title: Selective isolation of hydrophobin SC3 by solid-phase extraction with polytetrafluoroethylene microparticles and subsequent mass spectrometric analysis.
abstract: Hydrophobins are small proteins that play a role in a number of processes during the filamentous fungi growth and development. These proteins are characterized by the self-assembly of their molecules into an amphipathic membrane at hydrophilic-hydrophobic interfaces. Isolation and purification of hydrophobins generally present a challenge in their analysis. Hydrophobin SC3 from Schizophyllum commune was selected as a representative of class I hydrophobins in this work. A novel procedure for selective and effective isolation of hydrophobin SC3 based on solid-phase extraction with polytetrafluoroethylene microparticles loaded in a small self-made microcolumn is reported. The tailored binding of hydrophobins to polytetrafluoroethylene followed by harsh elution conditions resulted in a highly specific isolation of hydrophobin SC3 from the model mixture of ten proteins. The presented isolation protocol can have a positive impact on the analysis and utilization of these proteins including all class I hydrophobins. Hydrophobin SC3 was further subjected to reduction of its highly stable disulfide bonds and to chymotryptic digestion followed by mass spectrometric analysis. The isolation and digestion protocols presented in this work make the analysis of these highly hydrophobic and compact proteins possible.
authors.affiliation: Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Kupčík
authors.affiliation: Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic.
authors.forename: Miroslava
authors.initials: M
authors.lastname: Zelená
authors.affiliation: Department of Molecular Pathology and Biology, Faculty of Military Health Sciences, University of Defence, Hradec Králové, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Řehulka
authors.affiliation: Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Bílková
authors.affiliation: Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Česlová
mesh_terms: D000418:Albumins / Q000737:chemistry; D031230:Ananas / Q000737:chemistry; D000818:Animals; D001963:Bromelains / Q000737:chemistry; D032449:Canavalia / Q000737:chemistry; D002256:Carbonic Anhydrases / Q000737:chemistry; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D002645:Chickens; D002918:Chymotrypsin / Q000737:chemistry; D003208:Concanavalin A / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004912:Erythrocytes / Q000201:enzymology; D006736:Horses; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008863:Microspheres; D008892:Milk / Q000201:enzymology; D009206:Myocardium / Q000378:metabolism; D011138:Polytetrafluoroethylene / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D012567:Schizophyllum / Q000737:chemistry*; D052616:Solid Phase Extraction / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D013820:Thermolysin / Q000737:chemistry
chemical_list: D000418:Albumins; D002364:Caseins; D004220:Disulfides; D003208:Concanavalin A; D001963:Bromelains; D011138:Polytetrafluoroethylene; D045304:Cytochromes c; D002918:Chymotrypsin; D013820:Thermolysin; D002256:Carbonic Anhydrases
keywords: Hydrophobin SC3; Isolation; Mass spectrometry; Polytetrafluoroethylene microparticles; Protein analysis