，并且在室温下不会开始聚合。然后将填充的毛细管在水浴中加热，以良好控制的方式引发聚合。四种标准蛋白质的混合物用于评估涂层性能。与商业化的LPA毛细管相比，我们的LPA毛细管在CZE分析中具有更好的分离性能和出色的蛋白质峰形。我们还使用新的LPA毛细管通过CZE-MS分析了完整抗体（MW 150k），一式三份。完整的抗体产生了高斯型电泳峰，迁移时间的相对标准偏差为1.2％，碱基峰强度的为8.5％。自动化的CZE-MS系统用于在145小时内连续分离出97种BSA胰蛋白酶消化物。在此期间，分离效率平均超过100,000个理论塔板，没有系统变化。 LPA涂层方案具有极佳的批间重现性，迁移时间<7％和分离窗<1％的相对标准偏差。

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