%0 Journal Article
%T A diverse proteome is present and enzymatically active in metabolite extracts.
%A House RRJ
%A Soper-Hopper MT
%A Vincent MP
%A Ellis AE
%A Capan CD
%A Madaj ZB
%A Wolfrum E
%A Isaguirre CN
%A Castello CD
%A Johnson AB
%A Escobar Galvis ML
%A Williams KS
%A Lee H
%A Sheldon RD
%J Nat Commun
%V 15
%N 1
%D 2024 Jul 10
%M 38987243
%F 17.694
%R 10.1038/s41467-024-50128-z
%X Metabolite extraction is the critical first-step in metabolomics experiments, where it is generally regarded to inactivate and remove proteins. Here, arising from efforts to improve extraction conditions for polar metabolomics, we discover a proteomic landscape of over 1000 proteins within metabolite extracts. This is a ubiquitous feature across several common extraction and sample types. By combining post-resuspension stable isotope addition and enzyme inhibitors, we demonstrate in-extract metabolite interconversions due to residual transaminase activity. We extend these findings with untargeted metabolomics where we observe extensive protein-mediated metabolite changes, including in-extract formation of glutamate dipeptide and depletion of total glutathione. Finally, we present a simple extraction workflow that integrates 3 kDa filtration for protein removal as a superior method for polar metabolomics. In this work, we uncover a previously unrecognized, protein-mediated source of observer effects in metabolomics experiments with broad-reaching implications across all research fields using metabolomics and molecular metabolism.