Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis
Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis
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DOI：
10.1021/acs.analchem.8b05234
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发表时间：
2019-05-07
影响因子：
7.4
通讯作者：
Zhang, Hui
中科院分区：
文献类型：
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作者：
Clark, David J.;Hu, Yingwei;Zhang, Hui
基于质谱的尿蛋白质组学是发现用于诊断、预后、监测或预测涉及肾脏、前列腺和膀胱的泌尿系统疾病的治疗反应的蛋白质的最有吸引力的策略之一;然而，在尿液中发现的干扰化合物需要目前不适合于临床环境中的尿蛋白质组学的样品制备策略。在此，我们描述了C4-tip方法，包括一个简单的，自动化的策略，利用反相树脂tip为基础的格式和“上尖”消化检查尿蛋白质组。我们首先使用标准蛋白牛胎球蛋白确定了在C4尖端上进行蛋白质分离和蛋白酶消化的最佳条件。接下来，我们将C4-tip方法应用于尿蛋白质组学，使用LC MS/MS鉴定了总共813个蛋白质组，与从超滤制备方法鉴定的蛋白质相比，从C4-tip方法鉴定的蛋白质显示出类似的基因本体（GO）细胞组分分配分布。最后，我们评估了C4-tip方法的重现性，揭示了所有tip中鉴定的共享蛋白质的高斯皮尔曼相关性R值。总之，我们已经证明了C4-tip方法是一种简单，可靠的方法，用于在临床环境中通过质谱法进行尿蛋白质组的高通量分析。
Mass spectrometry-based urinary proteomics is one of the most attractive strategies to discover proteins for diagnosis, prognosis, monitoring, or prediction of therapeutic responses of urological diseases involving the kidney, prostate, and bladder; however, interfering compounds found in urine necessitate sample preparation strategies that are currently not suitable for urinary proteomics in the clinical setting. Herein, we describe the C4-tip method, comprising a simple, automated strategy utilizing a reverse-phase resin tip-based format and "on-tip" digestion to examine the urine proteome. We first determined the optimal conditions for protein isolation and protease digestion on the C4-tip using the standard protein bovine fetuin. Next, we applied the C4-tip method to urinary proteomics, identifying a total of 813 protein groups using LC MS/MS, with identified proteins from the C4-tip method displaying a similar distribution of gene ontology (GO) cellular component assignments compared to identified proteins from an ultrafiltration preparation method. Finally, we assessed the reproducibility of the C4-tip method, revealing a high Spearman correlation R-value for shared proteins identified across all tips. Together, we have shown the C4-tip method to be a simple, robust method for high-throughput analysis of the urinary proteome by mass spectrometry in the clinical setting.