Using high-concentration trypsin-immobilized magnetic nanoparticles for rapid in situ protein digestion at elevated temperature
Using high-concentration trypsin-immobilized magnetic nanoparticles for rapid in situ protein digestion at elevated temperature
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DOI：
10.1002/rcm.3191
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发表时间：
2007-01-01
影响因子：
2
通讯作者：
Shiea, Jentaie
中科院分区：
文献类型：
--
作者：
Jeng, Jingyueh;Lin, Mon-Fang;Shiea, Jentaie
我们描述了一种创新的方法-使用高浓度的胰蛋白酶修饰的磁性纳米颗粒（TMNP）-在高温下快速有效地消化蛋白质。所需的消化时间可以减少到小于10秒。消化后，从样品溶液中磁性收集TMNP用于重复使用，并使用基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱法表征消化的肽。当在57 ℃下使用TMNP（5 μ g/μ L）时，优化蛋白质消化;对于蛋白质鉴定实现了显著高的肽覆盖（例如，98%的溶菌酶）。虽然高浓度的TMNP用于消化，但短的消化时间导致通过自消化产生的胰蛋白酶肽的量低得多。结果，肽离子的质谱检测中的干扰显著降低。版权所有（c）2007约翰威利父子有限公司。
We describe an innovative approach - using a high concentration of trypsin-modified magnetic nanoparticles (TMNPs) - for the rapid and efficient digestion of proteins at elevated temperature. The required digestion time could be reduced to less than 10 s. After digestion, the TMNPs were collected magnetically from the sample solution for reuse and the digested peptides were characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Protein digestion was optimized when using the TMNPs (5 mu g/mu L) at 57 degrees C; a significantly high peptide coverage was achieved for protein identification (e.g., 98% for lysozyme). Although a high concentration of TMNPs was used for digestion, the short digestion time led to much lower amounts of trypsin peptides being produced through self-digestion. As a result, interference in the mass spectrometric detection of the peptide ions was reduced significantly. Copyright (c) 2007 John Wiley & Sons, Ltd.