Quantifying Protein Acetylation in Diabetic Nephropathy from FFPE Explants

Background: Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus and a leading cause of chronic kidney disease and end stage renal disease. One potential mechanism underlying cellular dysfunction contributing to kidney disease is aberrant post-translational modifications, such as lysine acetylation. Lysine acetylation is associated with cellular metabolic flux and is thought to be altered in patients with diabetes and dysfunctional renal metabolism. Methods: A novel technique was adapted to quantify sites of N-acetylation from formalin-fixed paraffin-embedded kidney explant tissue from patients with diabetic kidney disease and non-diseased donors (n=5 and n=7, respectively). Multiple methodologies were integrated to extract viable proteins from formalin-fixed paraffin-embedded kidney explant tissue followed by enrichment and isolation. Proteomic and acetylomic profiles were then quantified via LC-MS/MS analysis. Results: Quantitative proteomic analysis of FFPE tissues identified 840 total proteins with 260 of those significantly changing and our acetylomic analysis quantified 290 acetylated peptides with 98 of those significantly changing in human diabetic kidneys. Lysine acetylation is known to regulate protein function, providing a mechanism by which proteins respond to cellular metabolic status. Protein pathways found to be impacted in DKD patients revealed an association with numerous metabolic pathways, specifically mitochondrial function, EIF2 signaling, oxidative phosphorylation, lipid metabolism, sirtuin signaling, and LXR/RXR activation, each of which are intimated to play a significant role in the pathogenesis of DKD. Differential protein acetylation in DKD patients reflected biochemical processes associated with sirtuin signaling, valine, leucine, and isoleucine degradation, lactate metabolism, oxidative phosphorylation, and ketogenesis. Conclusions: Here, we establish a quantitative acetylomics platform for protein biomarker discovery in formalin-fixed and paraffin-embedded biopsies of kidney transplant patients suffering from diabetic kidney disease. Our analysis provides a novel platform for quantifying enriched post-translational modifications in preserved explant tissues and includes optimizations for addressing protein crosslinking, paraffin removal, and formalin-derived formaldehyde protein adducts combined with immunoprecipitation of acetyl-peptides and total protein quantitation.

背景：糖尿病肾病（Diabetic kidney disease, DKD）是糖尿病的严重并发症，亦是慢性肾病及终末期肾病的主要诱因。引发肾脏病变的细胞功能障碍潜在机制之一，为异常的翻译后修饰，如赖氨酸乙酰化（lysine acetylation）。赖氨酸乙酰化与细胞代谢通量密切相关，且被证实于糖尿病患者与肾功能代谢异常人群中发生紊乱。
方法：本研究适配了一种新型技术，对糖尿病肾病患者与非疾病供体（样本量分别为n=5与n=7）的福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）肾脏外植体组织的N-乙酰化位点开展定量分析。研究整合多种方法从上述福尔马林固定石蜡包埋肾脏外植体组织中提取活性蛋白，随后完成富集与分离。最终通过液相色谱-串联质谱（LC-MS/MS）分析对蛋白质组学与乙酰化蛋白质组学谱进行定量检测。
结果：对福尔马林固定石蜡包埋组织的定量蛋白质组学分析共鉴定出840种总蛋白，其中260种存在显著差异表达；乙酰化蛋白质组学分析则定量到290条乙酰化肽段，其中98条在人类糖尿病肾病肾脏组织中存在显著差异变化。已知赖氨酸乙酰化可调控蛋白功能，为蛋白响应细胞代谢状态提供了作用通路。在糖尿病肾病患者中受影响的蛋白质通路显示与多种代谢通路密切相关，具体包括线粒体功能、真核翻译起始因子2（EIF2）信号通路、氧化磷酸化、脂质代谢、去乙酰化酶（sirtuin）信号通路以及肝X受体/类视黄醇X受体（LXR/RXR）激活，上述通路均被提示在糖尿病肾病的发病机制中发挥关键作用。糖尿病肾病患者中存在差异的蛋白乙酰化模式，则反映出与去乙酰化酶信号通路、缬氨酸、亮氨酸与异亮氨酸降解、乳酸代谢、氧化磷酸化以及酮体生成相关的生化过程。
结论：本研究构建了一套定量乙酰化蛋白质组学平台，可用于在罹患糖尿病肾病的肾移植患者的福尔马林固定石蜡包埋活检组织中筛选蛋白质生物标志物。本分析为在保存的外植体组织中定量富集的翻译后修饰提供了全新技术方案，同时包含了针对蛋白交联、石蜡去除以及福尔马林衍生的甲醛蛋白加合物的优化策略，并结合了乙酰肽段免疫沉淀与总蛋白定量技术。