title: Fast and efficient proteolysis by reusable pepsin-encapsulated magnetic sol-gel material for mass spectrometry-based proteomics applications.
abstract: 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 α- and β-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 20min. 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.
authors.affiliation: Hacettepe University, Department of Chemistry, 06800 Ankara, Turkey; Çankırı Karatekin University, Department of Chemistry, 18100 Çankırı, Turkey.
authors.forename: H Mehmet
authors.initials: HM
authors.lastname: Kayili
authors.affiliation: Hacettepe University, Department of Chemistry, 06800 Ankara, Turkey. Electronic address: bekirsal@gmail.com.
authors.forename: Bekir
authors.initials: B
authors.lastname: Salih
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002214:Capsules; D002417:Cattle; D005782:Gels; D058185:Magnetite Nanoparticles; D013058:Mass Spectrometry; D010434:Pepsin A / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D013997:Time Factors
chemical_list: D002214:Capsules; D005782:Gels; D058185:Magnetite Nanoparticles; D011092:Polyethylene Glycols; D010434:Pepsin A
keywords: Encapsulation; Mass spectrometry; Pepsin; Proteolysis; Proteomics; Sol-gel