Evaluation of the SPEED Sample Preparation Protocol for Metabolomics

Abstract Multiomic (paired proteomic and metabolomic) analyses allow researchers to better decipher the underlying biology being studied. While sample preparation techniques for multiomic analyses exist, they are often automation-unfriendly or secondary to preferred techniques for proteomic analyses. The recently published, popular Sample Preparation by Easy Extraction and Digestion (SPEED) protocol offers a single sample preparation technique for near-universal sample preparation across sample types and is used routinely in many proteomics laboratories; however, this protocol has not previously been investigated as to its suitability for metabolomic analyses. We tested the compatibility of two variations of the SPEED protocol: trifluoroacetic acid (TFA) lysis followed by drying and methanol extraction and TFA lysis and neutralization followed with SP4 cleanup. We compared these protocols with standard metabolomics sample preparation protocols of direct extraction with methanol or ACN and bead-beating in ammonium acetate lysis. The results show that TFA lysis with drying and methanol extraction yielded quantification results that were comparable to or better than those of the other protocols tested. We additionally demonstrated the utility of this technique by performing multiomic analysis of iPSCs before and after differentiation into neurons and identified coordinated dysregulation of metabolites and proteins in key neuronal differentiation pathways. We also observed that ACN extraction performed similarly to the gold standard methanol extraction. Consequently, we conclude that laboratories that already routinely use the SPEED protocol for dedicated proteomics can obtain functional metabolomics samples for little to no additional effort or cost with regard to sample preparation.