Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706-1322, USA.
Analyst. 2019 Dec 16;145(1):61-69. doi: 10.1039/c9an01883b.
Neuropeptides are important signaling molecules responsible for a wide range of functions within the nervous and neuroendocrine system. However, they are difficult to study due to numerous challenges, most notably their large degree of variability and low abundance in vivo. As a result, effective separation methods with sensitive detection capabilities are necessary for profiling neuropeptides in tissue samples, particularly those of simplified model organisms such as crustaceans. In order to address these challenges, this study utilized a capillary electrophoresis (CE)-matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) platform, building upon our previous design for improved neuropeptidomic coverage. The capillary was coated with polyethylenimine (PEI) to reduce peptide adsorption and reverse the electroosmotic flow, and large volume sample stacking (LVSS) was used to load and pre-concentrate 1 μL of sample. The method demonstrated good reproducibility, with lower than 5% relative standard deviation for standards, and a limit of detection of approximately 100 pM for an allatostatin III peptide standard. The method was tested on brain and sinus gland (SG) tissue extracts and enabled detection of over 200 neuropeptides per run. When comparing the number detected in brain extracts in a direct spot, 60-second fractions, and 30-second fractions, the continuous trace collection afforded by the CE-MALDI-MSI platform yielded the largest number of detected neuropeptides. The method was compared to conventional LC-ESI-MS, and though the number of neuropeptides detected by LC-ESI-MS was slightly larger, the two methods were highly complementary, indicating the potential for the CE-MALDI-MSI method to uncover previously undetected neuropeptides in the crustacean nervous system. These results indicate the potential of CE-MALDI-MSI for routine use in neuropeptide research.
神经肽是重要的信号分子，负责神经系统和神经内分泌系统的广泛功能。然而，由于存在许多挑战，例如它们在体内的高度变异性和低丰度，因此很难对其进行研究。因此，需要有效的分离方法和具有敏感检测能力的方法来对组织样本中的神经肽进行分析，特别是对简化模型生物（如甲壳类动物）的组织样本。为了应对这些挑战，本研究利用了毛细管电泳（CE）-基质辅助激光解吸/电离（MALDI）-质谱成像（MSI）平台，该平台基于我们之前用于提高神经肽组学覆盖范围的设计。毛细管用聚乙烯亚胺（PEI）进行涂层处理，以减少肽的吸附并逆转电渗流，并且使用大体积样品堆积（LVSS）来加载和预浓缩 1 μL 的样品。该方法具有良好的重现性，标准品的相对标准偏差低于 5%，并且对一种 Allatostatin III 肽标准品的检测限约为 100 pM。该方法在脑和窦腺（SG）组织提取物上进行了测试，每次运行可检测到 200 多种神经肽。在直接点、60 秒馏分和 30 秒馏分中比较脑提取物中检测到的神经肽数量时，CE-MALDI-MSI 平台提供的连续痕量采集可获得最多的神经肽检测数量。将该方法与传统的 LC-ESI-MS 进行了比较，尽管 LC-ESI-MS 检测到的神经肽数量略多，但两种方法具有很强的互补性，表明 CE-MALDI-MSI 方法有可能在甲壳类动物神经系统中发现以前未检测到的神经肽。这些结果表明 CE-MALDI-MSI 有潜力在神经肽研究中常规使用。
