开放性实验:基于超高效液相色谱-串联质谱法的甲状腺癌组织切片蛋白质组定量分析

doi:10.3724/SP.J.1123.2024.07009

色谱 ›› 2025, Vol. 43 ›› Issue (3): 275-282.DOI: 10.3724/SP.J.1123.2024.07009

开放性实验:基于超高效液相色谱-串联质谱法的甲状腺癌组织切片蛋白质组定量分析

李一岚¹^,^*(), 袁辉明², 曹竞天³, 郑怡迪⁴, 李兰¹, 高培峰¹

1.北京理工大学分析测试中心, 北京 102488
2.中国科学院大连化学物理研究所, 中国科学院分离分析化学重点实验室, 辽宁大连 116023
3.北京理工大学求是书院, 北京 102488
4.北京理工大学北京书院, 北京 102488

收稿日期:2024-07-09 出版日期:2025-03-08 发布日期:2025-03-03
通讯作者: * Tel:(010)81381285,E-mail:liyilan@bit.edu.cn.
基金资助:
北京理工大学实验室研究项目(2023BITSYA04);北京理工大学实验室研究项目(2021BITSYC11);中国科学院分离分析化学重点实验室开放基金(KL2301)

Open experiment: quantitative proteomics analysis of thyroid-cancer tissue slices using ultra-high performance liquid chromatography-tandem mass spectrometry

LI Yilan¹^,^*(), YUAN Huiming², CAO Jingtian³, ZHENG Yidi⁴, LI Lan¹, GAO Peifeng¹

1. Analysis and Testing Center, Beijing Institute of Technology, Beijing 102488, China
2. CAS Key Laboratoryof Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
3. Qiushi College, Beijing Institute of Technology, Beijing 102488, China
4. School of Beijing, Beijing Institute of Technology, Beijing 102488, China

Received:2024-07-09 Online:2025-03-08 Published:2025-03-03
Supported by:
Laboratory Research Project of Beijing Institute of Technology(2023BITSYA04);Laboratory Research Project of Beijing Institute of Technology(2021BITSYC11);Open Fund Project of Key Laboratory of Separation and Analytical Chemistry, Chinese Academy of Sciences(KL2301)

摘要/Abstract

摘要：

开放性实验课程是培养本科生实践能力、创新思维和团队协作精神的重要手段。本项目基于超高效液相色谱-串联质谱法(UHPLC-MS/MS)在蛋白质组前沿领域的研究,设计了一个本科生开放性实验,即基于UHPLC-MS/MS完成甲状腺癌组织切片蛋白质组定量分析。首先,评价不同蛋白质裂解液对蛋白质的提取能力,以筛选适当的试剂用于蛋白质组样品预处理,可培养学生的动手能力和科研思维。其次,优化液相色谱-质谱采集方法,实现蛋白质组的深度覆盖分析,加深学生对于液相色谱-质谱技术的认识。最后,将该方法用于石蜡包埋甲状腺乳头状癌组织切片样品蛋白质组定量分析,发现了33个差异蛋白质,展示出在疾病分型方面的应用潜力。该开放性实验将仪器分析、分析化学和生物化学三门课程理论与实验相关知识交叉融合在一起,将科研思路和创新理念渗透给本科生。以进行大型仪器操作和创新性实验的形式帮助学生拓展巩固理论知识,有助于学生形成系统化知识体系,增强其对相关领域知识的整体把握,激发本科生科学研究的兴趣,开拓创新思维,增加团队合作精神。

关键词: 液相色谱, 质谱, 蛋白质组学, 无标记定量, 实验教学

Abstract:

