Metabolomic analysis of eukaryotic tissue and prokaryotes using negative mode MALDI time-of-flight mass spectrometry

被引:99
作者
Edwards, JL
Kennedy, RT [1 ]
机构
[1] Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA
[2] Univ Michigan, Dept Pharmacol, Ann Arbor, MI 48109 USA
关键词
D O I
10.1021/ac048323r
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
Metabolites in islets of Langerhans and Escherichia coli strain DH5-alpha were analyzed using negative-mode, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For analysis of anionic metabolites by MALDI, 9-aminoacridine as the matrix yielded a far superior signal in comparison to alpha-cyano-4-hydroxycinnamic acid, 2,5-dihydrobenzoic acid, 2,4,6,trihydroxyacetophenone, and 3-hydroxypicolinic acid. Limits of detection for metabolite standards were as low as 15 nM for GDP, GTP, ADP, and ATP and as high as 1 mu M for succinate in 1-mu L samples. Analysis of islet extracts allowed detection of 44 metabolites, 29 of which were tentatively identified by matching molecular weight to compounds in METLIN and KEGG databases. Relative quantification was demonstrated by comparing the ratio of selected di- and triphosphorylated nucleotides for islets incubated with different concentrations of glucose. For islets at 3 mM glucose, concentration ratios of ATP/ADP, GTP/GDP, and UTP/UDP were 1.9 +/- 1.39, 1.12 +/- 0.50, and 0.79 +/- 0.35 respectively, and at 20 mM glucose stimulation, the ratios increased to 4.13 +/- 1.89, 5.62 +/- 4.48, and 4.30 +/- 4.07 (n = 3). Analysis was also performed by placing individual, intact islets on a MALDI target plate with matrix and impinging the laser directly on the dried islet. Direct analysis of single islets allowed detection of 43 metabolites, 28 of which were database identifiable. A total of 43% of detected metabolites from direct islet analysis were different from those detected in islet extracts. The method was extended to prokaryotic cells by analysis of extracts from E. coli. Sixty metabolites were detected, 39 of which matched compounds in the MetaCyc database. A total of 27% of the metabolites detected from prokaryotes overlapped those found in islets. These results show that MALDI can be used for detection of metabolites in complex biological samples.
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收藏
页码:2201 / 2209
页数:9
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