Hill Jessica L, McIver Kara B, Katzer Kaleigh, Foster Michelle T
Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, CO 80523, USA.
Methods Protoc. 2022 Apr 15;5(2):34. doi: 10.3390/mps5020034.
Lipedema is a multifaceted chronic fat disorder characterized by the bilateral and disproportionate accumulation of fat predominantly in the lower body regions of females. Research strongly supports that estrogen factors likely contribute to the pathophysiology of this disease. We aim to help demonstrate this link by quantifying estrogen factor differences between women with and without lipedema. For time and lipedema adipose tissue conservation, the Protein Simple WES machine will be utilized in place of traditional western blotting. Here, we are interested in evaluating estrogen related factors, such as, but not limited to, estrogen receptors and enzymes involved in the successive conversions of cholesterol and androgens to estrogens in human subcutaneous adipose. Evaluation of these factors within adipose tissue, however, is novel for this instrument. Thus, we optimized tissue lysis and protein extraction for 11 proteins of interest. Antibodies and their working concentrations were determined based upon specific and distinguishable (signal-to-noise) peaks from electropherogram outputs across different tissue lysate concentrations. We found that overnight acetone precipitation proved to be the best procedure for extracting protein from lipid rich adipose tissue samples. Six of the eleven proteins were found to migrate to their expected molecular weights, however, five did not. For proteins that did not migrate as expected, overexpression lysates and empty vector controls were used to validate detection antibodies. Protein extract from subcutaneous adipose tissue and overexpression lysates were then combined to understand if migration was specifically altered by adipose tissue. From these results, we concluded that the lipid rich nature of adipose tissue in combination with the separation matrix designated for use with the WES were preventing the appropriate migration of some proteins rather than non-specific antibody binding or inappropriate preparation methods.
脂肪性水肿是一种多方面的慢性脂肪紊乱疾病，其特征是脂肪主要在女性下半身区域双侧且不成比例地堆积。研究有力地支持雌激素因素可能促成了这种疾病的病理生理过程。我们旨在通过量化有和没有脂肪性水肿的女性之间的雌激素因素差异来帮助证明这种联系。为了节省时间和保留脂肪性水肿的脂肪组织，将使用Protein Simple WES机器代替传统的蛋白质印迹法。在此，我们有兴趣评估雌激素相关因素，例如但不限于雌激素受体以及参与人体皮下脂肪中胆固醇和雄激素连续转化为雌激素过程的酶。然而，在脂肪组织内评估这些因素对于该仪器来说是新颖的。因此，我们针对11种感兴趣的蛋白质优化了组织裂解和蛋白质提取方法。根据不同组织裂解物浓度的电泳图输出中特定且可区分的（信噪比）峰来确定抗体及其工作浓度。我们发现过夜丙酮沉淀被证明是从富含脂质的脂肪组织样本中提取蛋白质的最佳方法。11种蛋白质中有6种迁移到了预期的分子量，但有5种没有。对于未按预期迁移的蛋白质，使用过表达裂解物和空载体对照来验证检测抗体。然后将皮下脂肪组织的蛋白质提取物和过表达裂解物合并，以了解迁移是否因脂肪组织而发生特异性改变。从这些结果中，我们得出结论，脂肪组织富含脂质的性质与指定用于WES的分离基质相结合，正在阻止一些蛋白质的适当迁移，而不是非特异性抗体结合或不适当的制备方法。
