Capillary LC-MS2 at the attomole level for monitoring and discovering endogenous peptides in microdialysis samples collected in vivo
Capillary LC-MS2 at the attomole level for monitoring and discovering endogenous peptides in microdialysis samples collected in vivo
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DOI：
10.1021/ac010774d
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发表时间：
2001-11-01
影响因子：
7.4
通讯作者：
Kennedy, RT
中科院分区：
文献类型：
--
作者：
Haskins, WE;Wang, ZQ;Kennedy, RT
熔融石英毛细管LC柱（25 μ m i.d.）3-mum-id。评价了与四极离子阱质谱仪连接的集成电喷雾发射器的高灵敏度LC-MS 2。柱制备涉及通过甲基丙烯酸缩水甘油酯和三甲基丙烯酸异丙酯的原位光聚合来构建熔料，通过用CO2激光器将柱出口拉到细尖来制备电喷雾发射器，并且用5 μ m反相颗粒浆料填充柱。大体积注射由自动双泵系统促进，该系统允许高流速的样品加载和低流速的洗脱。小的电喷雾发射器，低洗脱流速，和梯度陡度的优化允许4阿莫尔的检测限，相当于2 pM的1.8穆尔注射柱上，对于溶解在人工脑脊液中的肽的混合物。将该系统与微透析采样技术联用，用于监测和发现麻醉病人苍白球内源性神经肽。雄性Sprague-Dawley大鼠时间分段的MS 2扫描能够同时监测甲硫氨酸-脑啡肽、亮氨酸-脑啡肽和未知肽。甲硫氨酸脑啡肽和亮氨酸脑啡肽的基础透析液水平分别为60 +/- 30和70 +/- 20 pM，而K-（+）刺激水平分别为1900 +/- 500和1300 +/- 300 pM（n = 7）。数据依赖性和时间分段MS 2扫描显示透析液中存在几种未知肽。其中一个未知物被鉴定为肽I1-10（SPQLEDEAKE），这是前脑啡肽原A加工的新产物，使用MS 2，MS 3和数据库搜索。
Fused-silica capillary LC columns (25-mum i.d.) with 3-mum-i.d. integrated electrospray emitters interfaced to a quadrupole ion trap mass spectrometer were evaluated for high-sensitivity LC-MS2. Column preparation involved constructing frits by in situ photopolymerization of glycidyl methacrylate and trimethylolpropane trimethacrylate, preparing the electrospray emitter by pulling the column outlet to a fine tip with a CO2 laser puller, and slurry-packing the column with 5-mum reversed-phase particles. Large-volume injections were facilitated by an automated two-pump system that allowed high-flow rates for sample loading and low-flow rates for elution. Small electrospray emitters, low elution flow rates, and optimization of gradient steepness allowed a detection limit of 4 amol, corresponding to 2 pM for 1.8 muL injected on-column, for a mixture of peptides dissolved in artificial cerebral spinal fluid. The system was coupled on-line to microdialysis sampling and was used to monitor and discover endogenous neuropeptides from the globus pallidus of anesthetized. male Sprague-Dawley rats. Time-segmented MS2 scans enabled simultaneous monitoring of Met-enkephalin, Leu-enkephalin, and unknown peptides. Basal dialysate levels of Met-enkephalin and Leu-enkephalin were 60 +/- 30 and 70 +/- 20 pM while K-(+)-stimulated levels were 1900 +/- 500 and 1300 +/- 300 pM, respectively (n = 7). Data-dependent and time-segmented MS2 scans revealed several unknown peptides that were present in dialysate. One of the unknowns was identified as peptide I1-10 (SPQLEDEAKE), a novel product of preproenkephalin A processing, using MS2, MS3, and database searching.