title: A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance.
abstract: Absolute analyte quantification by nuclear magnetic resonance (NMR) spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. Here we report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (limit of detection [LOD] = 5-25 μM) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. The method does not require the use of databases and uses PULCON (pulse length-based concentration determination) quantitative NMR to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (Carr-Purcell-Meiboom-Gill) sequence or post-processing filtering approaches. Three practical applications of the method highlight its flexibility under different cell culture conditions. We identified and quantified (i) metabolic differences between genetically engineered human cell lines, (ii) alterations in cellular metabolism induced by differentiation of mouse myoblasts into myotubes, and (iii) metabolic changes caused by activation of neurotransmitter receptors in mouse myoblasts. Thus, the new protocol offers an easily implementable, efficient, and versatile tool for the investigation of cellular metabolism and signal transduction.
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia, 16163, Genova, Italy. Electronic address: luca.goldoni@iit.it.
authors.forename: Luca
authors.initials: L
authors.lastname: Goldoni
authors.affiliation: Department of Chemistry, University of Milan, 20133, Milano, Italy.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Beringhelli
authors.affiliation: CONCEPT Lab, Istituto Italiano di Tecnologia, 16163, Genova, Italy.
authors.forename: Walter
authors.initials: W
authors.lastname: Rocchia
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia, 16163, Genova, Italy.
authors.forename: Natalia
authors.initials: N
authors.lastname: Realini
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia, 16163, Genova, Italy; Departments of Anatomy and Neurobiology, Pharmacology, and Biological Chemistry, University of California, Irvine, CA, 92697, USA.
authors.forename: Daniele
authors.initials: D
authors.lastname: Piomelli
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D005110:Extracellular Space / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D051379:Mice; D032446:Myoblasts / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D044967:Serum / Q000737:chemistry
chemical_list: D001798:Blood Proteins
keywords: (1)H-NMR; Accuracy; CPMG; Deconvolution; PULCON; pH-controlled serum removal