Khudyakov Jane I
Department of Biological Sciences, University of the Pacific, Stockton, CA, USA.
Methods Mol Biol. 2025;2884:43-56. doi: 10.1007/978-1-0716-4298-6_4.
Adipose tissue (AT) is a complex, multifunctional endocrine organ that plays a significant role in animal evolution and human disease. Profiling of the proteome, or the set of proteins produced by a cell or tissue at a given time, can be used to explore the myriad functions of adipose tissue and understand its role in health and disease. The main challenges of adipose tissue proteomics include the high lipid and low protein content of the tissue and association of many proteins with lipid droplets. Here, we present a protocol for gel-free, label-free, bottom-up, relative quantitative proteomics of adipose tissue based on findings from the literature and our laboratory that yields reproducible protein and peptide identification rates while minimizing cost and processing time. This approach involves tissue homogenization, protein precipitation from homogenates, solubilization and denaturation of proteins in a buffer containing 5% sodium deoxycholate (SDC, an acid-insoluble detergent) and 5 mM tris(2-carboxyethyl)phosphine (TCEP, a reducing agent), alkylation with chloroacetamide, and in-solution tandem digestion with trypsin and Lys-C enzymes in the presence of 1% SDC. Acidification of peptides efficiently removes SDC prior to desalting and mass spectrometry. This method has been used successfully in our laboratory by both experienced researchers and those with limited technical backgrounds, including high school, undergraduate, and graduate students. We have identified >1500 proteins in adipose tissue of non-model mammals (e.g., blubber of marine mammals) spanning a dynamic range of 10 using this approach, including proteins of interest for comparative physiology such as adipokines, metabolic and antioxidant enzymes, lipid droplet proteins, metabolite transporters, and mitochondrial proteins, among others.
脂肪组织（AT）是一个复杂的多功能内分泌器官，在动物进化和人类疾病中发挥着重要作用。蛋白质组分析，即对细胞或组织在特定时间产生的蛋白质集合进行分析，可用于探索脂肪组织的多种功能，并了解其在健康和疾病中的作用。脂肪组织蛋白质组学的主要挑战包括组织中高脂质和低蛋白质含量，以及许多蛋白质与脂滴的结合。在此，我们基于文献和我们实验室的研究结果，提出了一种用于脂肪组织的无凝胶、无标记、自下而上的相对定量蛋白质组学方法，该方法能产生可重复的蛋白质和肽段鉴定率，同时将成本和处理时间降至最低。这种方法包括组织匀浆、从匀浆中沉淀蛋白质、在含有5%脱氧胆酸钠（SDC，一种酸不溶性去污剂）和5 mM三（2-羧乙基）膦（TCEP，一种还原剂）的缓冲液中溶解和变性蛋白质、用氯乙酰胺进行烷基化，以及在1% SDC存在下用胰蛋白酶和Lys-C酶进行溶液内串联消化。在脱盐和质谱分析之前，肽段的酸化能有效去除SDC。我们实验室的经验丰富的研究人员以及技术背景有限的人员，包括高中生、本科生和研究生，都已成功使用了这种方法。使用这种方法，我们在非模式哺乳动物（如海洋哺乳动物的鲸脂）的脂肪组织中鉴定出了超过1500种蛋白质，其动态范围为10，包括比较生理学中感兴趣的蛋白质，如脂肪因子、代谢和抗氧化酶、脂滴蛋白、代谢物转运蛋白和线粒体蛋白等。
