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Updated: Jul 5, 2026
09:49
Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor
Published on: April 6, 2016
Enzymatic digestion of proteins in solution
1OsteoArthritis Sciences, Inc., Cambridge, Massachusetts, USA.
Current Protocols in Protein Science
|April 23, 2008
Summary
This study details a protocol for generating peptide fragments from intact proteins using selective proteolysis. These peptide fragments are crucial for accurate protein structure analysis via mass spectrometry or sequencing.
Area of Science:
- Biochemistry
- Proteomics
- Analytical Chemistry
Background:
- Protein structure analysis is more accurate at the peptide level.
- Selective proteolysis using endoproteases is a common method for peptide generation.
- Many proteins are resistant to enzymatic digestion under nondenaturing conditions.
Purpose of the Study:
- To present a protocol for generating peptide fragments from intact, undenatured proteins.
- To enable direct analysis of fragments by mass spectrometry (MS) or after separation by reversed-phase High-Performance Liquid Chromatography (RP-HPLC).
- To provide methods for handling proteins resistant to digestion or insoluble in aqueous solutions.
Main Methods:
- Selective proteolysis of intact, undenatured proteins to generate peptide fragments.
- Digestion procedures in the presence of chaotropic agents and SDS for resistant proteins.
- Preparation of enzyme stocks, and reduction and alkylation of peptides.
Main Results:
- Generation of peptide fragments suitable for downstream analysis.
- Successful digestion of proteins resistant to proteolysis or with low aqueous solubility.
- Peptide fragments can be analyzed directly by MS or after RP-HPLC separation and automated sequencing.
Conclusions:
- The presented protocol facilitates accurate protein covalent structure analysis through peptide mapping.
- The methods described allow for the digestion of challenging protein samples.
- This approach enhances the utility of mass spectrometry and automated sequencing in proteomics.
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