Fridjonsdottir Elva, Nilsson Anna, Wadensten Henrik, Andrén Per E
Biomolecular Imaging and Proteomics, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden.
Methods Mol Biol. 2018;1719:41-49. doi: 10.1007/978-1-4939-7537-2_2.
Neuropeptides are bioactive peptides that are synthesized and secreted by neurons in signaling pathways in the brain. Peptides and proteins are extremely vulnerable to proteolytic cleavage when their biological surrounding changes. This makes neuropeptidomics challenging due to the rapid alterations that occur to the peptidome after harvesting of brain tissue samples. For a successful neuropeptidomic study the biological tissue sample analyzed should resemble the premortem state as much as possible. Heat stabilization has been proven to inhibit postmortem degradation by denaturing proteolytic enzymes, hence increasing identification rates of neuropeptides. Here, we describe a stabilization protocol of a frozen tissue specimen that increases the number of intact mature neuropeptides identified and minimizes interference of degradation products from abundant proteins. Additionally, we present an extraction protocol that aims to extract a wide range of hydrophilic and hydrophobic neuropeptides by using both an aqueous and an organic extraction medium.
神经肽是由大脑信号通路中的神经元合成和分泌的生物活性肽。当肽和蛋白质所处的生物环境发生变化时，它们极易受到蛋白水解切割的影响。由于脑组织样本采集后肽组会迅速发生变化，这使得神经肽组学研究具有挑战性。为了成功开展神经肽组学研究，所分析的生物组织样本应尽可能接近生前状态。事实证明，热稳定化可通过使蛋白水解酶变性来抑制死后降解，从而提高神经肽的鉴定率。在此，我们描述了一种冷冻组织标本的稳定化方案，该方案可增加所鉴定的完整成熟神经肽数量，并将丰富蛋白质降解产物的干扰降至最低。此外，我们还提出了一种提取方案，旨在通过使用水性和有机提取介质来提取广泛的亲水性和疏水性神经肽。
