In this study, we developed a fully integrated protein absolute quantification platform for simultaneous analysis of multiple tumor markers in human plasma, by which multiple target proteins (alpha-fetoprotein, prostate-specific antigen, carcino-embryonic antigen and mucin-1) were firstly enriched by aptamers immobilized capillary column using graphene oxide modified polymer microsphere as the separation matrix, and then the eluted target proteins were online denatured, reduced, desalted and digested by our developed fully automated sample treatment device (FAST), finally the resulting peptides were analyzed by parallel reaction monitoring (PRM) on LTQ-orbitrap velos mass spectrometry. Compared to traditional ELISA assay, the platform exhibited significant advantages such as short analysis time, low limit of detection, and ease of automation. Furthermore, our developed platform was also applied in the absolute quantification of tumor markers from clinical human plasma samples, and the results were comparable to those obtained by clinical immunoassay. All the results demonstrated that such a platform could provide a promising tool for achieving high sensitivity, high accuracy, and high throughput detection of disease related protein markers in the routine physical examination and clinical disease diagnosis.

在这项研究中，我们开发了一个完全集成的蛋白质绝对定量平台，用于同时分析人血浆中的多种肿瘤标志物，首先通过该目标蛋白，可以将多种目标蛋白（甲胎蛋白，前列腺特异性抗原，癌胚胚胎抗原和粘蛋白1）分离出来。用氧化石墨烯修饰的聚合物微球作为分离基质，通过适体固定化毛细管柱富集，然后用我们开发的全自动样品处理装置（FAST）在线对洗脱的目标蛋白进行变性，还原，脱盐和消化，最后对所得肽进行分析LTQ-orbitrap velos质谱仪上的平行反应监测（PRM）进行分析。与传统的ELISA分析相比，该平台具有显着的优势，例如分析时间短，检测限低和易于自动化。此外，我们开发的平台还应用于从人血浆中提取的肿瘤标志物的绝对定量，其结果与通过临床免疫测定获得的结果相当。所有结果表明，这种平台可以为在常规身体检查和临床疾病诊断中实现疾病相关蛋白标记物的高灵敏度，高精度和高通量检测提供有前途的工具。