# Simultaneous determination of free and total metabolite concentrations in proteinaceous specimens by 1D 1H CPMG NMR

## TL;DR

A new NMR method allows measuring total metabolite concentrations in protein-rich samples without disrupting proteins, making the process easier and more reliable.

## Contribution

A novel NMR-based approach to determine total metabolite concentrations without protein precipitation or additional steps.

## Key findings

- The method uses line broadening in NMR spectra to infer total metabolite concentrations.
- Validation on synthetic and biological samples showed accurate and reliable results.
- The approach preserves the sample for further analysis and avoids loss of volatile metabolites.

## Abstract

Nuclear magnetic resonance (NMR) spectroscopy is often used for the analysis of metabolites in proteinaceous biological specimens. However, the binding of metabolites to proteins impedes accurate quantitation of total metabolite concentrations by NMR, unless protein binding is disrupted by organic solvent precipitation, which increases variance and may result in the loss of volatile metabolites during post-extraction drying. Here, we present an approach for the inference of total metabolite concentrations from Carr-Purcell-Meiboom-Gill NMR spectra via computation of metabolite and sample-specific factors derived from the individual broadening of spectral peaks due to protein-metabolite binding. The method was validated on both synthetic proteinaceous samples and plasma and urine specimens including a certified reference plasma. Furthermore, results were compared with those obtained for methanol extracts of plasma specimens. In summary, our approach obviates the need for protein precipitation, is easy to use, and allows precise and reliable determination of total metabolite concentrations.

•Easy-to-use tool for quantification of total metabolite amounts in proteinaceous media•No need for additional experimental steps like ultrafiltration or protein precipitation•Leaves analyzed sample intact for further investigations•Applicable to different metabolites and different matrices like plasma or urine

Easy-to-use tool for quantification of total metabolite amounts in proteinaceous media

No need for additional experimental steps like ultrafiltration or protein precipitation

Leaves analyzed sample intact for further investigations

Applicable to different metabolites and different matrices like plasma or urine

The investigation of the metabolome can offer new insights into medical and/or biological topics. To this end, a popular method is nuclear magnetic resonance (NMR) spectroscopy. The presence of proteins makes this analysis more challenging due to protein-metabolite interactions. Most current methods to address this problem often need additional experimental steps or are only able to quantify the unbound fraction of a specific metabolite. Therefore, we developed an approach to obtain both free and total metabolite amounts from a single NMR spectrum based on the evaluation of the individual line broadenings of the metabolite NMR signals upon protein binding.

NMR spectroscopy is an important method for the study of the metabolome. Reindl et al. develop an easy-to-use tool to allow the accurate quantification of total metabolite concentrations in proteinaceous media like plasma without the need of additional experimental steps as already existing NMR spectra are utilized.