Zhao Cheng, Jiang Honghai, Smith Douglas R, Bruckenstein Stanley, Wood Troy D
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260-3000, USA.
Anal Biochem. 2006 Dec 15;359(2):167-75. doi: 10.1016/j.ab.2006.09.005. Epub 2006 Oct 5.
A method for integrating nanoelectrospray mass spectrometry with a microreactor for on-line digestion and fast peptide mass mapping from dilute protein samples is presented. Fused silica capillaries (i.d. 50 microm, o.d. 360 microm) are employed as the digestion microreactor and the nanoelectrospray emitter by immobilizing trypsin onto the surface of the inner wall of the fused silica capillary tubing. The procedure is demonstrated using solutions of 1pmol/mul angiotensin II, cytochrome c, hemoglobin, and beta-casein. Because the inner walls of the capillaries are modified by covalent chemical bonds, the adsorption of peptides and proteins to the inner walls of the capillaries is suppressed. This procedure was performed with solutions as dilute as 1fmol/mul (1nM) cytochrome c. This method shows generation of tryptic peptides with sequence coverage up to 90% within minutes; trypsin autolysis products are not detected. In addition, the immobilized enzyme can be cleaned easily, enabling the microreactor to be reused for nanoelectrospray.
本文介绍了一种将纳米电喷雾质谱与微反应器相结合的方法，用于对稀释蛋白质样品进行在线消化和快速肽质量图谱分析。通过将胰蛋白酶固定在熔融石英毛细管内壁表面，使用内径50微米、外径360微米的熔融石英毛细管作为消化微反应器和纳米电喷雾发射器。使用1皮摩尔/微升的血管紧张素II、细胞色素c、血红蛋白和β-酪蛋白溶液对该方法进行了验证。由于毛细管内壁通过共价化学键进行了修饰，抑制了肽和蛋白质在毛细管内壁的吸附。该方法在低至1飞摩尔/微升（1纳摩尔）的细胞色素c溶液中也能进行。该方法可在数分钟内产生序列覆盖率高达90%的胰蛋白酶肽；未检测到胰蛋白酶自溶产物。此外，固定化酶易于清洗，使微反应器能够重复用于纳米电喷雾。
