Immobilization of alpha-chymotrypsin to magnetic particles and their use for proteolytic cleavage of porcine pepsin A

Abstract

Alpha-Chymotrypsin was immobilized to CHO activated commercial magnetic particles via the protein free amino groups. Immobilized protease was used to study the phosphorylation degree of porcine pepsin A, as a model acidic protein and phosphoprotein.

The proteolytic digest obtained using the prepared immobilized alpha-chymotrypsin was separated using RP-HPLC and immobilized metal affinity chromatography (IMAC) with Fe(III) ions prior to MALDI-TOF analysis: the presence of phosphopeptide in porcine pepsin A was shown.

Keywords

• Metal-affinity-chromatography
• protein digestion
• saccharomyces-cerevisiae
• phosphoproteome analysis
• covalent immobilization
• mass-spectrometry
• trypsin
• stabilization
• phosphorylation
• reactor