Ivanov Yuri D, Pleshakova Tatyana, Malsagova Krystina, Kozlov Andrey, Kaysheva Anna, Kopylov Arthur, Izotov Alexander, Andreeva Elena, Kanashenko Sergey, Usanov Sergey, Archakov Alexander
Institute of Biomedical Chemistry RAMS, Moscow, Russia.
FEBS J. 2014 Oct;281(20):4705-17. doi: 10.1111/febs.13011. Epub 2014 Oct 4.
An approach combining atomic force microscopy (AFM) fishing and mass spectrometry (MS) analysis to detect proteins at ultra-low concentrations is proposed. Fishing out protein molecules onto a highly oriented pyrolytic graphite surface coated with polytetrafluoroethylene film was carried out with and without application of an external electric field. After that they were visualized by AFM and identified by MS. It was found that injection of solution leads to charge generation in the solution, and an electric potential within the measuring cell is induced. It was demonstrated that without an external electric field in the rapid injection input of diluted protein solution the fishing is efficient, as opposed to slow fluid input. The high sensitivity of this method was demonstrated by detection of human serum albumin and human cytochrome b5 in 10(-17) -10(-18) m water solutions. It was shown that an external negative voltage applied to highly oriented pyrolytic graphite hinders the protein fishing. The efficiency of fishing with an external positive voltage was similar to that obtained without applying any voltage.
提出了一种结合原子力显微镜（AFM）钓捕和质谱（MS）分析来检测超低浓度蛋白质的方法。在有和没有施加外部电场的情况下，将蛋白质分子钓捕到涂有聚四氟乙烯薄膜的高度定向热解石墨表面上。之后，通过原子力显微镜对它们进行可视化，并通过质谱进行鉴定。发现溶液注入会导致溶液中产生电荷，并在测量池中感应出电势。结果表明，与缓慢的流体输入相反，在稀释蛋白质溶液的快速注入输入中，没有外部电场时钓捕是有效的。通过检测10^(-17)-10^(-18) m水溶液中的人血清白蛋白和人细胞色素b5，证明了该方法的高灵敏度。结果表明，施加到高度定向热解石墨上的外部负电压会阻碍蛋白质钓捕。施加外部正电压时的钓捕效率与不施加任何电压时获得的效率相似。
