Metabolomic analysis of eukaryotic tissue and prokaryotes using negative mode MALDI time-of-flight mass spectrometry
Metabolomic analysis of eukaryotic tissue and prokaryotes using negative mode MALDI time-of-flight mass spectrometry
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DOI：
10.1021/ac048323r
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发表时间：
2005-04-01
影响因子：
7.4
通讯作者：
Kennedy, RT
中科院分区：
文献类型：
--
作者：
Edwards, JL;Kennedy, RT
采用负模式基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）分析了Langerhans和大肠杆菌dh5 - α菌株胰岛代谢产物。用MALDI分析阴离子代谢物时，9-氨基吖啶作为基质比α -氰基-4-羟基肉桂酸、2,5-二氢苯甲酸、2,4,6、三羟基苯乙酮和3-羟基吡啶酸的信号要好得多。代谢物标准物的检出限低至GDP、GTP、ADP和ATP的检出限低至15 nM，在1 μ L样品中琥珀酸盐的检出限高达1 μ M。对胰岛提取物的分析发现了44种代谢物，其中29种通过与METLIN和KEGG数据库中化合物的分子量匹配初步鉴定。通过比较不同浓度葡萄糖培养的胰岛所选择的二磷酸化和三磷酸化核苷酸的比例，证明了相对定量。葡萄糖浓度为3 mM时，胰岛ATP/ADP、GTP/GDP和UTP/UDP的浓度比分别为1.9 +/- 1.39、1.12 +/- 0.50和0.79 +/- 0.35，葡萄糖浓度为20 mM时，胰岛ATP/ADP、GTP/GDP和UTP/UDP的浓度比分别为4.13 +/- 1.89、5.62 +/- 4.48和4.30 +/- 4.07 （n = 3）。分析还通过将单个完整的胰岛放置在带有基质的MALDI靶板上并将激光直接撞击干燥的胰岛来进行。直接分析单个胰岛可检测43种代谢物，其中28种可在数据库中识别。直接胰岛分析检测到的代谢物中有43%与胰岛提取物检测到的代谢物不同。通过对大肠杆菌提取物的分析，将该方法扩展到原核细胞。检测到60种代谢物，其中39种与MetaCyc数据库中的化合物相匹配。从原核生物中检测到的代谢物中有27%与在胰岛中发现的代谢物重叠。这些结果表明MALDI可用于复杂生物样品中代谢物的检测。
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.