Enzymatic Digestion of Proteins in Solution

Abstract

Analysis of protein covalent structure is less complex and more accurate when performed on peptides derived from the larger protein. In contrast to acid-promoted total hydrolysis, peptides are typically generated by selective proteolysis, i.e., by specifically cleaving peptide bonds with endoproteases that have varying degrees of specificity. This unit presents a protocol that can be used to generate peptide fragments from intact, undenatured proteins. Fragments can be analyzed directly by mass spectrometry (MS) or, more often, are first separated by reversed-phase HPLC (RP-HPLC) and then analyzed by MS or automated sequencing. Most proteins are resistant to enzymatic proteolysis under nondenaturing conditions or are not soluble in aqueous solution. Digestion procedures performed in the presence of chaotropic agents and SDS are described, and support protocols provide instructions for preparing enzyme stocks and reducing and alkylating peptides prior to sequencing or HPLC analysis.

Secretome diversity and quantitative analysis of cellulolytic Aspergillus fumigatusZ5 in the presence of different carbon sources

“…After quantified by BCA method, 50 μg (RS), 22.1 μg (Av) and 13.5 μg (Gl) proteins were used for protein digestion and iTRAQ labeling. The protein digestion was performed following the method of Riviere et al [49]. iTRAQ labeling was carried out in accordance with Adav et al [50,51], and the peptide samples were labeled using the iTRAQ Reagent Multiplex Kit (Applied Bio systems, Foster City, CA) according to the manufacturer’s protocol.…”

Proteolysin, a Novel Highly Thermostable and Cosolvent-Compatible Protease from the Thermophilic Bacterium Coprothermobacter proteolyticus

“…Because of its thermostability, relaxed specificity, and resistance to routinely used protein denaturants and DTT, proteolysin is a candidate for proteomics studies when protein digestion under extreme conditions is required. Heat pretreatment and additives such as surfactants, organic solvents, and urea are commonly used to improve protein solubility and facilitate complete digestion for, e.g., peptide mapping (58,59). In conclusion, the convenient production in E. coli, the high thermostability, the broad pH range, and its high tolerance to surfactants and cosolvents make proteolysin an attractive candidate for proteolysis under demanding reaction conditions.…”

Integration of a membrane-based desalting step in a microfabricated disposable polymer injector for mass spectrometric protein analysis

“…Numerous protocols have been developed through years to recover proteins immobilized in a membrane for further characterization or assays, or to directly process them within the membrane [29]. The interaction between the proteins and the membrane has not been found to prevent further use or characterization of the proteins.…”