Open experiments play crucial roles in developing undergraduate students’ practical abilities, innovative thinking, and teamwork. This open experiment is designed to use ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in proteomics, a frontier field, by quantitatively analyzing thyroid-cancer-tissue-slice samples. Various reagents are first screened for further study by evaluating their abilities to extract proteins. This approach aims to foster students’ hands-on abilities and their scientific-research thinking. The liquid-chromatography method is subsequently optimized to enable deep-coverage of the proteome, thereby enhancing students’ understanding of liquid chromatography and mass spectrometry. Paraffin-embedded thyroid-cancer-tissue slices are finally subjected to quantitative proteomic analysis, resulting in the identification of 33 differentially expressed proteins, thereby demonstrating their potential use in disease-typing applications. This open experiment integrates theoretical and experimental knowledge gained through instrumental analysis, analytical chemistry, and biochemistry, thereby imparting scientific research thinking and innovative spirit to undergraduates. It also provides students with opportunities to expand and solidify theoretical knowledge through hands-on instrumental operation and experiments, which helps to build a systematic knowledge framework and develop a comprehensive understanding of relevant fields. Moreover, it stimulates undergraduate interest in scientific research, cultivates innovative thinking, and fosters team cooperation.

Key words: liquid chromatography (LC), mass spectrometry (MS), proteomics, label-free quantification, experimental teaching

中图分类号:

O658

李一岚, 袁辉明, 曹竞天, 郑怡迪, 李兰, 高培峰. 开放性实验:基于超高效液相色谱-串联质谱法的甲状腺癌组织切片蛋白质组定量分析[J]. 色谱, 2025, 43(3): 275-282.

LI Yilan, YUAN Huiming, CAO Jingtian, ZHENG Yidi, LI Lan, GAO Peifeng. Open experiment: quantitative proteomics analysis of thyroid-cancer tissue slices using ultra-high performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2025, 43(3): 275-282.

图/表 8

图1 不同蛋白质提取试剂鉴定到的(a)蛋白质数量与 (b)蛋白质的韦恩图

Fig. 1 (a) Numbers and (b) Venn diagram of identified proteins under different protein extraction buffers GuHCl: guanidine hydrochloride; SDS: sodium dodecyl sulfate.

图2 不同分离梯度下肽段的总离子流色谱图

Fig. 2 Total ion chromatograms of peptides with different separation gradients

图3 同一离子(m/z 855.46747)在不同分离梯度下的提取离子色谱图

Fig. 3 Extracted ion chromatograms of the ion (m/z 855.46747) under different separation gradients

图4 不同分离梯度下(a)蛋白质和肽段的鉴定数量与 (b)蛋白质的韦恩图

Fig. 4 (a) Numbers of identified proteins and peptides under different separation gradients; (b) Venn diagram of identified proteins

图5 (a)在85 min的分离梯度下离子(m/z 855.46747)的色谱峰,(b)3次重复实验鉴定蛋白质的韦恩图及(c)峰强度的皮尔森相关性分析

Fig. 5 (a) Chromatographic peaks of the ion (m/z 855.46747) under the separation gradient of 85 min, (b) Venn diagram of identified proteins and (c) Pearson correlation coefficients of the intensity within three replicated experiments R1: replicate 1; R2: replicate 2; R3: replicate 3.

图6 组织切片样品定量到的蛋白质的无标记定量强度分析的重复性

Fig. 6 Repeatability of the proteins quantified from tissue slices a. goiter (G); b. thyroid papillary carcinoma T1N0M0 (T).

图7 (a)甲状腺肿和甲状腺乳头状癌组织切片样品定量到的蛋白质的火山图和(b)差异蛋白质分级聚类分析图

Fig. 7 (a) Volcano plots of proteins identified from thyroid papillary carcinoma and goiter tissue slices, and (b) unsupervised clustering of differential proteins T/G: the ratio of intensities of proteins identified in thyroid papillary carcinoma patients and goiter patients.

图8 甲状腺肿和甲状腺乳头状癌组织切片样品差异蛋白质的基因本体论(a)生物学过程、(b)KEGG通路和(c)分子学功能分析

Fig. 8 Gene ontology (a) biological process, (b) KEGG pathway, and (c) molecular function analysis of differential proteins between goiter and thyroid papillary carcinoma tissue slices

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开放性实验:基于超高效液相色谱-串联质谱法的甲状腺癌组织切片蛋白质组定量分析

Open experiment: quantitative proteomics analysis of thyroid-cancer tissue slices using ultra-high performance liquid chromatography-tandem mass spectrometry

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