Stable isotope labeling by essential nutrients in cell culture (SILEC) for accurate measurement of nicotinamide adenine dinucleotide metabolism.
Stable isotope labeling by essential nutrients in cell culture (SILEC) for accurate measurement of nicotinamide adenine dinucleotide metabolism.
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DOI：
10.1039/c7an01378g
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发表时间：
2017-11-20
期刊：
影响因子：
--
通讯作者：
Snyder NW
中科院分区：
文献类型：
--
作者：
Frederick DW;Trefely S;Buas A;Goodspeed J;Singh J;Mesaros C;Baur JA;Snyder NW
Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are conserved metabolic cofactors that mediate reduction-oxidation (redox) reactions throughout all domains of life. The diversity of synthetic routes and cellular processes involving the transfer of reducing equivalents to and from these cofactors makes the accurate quantitation and metabolic tracing of NAD(H) and NADP(H) of broad interest. However, current analytical techniques typically rely on standard curves that do not incorporate confounding effects of the sample matrix. We utilized the essential requirement of niacin and tryptophan for NAD synthesis in mammalian cells to devise a stable isotope labeling by essential nutrients in cell culture (SILEC) method for efficient labeling of intracellular NAD(H) and NADP(H) pools. Coupling this approach with detection by liquid chromatography-high resolution mass spectrometry (LC-HRMS), we demonstrate the utility of incorporating a [13C315N1]-nicotinamide moiety into a library of NAD-derived metabolites for use as internal standards in matrixed samples. Using a two-label system incorporating [13C315N1]-nicotinamide and [13C11]-tryptophan, we quantify the relative contribution of salvage and de novo NAD synthesis, respectively, in S. cerevisiae and HepG2 human hepatocellular carcinoma cells under basal conditions. As a further proof-of-principle, we demonstrate an improvement in the linear range for quantification of NAD and apply this method to analysis of NAD(H) in mouse liver. This method demonstrates the generalizability of SILEC, and provides a simple method for generating a library of stable isotope labeled internal standards for quantifying and tracing the metabolism of cellular and tissue NAD(H) and NADP(H).
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DOI：
10.1126/science.1171641
发表时间：
2009-05-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Ramsey KM;Yoshino J;Brace CS;Abrassart D;Kobayashi Y;Marcheva B;Hong HK;Chong JL;Buhr ED;Lee C;Takahashi JS;Imai S;Bass J
通讯作者：
Bass J
DOI：
10.1146/annurev.anchem.1.031207.112945
发表时间：
2008-01-01
影响因子：
8
作者：
Marshall, Alan G.;Hendrickson, Christopher. L.
通讯作者：
Hendrickson, Christopher. L.
影响因子：
6
作者：
Trammell SA;Brenner C
通讯作者：
Brenner C
影响因子：
7
作者：
Ong, SE;Blagoev, B;Mann, M
通讯作者：
Mann, M
影响因子：
2
作者：
Snyder, Nathaniel W.;Basu, Sankha S.;Zhou, Zinan;Worth, Andrew J.;Blair, Ian A.
通讯作者：
Blair, Ian A.