Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
Anal Chem. 2017 Jun 20;89(12):6774-6780. doi: 10.1021/acs.analchem.7b01147. Epub 2017 Jun 7.
A surface-confined aqueous reversible addition-fragmentation chain transfer (SCARAFT) polymerization method was developed to coat capillaries for use in capillary zone electrophoresis (CZE). SCARAFT polymerization primarily takes place on the inner surface of the capillary instead of in solution, which greatly improves the homogeneity of the coating. Capillaries treated with this coating produced an electroosmotic mobility of 2.8 ± 0.2 × 10 cm·V·s (N = 3), which is roughly an order of magnitude lower than that of commercial linear polyacrylamide (LPA)-coated capillaries. Coated capillaries were evaluated for bottom-up proteomic analysis using CZE. The very low electroosmotic mobility results in a 200 min separation and improved single-shot analysis. An average of 977 protein groups and 5605 unique peptides were identified from 50 ng of an E. coli digest, and 2158 protein groups and 10 005 peptides were identified from 25 ng of a HeLa digest using single-shot analysis with a SCARAFT-acrylamide capillary coupled to a Q Exactive HF mass spectrometer. The coating is stable. A single capillary was used for over 200 h (8.4 days) of continuous operation. RSD in migration time was between 2 and 3% for selected ion electropherograms (SIEs) generated for six ions; median theoretical plate counts ranged from 240 000 to 600 000 for these SIEs. Various types of coatings could be prepared by simply changing the functional vinyl monomers in the polymerization mixture. Positively charged coatings using direct attachment and formation of a block copolymer were prepared and demonstrated for the separation of mixtures of intact proteins.
一种表面受限的水溶液可逆加成-断裂链转移（SCARAFT）聚合方法被开发用于涂覆用于毛细管区带电泳（CZE）的毛细管。SCARAFT 聚合主要发生在毛细管的内表面上，而不是在溶液中，这极大地提高了涂层的均一性。用这种涂层处理过的毛细管产生的电渗流迁移率为 2.8 ± 0.2 × 10 cm·V·s（N = 3），大致是商业线性聚丙烯酰胺（LPA）涂层毛细管的一个数量级。用 CZE 对涂覆的毛细管进行了自上而下的蛋白质组分析评估。非常低的电渗流迁移率导致 200 分钟的分离和改善的单次分析。从 50ng 的大肠杆菌消化物中鉴定出 977 个蛋白质组和 5605 个独特肽段，从 25ng 的 HeLa 消化物中鉴定出 2158 个蛋白质组和 10005 个肽段，使用单次分析，SCARAFT-丙烯酰胺毛细管与 Q Exactive HF 质谱仪相连。该涂层稳定。一根毛细管可连续运行超过 200 小时（8.4 天）。对于为六个离子生成的选择离子电泳图（SIE），迁移时间的 RSD 在 2%到 3%之间；对于这些 SIE，理论板数中位数范围从 240000 到 600000。通过简单地改变聚合混合物中的功能乙烯基单体，可以制备各种类型的涂层。使用直接附着和形成嵌段共聚物制备了带正电荷的涂层，并用于分离完整蛋白质混合物。
