A diverse proteome is present and enzymatically active in metabolite extracts

Area of Science:

• Biochemistry
• Proteomics
• Metabolomics

Background:

• Metabolite extraction is a crucial initial step in metabolomics.
• Proteins are generally expected to be inactivated or removed during this process.

Purpose of the Study:

• To investigate the presence and impact of proteins in metabolite extracts.
• To identify protein-mediated metabolite changes and develop improved extraction methods.

Main Methods:

• Analysis of protein content in various metabolite extracts.
• Use of stable isotope labeling and enzyme inhibitors to study in-extract reactions.
• Application of untargeted metabolomics to assess metabolite alterations.
• Development and testing of a 3 kDa filtration workflow.

Main Results:

• Over 1000 proteins were identified in common metabolite extracts.
• Residual transaminase activity caused in-extract metabolite interconversions.
• Protein activity led to significant metabolite changes, including glutamate dipeptide formation and glutathione depletion.
• A 3 kDa filtration method effectively removed proteins and improved polar metabolomics.

Conclusions:

• Proteins in metabolite extracts represent a significant, previously unrecognized source of observer effects in metabolomics.
• The findings have broad implications for all research fields utilizing metabolomics.
• A simple protein removal workflow using 3 kDa filtration enhances polar metabolomics accuracy.