Chen Wei-Yu, Chen Yu-Chie
Department of Applied Chemistry and Institute of Molecular Science, National Chiao Tung University, Hsinchu 300, Taiwan.
Anal Chem. 2007 Mar 15;79(6):2394-401. doi: 10.1021/ac0614893. Epub 2007 Feb 7.
In this study, we demonstrated that microwave-assisted enzymatic digestion could be greatly accelerated by multifunctional magnetite beads. The acceleration of microwave-assisted enzymatic digestion by the presence of the magnetite beads was attributable to several features of the beads. Their capacity to absorb microwave radiation leads to rapid heating of the beads. Furthermore, their negatively charged functionalities cause adsorption of proteins with opposite charges onto their surfaces by electrostatic interactions, leading to a concentration on the surfaces of the beads of proteins present in trace amounts in the solution. The adsorbed proteins are denatured and hence rendered vulnerable to enzymatic digestion and are digested on the beads. For microwave heating, 30 s was sufficient for carrying out the tryptic digestion of cytochrome c, in the presence of magnetite beads, while 1 min was adequate for tryptic digestion of myoglobin. The digestion products were characterized by MALDI-MS. This rapid enzymatic digestion allowed the entire time for identification of proteins to be greatly reduced. Furthermore, specific proteins present in trace quantities were enriched from the sample on the magnetite beads and could be rapidly isolated from the sample by employing an external magnetic field. These multiple roles of magnetite beads, as the absorber for microwave irradiation, the concentrating probe, and the agent for unfolding proteins, contributed to their capability of accelerating microwave-assisted enzymatic digestion. We also demonstrated that trypsin immobilized magnetite beads were suitable for use in microwave-assisted enzymatic digestion.
在本研究中，我们证明了多功能磁铁矿珠可极大地加速微波辅助酶解。磁铁矿珠的存在对微波辅助酶解的加速作用归因于其几个特性。它们吸收微波辐射的能力导致珠子快速升温。此外，其带负电的官能团通过静电相互作用使带相反电荷的蛋白质吸附在其表面，从而使溶液中痕量存在的蛋白质在珠子表面富集。吸附的蛋白质会变性，因此易于酶解，并在珠子上被消化。对于微波加热，在存在磁铁矿珠的情况下，30秒就足以对细胞色素c进行胰蛋白酶消化，而1分钟则足以对肌红蛋白进行胰蛋白酶消化。消化产物通过基质辅助激光解吸电离质谱（MALDI-MS）进行表征。这种快速酶解使蛋白质鉴定的整个时间大大缩短。此外，痕量存在的特定蛋白质在磁铁矿珠上从样品中得到富集，并可通过施加外部磁场从样品中快速分离出来。磁铁矿珠作为微波辐射的吸收体、富集探针和蛋白质解折叠剂的这些多重作用，促成了它们加速微波辅助酶解的能力。我们还证明了固定化胰蛋白酶的磁铁矿珠适用于微波辅助酶解。
