Measuring D-amino acid-containing neuropeptides with capillary electrophoresis.
Measuring D-amino acid-containing neuropeptides with capillary electrophoresis.
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DOI：
10.1039/b504717j
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发表时间：
2005-07
期刊：
影响因子：
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通讯作者：
Sarah A. Sheeley;Hai Miao;Michael A. Ewing;S. Rubakhin;J. Sweedler
中科院分区：
文献类型：
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作者：
Sarah A. Sheeley;Hai Miao;Michael A. Ewing;S. Rubakhin;J. Sweedler
神经肽是经过大量翻译后修饰 (PTM) 的基因产物，通常通过多种质谱方法进行表征。最近，已经记录了几种神经肽中存在 D 型氨基酸。由于这种修饰没有相关的质量转移，因此很难单独使用质谱 (MS) 来评估这种特定的 PTM。在这里，我们展示了几种使用毛细管电泳 (CE) 结合吸光度和激光诱导荧光 (LIF) 来分离含有 D-氨基酸的天然和衍生软体动物肽的方法。肽衍生化和 CE/LIF 的组合非常适合单细胞测量，因为它能够表征如此小样品中的肽。为了验证这种方法，对海洋软体动物加州海兔个体神经元中存在的含有 D-Trp 的肽 NdWFa (NH2-Asn-D-Trp-Phe-CONH2) 进行了表征。质谱显示NdWFa和/或NWFa存在于特定神经元中；这些细胞的 CE/LIF 分析表明 NdWFa 是肽的主要形式。
Neuropeptides are heavily posttranslationally modified (PTM) gene products that are often characterized by a variety of mass spectrometric approaches. Recently, the occurrence of amino acids in the D-form has been documented in several neuropeptides. As this modification has no associated mass shift, this particular PTM is difficult to evaluate using mass spectrometry (MS) alone. Here we demonstrate several approaches using capillary electrophoresis (CE) with absorbance and laser-induced fluorescence (LIF) for the separation of native and derivatized molluscan peptides containing D-amino acids. The combination of peptide derivatization followed by CE/LIF is well suited for single cell measurements because of its ability to characterize the peptides in such small samples. In order to verify this approach, the D-Trp-containing peptide NdWFa (NH2-Asn-D-Trp-Phe-CONH2), present in individual neurons from the marine mollusk Aplysia californica, has been characterized. The mass spectra show that NdWFa and/or NWFa are present in specific neurons; CE/LIF analysis of these cells demonstrates that NdWFa is the dominant form of the peptide.