This is a three-in-one workflow, from automated sample prep to LC/MS analysis, that measures metabolites, lipids and proteins from individual plasma samples. LC/MS omics analyses, comprising metabolomics, lipidomics, and proteomics, offer a way to measure metabolism. This includes measuring components of the three main metabolic branches: (1) conversion of nutrients into energy, predominantly ATP, (2) conversion of nutrients into building blocks, including those used to build proteins and nucleic acids, and (3) conversion of nutrients into waste products.
Comprehensive LC/MS metabolomics, lipidomics and proteomics analysis requires extraction, separation, and detection of all three compound classes from individual samples. We developed a novel automated workflow for extraction of metabolites, lipids and proteins from 20 µl plasma samples and analyzed each extracted fraction with a unique LC/MS method. The coverage and reproducibility across metabolites, lipids, and proteins for this three-in-one omics workflow were assessed.
Metabolite, lipid and protein fractions were extracted from 20 µl pooled mouse plasma samples using a liquid handler with commercial accessories. Proteins were precipitated and collected as a pellet that was prepared via tryptic digestion. Metabolites and lipids were separated by passing the supernatant through a solid phase extraction plate, metabolites being collected in the flow-through and lipids being captured by the sorbent and subsequently eluted.
Each separate fraction was analyzed using targeted or untargeted LC/MS analysis. Polar metabolite analysis used HILIC chromatography and an LC/TQ. Lipid analysis used RP C18 chromatography and a LC/TQ. Protein analysis used RP chromatography and an LC/Q-TOF. Data analysis utilized commercially available quantitative software and Spectrum Mill software.
Targeted metabolomics analysis detected 437 metabolites, covering major metabolite classes and metabolic pathways. A subset of 68 metabolites were selected for reproducibility analysis. Targeted lipidomics analysis detected 615 lipids across commonly analyzed lipid classes, including ceramides, phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, sphingomyelins, and triacylglycerols. Untargeted protein analysis detected an average of 1479 proteins from each 20 µl plasma sample when using a 2 peptide per protein guidance and 1% FDR rate.
Overall, this three-in-one LC/MS omics workflow offers great coverage and reproducibility for metabolites, lipids and proteins. This end-to-end workflow achieves reliable results with small plasma volumes and is a fast and efficient solution for multi-omics analyses. Finally, the workflow offers flexibility for those who may only want to collect and monitor a single type of biomolecule (metabolite, lipid or protein) or those who are interested in measuring two of the three types of biomolecules.