Discovery and neurochemical screening of peptides in brain extracellular fluid by chemical analysis of in vivo microdialysis samples
Discovery and neurochemical screening of peptides in brain extracellular fluid by chemical analysis of in vivo microdialysis samples
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DOI：
10.1021/ac049363y
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发表时间：
2004-09-15
影响因子：
7.4
通讯作者：
Kennedy, RT
中科院分区：
文献类型：
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作者：
Haskins, WE;Watson, CJ;Kennedy, RT
采用毛细管液相色谱(LC)-串联质谱仪(MS2)对活体大鼠脑细胞外液中的内源性多肽进行分析。将4 mm长的微透析探头以0.6微升/分的速度注入麻醉雄性大鼠纹状体，采集3.6微升的透析液，在线注入毛细管LC-MS2系统进行分析。在基础状态下和通过微透析探头注入高K+溶液引起的局部去极化过程中，共收集了13种不同动物的3349个MS2频谱。消减分析显示，总共有859个MS2光谱，这些光谱只在去极化过程中观察到。从这些光谱中，用数据库搜索软件鉴定了来自6个不同蛋白质前体的29个肽序列(其中25个是以前没有观察到的肽)。识别的蛋白质包括神经肽的前体、突触蛋白、血液蛋白和转运蛋白。已鉴定的多肽代表神经递质、神经调节剂和突触活动或脑组织损伤的标记。对新发现的脑啡肽原片段的神经活性进行筛选，方法是将这些肽注入大脑，同时通过微透析采样结合毛细管电泳法监测氨基酸神经递质。在测试的六种多肽中，有三种引起各种神经活性氨基酸的显著增加。这些结果表明，这种方法的组合可以识别新的神经递质候选者，并筛选潜在的神经活性。
Endogenous peptides from brain extracellular fluid of live rats were analyzed using capillary liquid chromatography (LC)-tandem mass spectrometry (MS2). A 4-mm-long microdialysis probe perfused at 0.6 muL/min implanted into the striatum of anesthetized male rats was used to collect 3.6 muL dialysate fractions that were injected online into the capillary LC-MS2 system for analysis. A total of 3349 MS2 spectra were collected from 13 different animals under basal conditions and during localized depolarization evoked by infusion of a high-K+ solution through the microdialysis probe. Subtractive analysis revealed a total of 859 MS2 spectra that were observed only during depolarization. From these spectra, 29 peptide sequences (25 were peptides not previously observed) from 6 different protein precursors were identified using database searching software. Proteins identified include precursors to neuropeptides, synaptic proteins, blood proteins, and transporters. The identified peptides represent candidates for neurotransmitters, neuromodulators, and markers of synaptic activity or brain tissue damage. A screen for neuroactivity of novel proenkephalin fragments that were found was performed by infusing the peptides into the brain while monitoring amino acid neurotransmitters by microdialysis sampling combined with capillary electrophoresis. Three of the six tested peptides evoked significant increases in various neuroactive amino acids. These results demonstrate that this combination of methods can identify novel neurotransmitter candidates and screen for potential neuroactivity.