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Quantitating metabolites in protein precipitated serum using NMR spectroscopy
G A Nagana Gowda1, Daniel Raftery
1Northwest Metabolomics Research Center, Anesthesiology and Pain Medicine, and ‡Department of Chemistry, University of Washington , Seattle, Washington 98109, United States.
Analytical Chemistry
|May 7, 2014
Summary
Protein precipitation is a reliable method for NMR metabolomics of blood serum, offering comparable results to ultrafiltration but with better metabolite recovery. This approach facilitates direct comparison with mass spectrometry data.
Area of Science:
- Metabolomics
- Biochemistry
- Analytical Chemistry
Background:
- Quantitative NMR-based metabolite profiling of blood is hindered by abundant proteins.
- Protein removal is crucial, with ultrafiltration being a standard but potentially lossy method.
- Protein precipitation's quantitative performance in NMR metabolomics requires thorough evaluation.
Purpose of the Study:
- To comprehensively evaluate protein precipitation methods (methanol, acetonitrile, PCA, TCA) against ultrafiltration for NMR-based metabolomics.
- To identify and quantify metabolites in human blood serum using 1D and 2D NMR.
- To assess the impact of a novel isotope tag ((15)N-cholamine) on metabolite detection.
Main Methods:
- Comparison of protein precipitation (methanol, acetonitrile, perchloric acid, trichloroacetic acid) and ultrafiltration techniques.
- 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy for metabolite identification and absolute quantitation.
- Analysis of 44 human blood metabolites and evaluation of reproducibility (CV).
Main Results:
- Both protein precipitation and ultrafiltration detected 44 metabolites with similar reproducibility (avg. CV 3.7% vs. 3.6%).
- Ultrafiltration resulted in 10-74% lower concentrations for nearly half of the quantified metabolites.
- Methanol-based protein precipitation showed higher recovery for specific metabolites like tryptophan and benzoate.
Conclusions:
- Protein precipitation, particularly using methanol, is a reliable and effective alternative to ultrafiltration for routine NMR-based metabolomics of human blood serum/plasma.
- Protein precipitation enhances metabolite recovery compared to ultrafiltration, preserving crucial metabolite concentrations.
- This method facilitates direct correlation of NMR and mass spectrometry data, leveraging the strengths of both platforms for comprehensive blood metabolomics.
