A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

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• 作者：Luca Goldoni^a ; ^{luca.goldoni@iit.it" class="auth_mail" title="E-mail the corresponding author} ; Tiziana Beringhelli^b ; Walter Rocchia^c ; Natalia Realini^a ; Daniele Piomelli^a ; ^d
• 关键词：1H-NMR ; pH-controlled serum removal ; PULCON ; Accuracy ; CPMG ; Deconvolution
• 刊名：Analytical Biochemistry
• 出版年：2016
• 出版时间：15 May 2016
• 年：2016
• 卷：501
• 期：Complete
• 页码：26-34
• 全文大小：1038 K

文摘

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.