Department of Pharmaceutical Biosciences, Spatial Mass Spectrometry, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA.
Methods Mol Biol. 2024;2758:49-60. doi: 10.1007/978-1-0716-3646-6_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 living 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 two different stabilization protocols for rodent brain samples that increase the number of intact mature neuropeptides and minimize interference from degradation products of abundant proteins. Additionally, we present an extraction protocol that aims to extract a wide range of hydrophilic and hydrophobic neuropeptides by sequentially using an aqueous and an organic extraction medium.
神经肽是由神经元在大脑信号通路中合成和分泌的生物活性肽。当生物环境发生变化时，肽和蛋白质极易受到蛋白水解的影响。这使得神经肽组学具有挑战性，因为在采集脑组织样本后，肽组会迅速发生变化。为了成功进行神经肽组学研究，分析用的生物组织样本应尽可能接近活体状态。热稳定化已被证明可以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。在这里，我们描述了两种不同的稳定化方案，可增加成熟完整神经肽的数量，并最大限度减少丰富蛋白质的降解产物的干扰。此外，我们还介绍了一种提取方案，旨在通过顺序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。
