Immobilized endoproteinase Glu-C to magnetic bead cellulose as a tool in proteomic analysis

• Published in: Journal of separation science Vol. 36; no. 12; pp. 2043 - 2048
• Main Authors: Prikryl, Petr, Ticha, Marie, Kucerova, Zdenka
• Format: Journal Article
• Language: English
• Published: Weinheim Blackwell Publishing Ltd 01.06.2013; Wiley; Wiley Subscription Services, Inc
• Subjects: Analytical, structural and metabolic biochemistry; Bacterial Proteins - chemistry; Beads; Biological and medical sciences; BSA; Buffers; Caseins - chemistry; Cellulose; Cellulose - chemistry; Chemical reactions; Cleavage; Enzyme immobilization; Enzyme Stability; Enzymes; Enzymes, Immobilized - chemistry; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Glu-C; Hydrogen-Ion Concentration; Immobilized endoproteinase; Magnetic bead cellulose; Magnetics; Proteinase; Proteins; Proteomics; Proteomics - instrumentation; Proteomics - methods; Serine Endopeptidases - chemistry; Serum Albumin, Bovine - chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Staphylococcus aureus; Staphylococcus aureus - enzymology; α-Casein; Ball; Peptides; a-Casein; Matrix assisted laser desorption ionization; Thermal stability; Glutamyl endopeptidase; BSA; Milk protein; Casein; Phosphoproteins; Proteolysis; Enzyme immobilization; Whey protein; Glu-C; Magnetic bead cellulose; Serine endopeptidases; Enzyme; Immobilized endoproteinase; Serum albumin; Serum protein; Peptidases; Magnetic particles; Analysis method; Peptide fragment; Time of flight method; Storage stability; Cellulose derivatives; Proteomics; Hydrolases; Immobilized enzyme; Mass spectrometry
• ISSN: 1615-9306; 1615-9314
• DOI: 10.1002/jssc.201300118

Magnetic bead cellulose activated with divinyl sulfone was used for the immobilization of Staphylococcus aureus endoproteinase Glu‐C (EC 3.4.21.19). The immobilized proteinase was characterized by increased thermostability, by decreased self‐cleavage activity, and a possibility of repeated use. The prepared immobilized enzyme was applied for the proteolytic cleavage of α‐casein and BSA under different conditions (different composition of buffers, different pH, and different time of digestion). The possibilities of the direct use of enzyme reaction products for MALDI TOF MS analysis were shown.