Fast and efficient proteolysis by reusable pepsin-encapsulated magnetic sol-gel material for mass spectrometry-based proteomics applications

作者：Kayili H Mehmet; Salih Bekir^*

来源：Talanta, 2016, 155: 78-86.

DOI：10.1016/j.talanta.2016.04.014

• Sol-gel
• Encapsulation
• Pepsin
• Proteolysis
• Mass spectrometry
• Proteomics

Hydrophobic silicon-based material having magnetic properties was fairly synthesized by a classical sol-gel approach. Pepsin enzyme was encapsulated in the sol-gel material and the enzyme activity was evaluated in consequence of the digestion of some common proteins such as alpha- and beta-casein, cytochrome c, myoglobin, and bovine serum albumin (BSA) both in a single protein batch and in the protein mixture. The optimum digestion time of the studied proteins using pepsin-encapsulated magnetic sol-gel material was found to be 20 min. To produce the magnetic sol-gel material for convenient and easy proteomics applications, Fe3O4 was doped inside sol-gel material during the gelation step. It was observed that the activity of encapsulated pepsin was not affected by the amount of Fe3O4. Poly(ethylene glycol) was also inserted in sol-gel bulk to obtain suitable roughness and increase the hydrophilicity of the material surface to let protein molecules reach to the sol-gel material easily. The digestion of the protein mixture and non-fat bovine milk was performed with the pepsin-encapsulated magnetic sol-gel material and the digested solutions were analyzed using SDS-PAGE, MALDI-TOF-MS and LC-MS/MS for the protein identification. Reusability of the pepsin-encapsulated sol-gel material was examined and it was determined that they could be used at least 20 times. Finally, IgG digestions with a fast incubation time period were carried out using pepsin-encapsulated sol-gel material for generation of (Fab)(2) product to evaluate the kinetic performance of the material.

• 出版日期2016-8-1