Extraction optimization for combined metabolomics, peptidomics, and proteomics analysis of gut microbiota samples

J Mass Spectrom. 2021 Apr;56(4):e4625. doi: 10.1002/jms.4625. Epub 2020 Sep 3.


Multiomic studies are increasingly performed to gain a deeper understanding of molecular processes occurring in a biological system, such as the complex microbial communities (i.e., microbiota) that reside the distal gut. While a combination of metabolomics and proteomics is more commonly used, multiomics studies including peptidomcis characterization are less frequently undertaken. Here, we investigated three different extraction methods, chosen for their previous use in extracting metabolites, peptides, and proteins, and compared their ability to perform metabolomic, peptidomic, and proteomic analysis of mouse cecum content. The methanol/chloroform/water extraction performed the best for metabolomic and peptidomic analysis as it detected the largest number of small molecules and identified the largest number of peptides, but the acidified methanol extraction performed best for proteomics analysis as it had the highest number of protein identifications. The methanol/chloroform/water extraction was further analyzed by identifying metabolites with tandem mass spectrometry (MS/MS) analysis and by gene ontology analysis for the peptide and protein results to provide a multiomics analysis of the gut microbiota.

Keywords: metabolomics; microbiome; multiomics; peptidomics; proteomics.

MeSH terms

  • Animals
  • Cecum / microbiology
  • Chloroform / chemistry
  • Chromatography, High Pressure Liquid
  • Complex Mixtures / analysis*
  • Gastrointestinal Microbiome / physiology*
  • Male
  • Metabolomics / methods*
  • Methanol / chemistry
  • Mice, Inbred C57BL
  • Microbiota / physiology
  • Peptides / analysis*
  • Peptides / metabolism
  • Proteomics / methods*
  • Tandem Mass Spectrometry
  • Water


  • Complex Mixtures
  • Peptides
  • Water
  • Chloroform
  • Methanol