Lin Shuang, Yun Dong, Qi Dawei, Deng Chunhui, Li Yan, Zhang Xiangmin
Department of Chemistry & Institute of Biomedical Sciences, Fudan University, Shanghai, China.
J Proteome Res. 2008 Mar;7(3):1297-307. doi: 10.1021/pr700586j. Epub 2008 Feb 8.
In this study, a novel microwave-assisted protein digestion method was developed using trypsin-immobilized magnetic nanoparticles (TIMNs). The magnetic nanoparticles worked as not only substrate for enzyme immobilization, but also excellent microwave irradiation absorber and, thus, improved the efficiency of microwave-assisted digestion greatly. Three standard proteins, bovine serum albumin (BSA), myoglobin, and cytochrome c, were used to optimize the conditions of this novel digestion method. With the optimized conditions, peptide fragments produced in very short time (only 15 s) could be identified successfully by MALDI-TOF-MS. When it was compared to the conventional in-solution digestion (12 h), equivalent or better digestion efficiency was observed. Even when protein quantity was as low as micrograms, this novel digestion method still could digest proteins successfully, while the same samples by conventional in-solution digestion failed. Moreover, with an external magnetic field, the enzyme could be removed easily and reused. It was verified that, after 4 replicate runs, the TIMNs still kept high activity. To further confirm the efficiency of this rapid digestion method for proteome analysis, it was applied to the protein extract of rat liver. Without any preparation and prefractionation processing, the entire proteome digested by TIMNs in 15 s went through LC-ESI-MS/MS direct analysis. The whole shotgun proteomic experiment was finished in only 1 h with the identification of 313 proteins ( p < 0.01). This new application of TIMNs in microwave-assisted protein digestion really opens a route for large-scale proteomic analysis.
在本研究中，开发了一种使用固定化胰蛋白酶磁性纳米颗粒（TIMNs）的新型微波辅助蛋白质消化方法。磁性纳米颗粒不仅作为酶固定化的底物，而且还是出色的微波辐射吸收剂，从而大大提高了微波辅助消化的效率。使用三种标准蛋白质，即牛血清白蛋白（BSA）、肌红蛋白和细胞色素c来优化这种新型消化方法的条件。在优化条件下，通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）能够成功鉴定在极短时间内（仅15秒）产生的肽片段。与传统的溶液内消化（12小时）相比，观察到了相当或更好的消化效率。即使蛋白质数量低至微克级，这种新型消化方法仍能成功消化蛋白质，而相同样品采用传统溶液内消化则失败。此外，借助外部磁场，酶可以轻松去除并重复使用。经证实，经过4次重复运行后，TIMNs仍保持高活性。为了进一步证实这种快速消化方法用于蛋白质组分析的效率，将其应用于大鼠肝脏的蛋白质提取物。无需任何制备和预分级处理，TIMNs在15秒内消化的整个蛋白质组直接进行液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）分析。整个鸟枪法蛋白质组学实验仅在1小时内完成，鉴定出313种蛋白质（p<0.01）。TIMNs在微波辅助蛋白质消化中的这种新应用确实为大规模蛋白质组分析开辟了一条途径。
