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解析肝癌发生、发展的分子机制并寻找高灵敏度的早期诊断标志物，是提升医疗质量的基础。该文首先针对血浆蛋白质组成复杂、高低丰度差异较大造成的蛋白质鉴定覆盖度低的问题，优化了液相色谱的梯度洗脱条件，将分离梯度从56 min延长至130 min，使血浆蛋白质鉴定数量从588个提升至1 261个；然后构建标准化研究流程，对肝癌患者与健康人的血浆进行无标记定量蛋白质组学分析，在两类样品中共定量到2 062个蛋白质，其中包含56个差异蛋白质（t检验，显著性水平p<0.05,1.5倍变化）。生物信息学分析结果显示，差异蛋白质涉及与癌症发生、发展紧密相关的多种功能和过程，实验得到的血浆蛋白质组定量结果可为后续肝癌发展机制及疾病潜在生物标志物的研究提供参考。
Abstract：[Objective] Early symptoms of hepatocellular carcinoma(HCC) are often either absent or very vague; thus, most patients are diagnosed at advanced stages with poor prognoses. Therefore, it is particularly important to elucidate the molecular mechanism underlying the onset and progression of HCC, and to identify sensitive early diagnostic biomarkers. Ultra-performance liquid chromatography-tandem mass spectrometry plays a pivotal role in proteomic research. The significant differences in the abundance and complex composition of plasma proteins result in low protein identification. Here, the influence of different liquid chromatography gradient separation conditions on the identification of plasma proteins was investigated. Furthermore, the developed method was applied for proteomic analysis of plasma samples from HCC patients and healthy individuals to identify early diagnostic biomarkers for HCC. [Methods] The effectiveness of different liquid chromatography separation gradients(56, 85, and 130 min) to identify plasma proteins was investigated. A standardized plasma analytical workflow suitable for proteomics research was established, which was combined with label-free quantitative proteomics technology, to screen differentially expressed proteins(DEPs) in plasma samples from HCC patients and healthy individuals. Bioinformatics analyses were performed to elucidate the molecular functions and biological processes of these DEPs associated with the pathogenesis of HCC. [Results] Extending the separation gradient from 56 to 85 and 130 min increased the number of identified plasma proteins from 588 to 988 and 1 261, respectively. Also, 64.1% and 63.6% of the proteins identified with separation gradients of 56 and 85 min were also identified at 130 min. In total, 563 proteins were identified, approximately 3.8 times more than individually obtained with a separation gradient of 130 min, which was attributed to the superior separation of complex peptides and greater protein identification coverage than achieved with the long separation gradient. Label-free quantitative analysis of the proteins from HCC patients and healthy individuals successfully quantified 2 062 protein groups. With cutoffs of p < 0.05(t-test) and 1.5-fold difference, 56 DEPs(33 up-regulated and 23 down-regulated) were identified in HCC patients. As many as 69.6% of the DEPs are reportedly associated with HCC, highly consistent with the results of previous studies. In addition, other DEPs not previously reported as biomarkers of HCC were significantly associated with liver cirrhosis, fatty liver, and liver fibrosis. Bioinformatics analysis indicated that many of the DEPs were closely associated with the onset and progression of HCC, such as serine-type endopeptidase inhibitor activity, haptoglobin binding action, immune response, and lipid transport. These results indicate that the identified DEPs may play crucial roles in the development of HCC Further research is ongoing to explore the potential of these DEPs as biomarkers of HCC. [Conclusions] The standardized plasma proteomics workflow established in this study not only provides a valuable methodological framework for subsequent investigations in this field but also offers diverse technical solutions for plasma proteome profiling. The quantitative results of the plasma protein profile obtained in this study provide data support for subsequent research on the mechanism of HCC development and the identification of potential biomarkers.
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基本信息:
DOI：10.16791/j.cnki.sjg.2025.12.008
中图分类号:O657.63;R735.7
引用信息:
[1]李一岚,虞雯静,施文超,等.基于非标记定量技术的肝癌血浆蛋白质组研究[J].实验技术与管理,2025,42(12):71-78.DOI:10.16791/j.cnki.sjg.2025.12.008.
基金信息:
北京理工大学实验室研究项目（2023BITSYA04）; 中国分析测试协会高校分析测试分会第一届“皖仪科技”卓越工程师提升计划项目（测协发字[2024]19号）
2025-10-23
2025-10-23
2025-10-23