Measuring Complete Isotopomer Distribution of Aspartate Using Gas Chromatography/Tandem Mass Spectrometry

被引:63
作者
Choi, Jungik [1 ]
Grossbach, Matthew T. [1 ]
Antoniewicz, Maciek R. [1 ]
机构
[1] Univ Delaware, Dept Chem & Biomol Engn, Metab Engn & Syst Biol Lab, Newark, DE 19716 USA
基金
美国国家科学基金会;
关键词
METABOLIC FLUX ANALYSIS; UNITS EMU; TRACERS; FRAMEWORK; GLUTAMATE;
D O I
10.1021/ac300611n
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
We have developed a simple and accurate method for determining the complete positional isotopomer distribution of aspartate carbon atoms by gas chromatography/tandem mass spectrometry for C-13-metabolic flux analysis. First, we screened tandem mass spectrometry (MS) spectra of the tert-butyldimethylsilyl (TBDMS) derivative of aspartate for daughter fragments with the necessary carbon atom fragmentations to fully resolve all 16 isotopomers of aspartate. Tandem MS scanning parameters were optimized for each daughter fragment, and the accuracy of tandem MS measurements were evaluated. We selected five accurate fragments that provided a redundant set of 47 labeling measurements to quantify the complete isotopomer distribution of aspartate by least-squares regression. The validity of the approach was demonstrated using six C-13-labeled aspartate standards and natural aspartate.
引用
收藏
页码:4628 / 4632
页数:5
相关论文
共 27 条
[1]   Towards dynamic metabolic flux analysis in CHO cell cultures [J].
Ahn, Woo Suk ;
Antoniewicz, Maciek R. .
BIOTECHNOLOGY JOURNAL, 2012, 7 (01) :61-74
[2]   Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry [J].
Ahn, Woo Suk ;
Antoniewicz, Maciek R. .
METABOLIC ENGINEERING, 2011, 13 (05) :598-609
[3]   Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis [J].
Antoniewicz, Maciek R. ;
Kelleher, Joanne K. ;
Stephanopoulos, Gregory .
ANALYTICAL CHEMISTRY, 2007, 79 (19) :7554-7559
[4]   Elementary metabolite units (EMU): A novel framework for modeling isotopic distributions [J].
Antoniewicz, Maciek R. ;
Kelleher, Joanne K. ;
Stephanopoulos, Gregory .
METABOLIC ENGINEERING, 2007, 9 (01) :68-86
[5]   Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements [J].
Antoniewicz, Maciek R. ;
Kelleher, Joanne K. ;
Stephanopoulos, Gregory .
METABOLIC ENGINEERING, 2006, 8 (04) :324-337
[6]   Measuring Deuterium Enrichment of Glucose Hydrogen Atoms by Gas Chromatography/Mass Spectrometry [J].
Antoniewicz, Maciek R. ;
Kelleher, Joanne K. ;
Stephanopoulos, Gregory .
ANALYTICAL CHEMISTRY, 2011, 83 (08) :3211-3216
[7]   Tandem mass spectrometry: A novel approach for metabolic flux analysis [J].
Choi, Jungik ;
Antoniewicz, Maciek R. .
METABOLIC ENGINEERING, 2011, 13 (02) :225-233
[8]   Selection of tracers for 13C-Metabolic Flux Analysis using Elementary Metabolite Units (EMU) basis vector methodology [J].
Crown, Scott B. ;
Antoniewicz, Maciek R. .
METABOLIC ENGINEERING, 2012, 14 (02) :150-161
[9]   Resolving the TCA cycle and pentose-phosphate pathway of Clostridium acetobutylicum ATCC 824: Isotopomer analysis, in vitro activities and expression analysis [J].
Crown, Scott B. ;
Indurthi, Dinesh C. ;
Ahn, Woo Suk ;
Choi, Jungik ;
Papoutsakis, Eleftherios T. ;
Antoniewicz, Maciek R. .
BIOTECHNOLOGY JOURNAL, 2011, 6 (03) :300-305
[10]   13C isotopomer analysis of glutamate by tandem mass spectrometry [J].
Jeffrey, FMH ;
Roach, JS ;
Storey, CJ ;
Sherry, AD ;
Malloy, CR .
ANALYTICAL BIOCHEMISTRY, 2002, 300 (02) :192-205