Histone acetylation and methylation play a critical role in transcription and gene regulation. Identification of sites of lysine acetylation and methylation in histones or other proteins by mass spectrometry (MS) is of increasing interest. In this paper, we report the use of MS to differentiate between peptides containing acetylated or tri-methylated lysines. High accuracy matrix-assisted laser desorption/ionization-time of flight MS gives better than five parts per million measurement accuracy, which is sufficient to verify acetylation and/or methylation. Electrospray ionization tandem mass spectrometry was used to assign modification sites and to differentiate acetylation from methylation. Typically, an immonium ion at m/z 98 corresponds to a mono-methylated lysine and an immonium ion at m/z 126 corresponds to an acetylated lysine. The neutral loss ion (MH(+)-59) is unique for a tri-methylated lysine. For a peptide with two or more modification sites of acetylation or tri-methylation or one site containing partial acetylation and tri-methylation, the a(2)-, b(2)-type ion is the characteristic index for an acetylated lysine whereas the b(2)-59 ion is indicative of a tri-methylated lysine in the N-terminus. The y-type ions and y-59 ions are characteristic of an acetylated lysine and a tri-methylated lysine at the C-terminus, respectively. We demonstrated that a lysine in a peptide modified by methylation or acetylation can be differentiated by MS using our method. Even if more then one lysine is present in a peptide and different modifications of this amino acid occur, they can be distinguished. This method was successful for the determination of the acetylation and methylation status of lysine 9 of histone H3 in chicken erythrocytes and human HeLa cell lines.

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Abstract:The rate of glutamate synthesis from leucine by the branched-chain aminotransferase was measured in rat brain in vivo at steady state. The rats were fed exclusively by intravenous infusion of a nutrient solution containing [15N]leucine. The rate of glutamate synthesis from leucine, determined from the rate of increase of brain [15N]glutamate measured by 15N NMR and the 15N enrichments of brain and blood leucine analyzed by gas chromatography-mass spectrometry, was 0.7-1.8 j.tmol/g/ h at a steady-state brain leucine concentration of 0.25 mol/g. A comparison of the observed fractional 15N enrichments of brain leucine (0.42 ±0.03) and glutamate (0.21 ±0.015) showed that leucine provides '-.~50%of glutamate nitrogen under our experimental condition. From the observed rate (0.7-1.8 j.imol/g) and the known Km of the branched-chain aminotransferase for leucine (1.2 mM), the rate of glutamate synthesis from leucine at physiological brain leucine concentration (0.11~mol/ g) was estimated to be 0.35-0.9~mol/g/h, with leucine providing~25% of glutamate nitrogen. The results strongly suggest that plasma leucine from dietary source, transported into the brain, is an important external source of nitrogen for replenishment of brain glutamate in vivo. Implications of the results for treatment of maple-syrup urine disease patients with leucine-restricted diet are discussed. Key Words: Leucine-Glutamate-BrainBranched-chain aminotransferase-1 5N NMR-In vivo. J. Neurochem. 70, 1304Neurochem. 70, -1315Neurochem. 70, (1998.The branched-chain amino acids (BCAA) -leucine, isoleucine, and valine-are essential amino acids that readily cross the blood-brain barrier. Leucine is the predominant BCAA in most dietary proteins (Berry et al., 1989) and has the highest influx rate into the brain (Smith et al., 1987). In the brain, leucine has two metabolic fates: (a) incorporation into proteins and (b) transamination to a-ketoglutarate to form glutamate, catalyzed by branched-chain aminotransferase (BCAT; EC 2.6.1.42):

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Glial uptake of neurotransmitter glutamate (GLU) from the extracellular fluid was studied in vivo in rat brain by 13 C NMR and microdialysis combined with gas-chromatography/massspectrometry. Brain GLU C5 was 13 C enriched by intravenous [2,5-13 C]glucose infusion, followed by [ 12 C]glucose infusion to chase 13 C from the small glial GLU pool. This leaves [5-13 C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. During the chase, the 13 C enrichment of whole-brain GLU C5 was significantly lower than that of extracellular GLU (GLU ECF ) derived from exocytosis of vesicular GLU. Glial uptake of neurotransmitter N enrichment of precursor NH 3 (0.87 ± 0.014), the rate of synthesis of GLN (V¢ GLN ), derived from neurotransmitter GLU ECF , was determined to be 6.4 ± 0.44 lmol/g/h. Comparison with V GLN measured previously by an independent method showed that the neurotransmitter provides 80-90% of the substrate GLU pool for GLN synthesis. Hence, under our experimental conditions, the rate of 6.4 ± 0.44 lmol/g/h also represents a reasonable estimate for the rate of glial uptake of GLU ECF , a process that is crucial for protecting the brain from GLU excitotoxicity.

A new approach to mixture analysis has been applied to the direct detection of various alkaloids in plant materials. The method requires absolutely no sample treatment. Results are presented for cocaine, morphine, papaverine, coniine, and atropine. The signal-to-background characteristics are superior to those of conventional mass spectrometry. Sensitivity is sufficient to detect and identify between 1 and 10 nanograms of alkaloid.

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