Edwards James L, Kennedy Robert T
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109, USA.
Anal Chem. 2005 Apr 1;77(7):2201-9. doi: 10.1021/ac048323r.
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 muM for succinate in 1-muL 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.
使用负模式基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）分析了胰岛和大肠杆菌DH5-α菌株中的代谢物。为了通过MALDI分析阴离子代谢物，与α-氰基-4-羟基肉桂酸、2,5-二羟基苯甲酸、2,4,6-三羟基苯乙酮和3-羟基吡啶甲酸相比，9-氨基吖啶作为基质产生的信号要好得多。在1μL样品中，代谢物标准品的检测限对于GDP、GTP、ADP和ATP低至15 nM，对于琥珀酸高达1μM。胰岛提取物分析可检测到44种代谢物，其中29种通过将分子量与METLIN和KEGG数据库中的化合物匹配进行了初步鉴定。通过比较不同葡萄糖浓度孵育的胰岛中选定的二磷酸和三磷酸核苷酸的比例进行了相对定量。对于3 mM葡萄糖浓度下的胰岛，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，在20 mM葡萄糖刺激下，这些比例分别增加到4.13±1.89,5.62±4.48和4.30±4.07（n = 3）。还通过将单个完整的胰岛置于带有基质的MALDI靶板上并将激光直接照射在干燥的胰岛上来进行分析。单个胰岛的直接分析可检测到43种代谢物，其中28种可通过数据库识别。直接胰岛分析中检测到的代谢物中共有43%与胰岛提取物中检测到的不同。通过分析大肠杆菌提取物，该方法扩展到了原核细胞。检测到60种代谢物，其中39种与MetaCyc数据库中的化合物匹配。原核生物中检测到的代谢物共有27%与胰岛中发现的代谢物重叠。这些结果表明，MALDI可用于检测复杂生物样品中的代谢物。
