Jeng Jingyueh, Lin Mon-Fang, Cheng Fong-Yu, Yeh Chen-Sheng, Shiea Jentaie
Department of Biotechnology, Chia Nan University of Pharmacy & Science, 60, Erh-Jen RD., Sec.1, Jen-Te, Tainan 717, Taiwan.
Rapid Commun Mass Spectrom. 2007;21(18):3060-8. doi: 10.1002/rcm.3191.
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 microg/microL) 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.
我们描述了一种创新方法——使用高浓度的胰蛋白酶修饰磁性纳米颗粒（TMNP）——在高温下快速高效地消化蛋白质。所需的消化时间可缩短至不到10秒。消化后，通过磁性从样品溶液中收集TMNP以便重复使用，并使用基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱对消化后的肽进行表征。使用TMNP（5微克/微升）在57摄氏度下进行蛋白质消化时得到了优化；在蛋白质鉴定方面实现了显著高的肽覆盖率（例如，溶菌酶的覆盖率为98%）。尽管使用了高浓度的TMNP进行消化，但短的消化时间导致通过自消化产生的胰蛋白酶肽的量要低得多。结果，肽离子质谱检测中的干扰显著降低。
