Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus.
Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus.
复制标题
DOI：
10.3791/63322
复制
发表时间：
2022-05-31
影响因子：
1.2
通讯作者：
Li, Lingjun
中科院分区：
文献类型：
--
作者：
Phetsanthad, Ashley;Vu, Nhu Q.;Li, Lingjun
神经肽是调节几乎所有生理和行为过程的信号分子，如发育、繁殖、食物摄入和对外部应激源的反应。然而，人们对神经肽的生物化学机制、全面性及其功能作用仍知之甚少。这些内源性肽的表征被这类信号分子的巨大多样性所阻碍。此外，神经肽在比神经递质低100 - 1000倍的浓度下具有生物活性，并且在突触释放后容易被酶降解。质谱（MS）是一种高度敏感的分析工具，可以在没有全面的先验知识的情况下识别，量化和定位分析物。它非常适合全球分析神经肽和帮助发现新的肽。由于这类肽的低丰度和高化学多样性，一些样品制备方法、质谱采集参数和数据分析策略已经从蛋白质组学技术中改编，以实现最佳的神经肽表征。本文描述了从复杂生物组织中分离神经肽的方法，并利用液相色谱(LC)-MS和基质辅助激光解吸/电离(MALDI)-MS进行序列表征、定量和定位。从蓝蟹（Callinectes sapidus）这种没有全面基因组信息的生物中制备神经肽数据库的程序。这些工作流程可以适应研究其他类别的内源性肽在不同的物种使用各种仪器。质谱表征神经肽提供序列、定量和定位信息。这种优化的工作流程不仅适用于神经肽的研究，也适用于其他内源性肽的研究。本文提供的方案描述了使用LC-ESI-MS、MALDI-MS斑点和MALDI-MS成像的样品制备、质谱采集、质谱分析和神经肽数据库生成。
Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and response to external stressors. Yet, the biochemical mechanisms and full complement of neuropeptides and their functional roles remain poorly understood. Characterization of these endogenous peptides is hindered by the immense diversity within this class of signaling molecules. Additionally, neuropeptides are bioactive at concentrations 100x – 1000x lower than that of neurotransmitters and are prone to enzymatic degradation after synaptic release. Mass spectrometry (MS) is a highly sensitive analytical tool that can identify, quantify, and localize analytes without comprehensive a priori knowledge. It is well-suited for globally profiling neuropeptides and aiding in the discovery of novel peptides. Due to the low abundance and high chemical diversity of this class of peptides, several sample preparation methods, MS acquisition parameters, and data analysis strategies have been adapted from proteomics techniques to allow optimal neuropeptide characterization. Here, methods are described for isolating neuropeptides from complex biological tissues for sequence characterization, quantitation, and localization using liquid chromatography (LC)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS. A protocol for preparing a neuropeptide database from the blue crab, Callinectes sapidus, an organism without comprehensive genomic information, is included. These workflows can be adapted to study other classes of endogenous peptides in different species using a variety of instruments. Mass spectrometric characterization of neuropeptides provides sequence, quantitation, and localization information. This optimized workflow is not only useful for neuropeptide studies, but also other endogenous peptides. The protocols provided here describe sample preparation, MS acquisition, MS analysis, and database generation of neuropeptides using LC-ESI-MS, MALDI-MS spotting, and MALDI-MS imaging.
登录
查看更多内容
DOI：
10.3791/1207
发表时间：
2009-03-23
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Gutierrez, Gabrielle J;Grashow, Rachel G
通讯作者：
Grashow, Rachel G
影响因子：
4.4
作者：
Pratavieira, Marcel;da Silva Menegasso, Anally Ribeiro;Palma, Mario Sergio
通讯作者：
Palma, Mario Sergio
影响因子：
--
作者：
Lee JE
通讯作者：
Lee JE
影响因子：
4.1
作者：
Maes, Katrien;Van Liefferinge, Joeri;Van Eeckhauta, Ann
通讯作者：
Van Eeckhauta, Ann
DOI：
10.1016/j.chemolab.2021.104333
发表时间：
2021-06-01
影响因子：
3.9
作者：
Perez-Cova, Miriam;Bedia, Carmen;Jaumot, Joaquim
通讯作者：
Jaumot, Joaquim