Expanding Coverage of the Metabolome for Global Metabolite Profiling

• Published in: Analytical chemistry (Washington) Vol. 83; no. 6; pp. 2152 - 2161
• Main Authors: Yanes, Oscar, Tautenhahn, Ralf, Patti, Gary J, Siuzdak, Gary
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15-03-2011; American Chemical Society (ACS)
• Subjects: Acetates - chemistry; Ammonia; Analytical chemistry; Anions; BASIC BIOLOGICAL SCIENCES; Bias; Cations; Chemical Fractionation; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Culture Techniques; E coli; Escherichia coli - growth & development; Escherichia coli - metabolism; Ethanol - chemistry; Exact sciences and technology; Formates - chemistry; Hydrogen-Ion Concentration; INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Ionization; Mass spectrometry; Metabolism; Metabolome; Metabolomics - methods; Molecular Weight; Organic compounds; Other chromatographic methods; Solvents; Solvents - chemistry; Spectrometric and optical methods; Spectrometry, Mass, Electrospray Ionization; Temperature; Water - chemistry; Metabolomics; Temperature; Metabolite; Bias; Escherichia coli; Standard; Mobile phase; Electrospray; Mixture; Molecules; Signal; Endogenous; Sample preparation; Efficiency; pH; Bacteria; Polarity; Enterobacteriaceae; Polar solvent; Use; Hydrophobic compound; Concentration; Method; Chemical ionization; Stationary condition; Phase separation; Extraction chromatography; Mass spectrometry; Ammonium fluorides
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac102981k

Mass spectrometry-based metabolomics is the comprehensive study of naturally occurring small molecules collectively known as the metabolome. Given the vast structural diversity and chemical properties of endogenous metabolites, biological extraction and chromatography methods bias the number, property, and concentration of metabolites detected by mass spectrometry and creates a challenge for global untargeted studies. In this work, we used Escherichia coli bacterial cells to explore the influence of solvent polarity, temperature, and pH in extracting polar and nonpolar metabolites simultaneously. In addition, we explored chromatographic conditions involving different stationary and mobile phases that optimize the separation and ionization of endogenous metabolite extracts as well as a mixture of synthetic standards. Our results reveal that hot polar solvents are the most efficient in extracting both hydrophilic and hydrophobic metabolites simultaneously. In addition, ammonium fluoride in the mobile phase substantially improved ionization efficiency in negative electrospray ionization mode by an average increase in signal intensity of 5.7 and over a 2-fold increase in the total number of features detected. The improvement in sensitivity with ammonium fluoride resulted in 3.5 times as many metabolite hits in databases compared to ammonium acetate or formic acid enriched mobile phases and allowed for the identification of unique metabolites involved in fundamental cellular pathways.