Slovakova Marcela, Minc Nicolas, Bilkova Zuzana, Smadja Claire, Faigle Wolfgang, Fütterer Claus, Taverna Myriam, Viovy Jean-Louis
Laboratoire Physicochimie-Curie, UMR/CNRS 168, Institut Curie, 75248 Paris Cedex 5, France.
Lab Chip. 2005 Sep;5(9):935-42. doi: 10.1039/b504861c. Epub 2005 Jul 22.
The use of grafted trypsin magnetic beads in a microchip for performing protein digestion is described. The PDMS device uses strong magnets to create a magnetic field parallel to the flow with a strong gradient pointing through the center of the chip channel. This allows for the formation of a low-hydrodynamic resistance plug of magnetic trypsin beads that serves as a matrix for protein digestion. This device represents an inexpensive way of fabricating a multi open-tubular-like column with an appropriate pore size for proteins. Kinetics studies of the hydrolysis of a model peptide show a 100-fold increase in digestion speed obtained by the microsystem when compared to a batch wise system. This system also offers the great advantage of easy replacement, as the bead matrix is easily washed out and replaced. High performance and reproducibility for digesting recombinant human growth hormone are confirmed by analysing the digest products in both CE and MALDI-TOF MS. Similar sequence coverage (of about 44%) is obtained from MS analysis of products after 10 minutes on-chip and 4 h with soluble trypsin in bulk.
本文描述了在微芯片中使用接枝胰蛋白酶磁珠进行蛋白质消化的方法。聚二甲基硅氧烷（PDMS）装置利用强磁体产生与流动方向平行的磁场，且磁场梯度较强，指向芯片通道中心。这使得能够形成低流体动力学阻力的磁性胰蛋白酶珠塞，作为蛋白质消化的基质。该装置代表了一种廉价的制造具有适合蛋白质的适当孔径的多开口管状柱的方法。对模型肽水解的动力学研究表明，与分批系统相比，微系统获得的消化速度提高了100倍。该系统还具有易于更换的巨大优势，因为珠状基质很容易冲洗掉并更换。通过在毛细管电泳（CE）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）中分析消化产物，证实了该系统在消化重组人生长激素方面具有高性能和可重复性。在芯片上消化10分钟后和使用可溶性胰蛋白酶在批量中消化4小时后，通过质谱分析获得了相似的序列覆盖率（约44%）。
