| Abstract
| - Metabolites in islets of Langerhans and Escherichia colistrain DH5-α were analyzed using negative-mode, matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF-MS). For analysis of anionicmetabolites by MALDI, 9-aminoacridine as the matrixyielded a far superior signal in comparison to α-cyano-4-hydroxycinnamic acid, 2,5-dihydrobenzoic acid, 2,4,6,-trihydroxyacetophenone, and 3-hydroxypicolinic acid.Limits of detection for metabolite standards were as lowas 15 nM for GDP, GTP, ADP, and ATP and as high as 1μM for succinate in 1-μL samples. Analysis of islet extractsallowed detection of 44 metabolites, 29 of which weretentatively identified by matching molecular weight tocompounds in METLIN and KEGG databases. Relativequantification was demonstrated by comparing the ratioof selected di- and triphosphorylated nucleotides for isletsincubated with different concentrations of glucose. Forislets 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 glucosestimulation, the ratios increased to 4.13 ± 1.89, 5.62 ±4.48, and 4.30 ± 4.07 (n = 3). Analysis was alsoperformed by placing individual, intact islets on a MALDItarget plate with matrix and impinging the laser directlyon the dried islet. Direct analysis of single islets alloweddetection of 43 metabolites, 28 of which were databaseidentifiable. A total of 43% of detected metabolites fromdirect islet analysis were different from those detected inislet extracts. The method was extended to prokaryoticcells by analysis of extracts from E. coli. Sixty metaboliteswere detected, 39 of which matched compounds in theMetaCyc database. A total of 27% of the metabolitesdetected from prokaryotes overlapped those found inislets. These results show that MALDI can be used fordetection of metabolites in complex biological samples.
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