色谱

色谱 ›› 2021, Vol. 39 ›› Issue (10): 1094-1101.DOI: 10.3724/SP.J.1123.2021.06018

• 研究论文 • 上一篇    下一篇

石蜡包埋组织中组蛋白的提取分离和翻译后修饰的定量分析

田姗姗1, 刘冉冉1, 钱晓龙2, 郭晓静2,*(), 张锴1,*()   

  1. 1.天津医科大学基础医学院, 天津市医学表观遗传学重点实验室, 天津 300070
    2.天津医科大学肿瘤医院乳腺病理研究室, 教育部乳腺癌防治重点实验室, 国家肿瘤临床医学研究中心, 天津 300060
  • 收稿日期:2021-06-10 出版日期:2021-10-08 发布日期:2021-09-10
  • 通讯作者: 郭晓静,张锴
  • 作者简介:E-mail: guoxiaojing@tjmuch.com(郭晓静).
    *E-mail: kzhang@tmu.edu.cn(张锴);
    第一联系人：#共同第一作者.
  • 基金资助:
    国家自然科学基金(21874100);国家自然科学基金(22074103);天津市自然科学基金(19JCZDJC35000);天津市自然科学基金(19JCQNJC08900)

Extraction and isolation of histones from paraffin-embedded tissues and quantitative analysis of post-translational modifications

TIAN Shanshan1, LIU Ranran1, QIAN Xiaolong2, GUO Xiaojing2,*(), ZHANG Kai1,*()   

  1. 1. School of Basic Medical Sciences, Tianjin Medical University, Tianjin Key Lab for Medical Epigenetics, Tianjin 300070, China
    2. Department of Breast Pathology and Lab, Tianjin Medical University Cancer Institute and Hospital, Key Laboratory of Breast Cancer Prevention and Therapy, Ministry of Education; National Clinical Research Center of Cancer; Tianjin 300060, China
  • Received:2021-06-10 Online:2021-10-08 Published:2021-09-10
  • Contact: GUO Xiaojing,ZHANG Kai
  • Supported by:
    National Natural Science Foundation of China(21874100);National Natural Science Foundation of China(22074103);Tianjin Natural Science Foundation(19JCZDJC35000);Tianjin Natural Science Foundation(19JCQNJC08900)

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摘要：

组蛋白翻译后修饰(HPTMs)参与基因转录调控,其异常与肿瘤等重大疾病的发生发展密切相关。石蜡包埋组织是当前疾病研究的重要样本资源,对肿瘤机制和标志物研究具有重要意义。目前基于质谱的蛋白质组学技术已成为HPTMs分析的有力工具,而针对福尔马林固定石蜡包埋(FFPE)组织样品的HPTMs分析还十分有限。该研究发展了一种基于高效液相色谱-串联质谱的FFPE组织样本HPTMs分离分析新方法。通过研究并优化组蛋白的提取策略,建立了FFPE组织样本脱蜡水化处理、蛋白质提取与聚丙烯酰胺凝胶电泳分离相结合的组蛋白提取和分离方法。通过研究FFPE切片数量、组蛋白化学衍生化方法等对组蛋白鉴定的影响,确定了组蛋白处理的具体步骤。通过HPLC分离结合非依赖性采集模式的质谱分析,鉴定了组蛋白修饰的类型、位点和丰度。最后,将优化的实验方法应用于FFPE临床样本的HPTMs分析,鉴定了2例人乳腺浸润性癌和癌旁正常组织的HPTMs图谱,均获得了100种以上的不同组蛋白修饰形式的多肽。定量分析了他们的差异性水平,通过主成分降维分析,发现浸润性癌和癌旁正常组织之间组蛋白修饰丰度存在明显的差异,且差异性具有一定的规律,特别是涉及转录调控的组蛋白修饰与乳腺癌的预后和治疗靶点具有相关性,进而探讨了乳腺癌中异常HPTMs的生物学意义。该研究对临床石蜡样本中组蛋白修饰的分离分析以及肿瘤表观遗传标志物的检测进行了有益的探索。

关键词: 高效液相色谱-串联质谱, 组蛋白翻译后修饰, 临床组织样本, 石蜡包埋, 乳腺癌

Abstract:

Histone post-translational modifications (HPTMs) have been believed to play crucial roles in the regulation of gene transcription. Thus, aberrant modification of histone can contribute to the occurrence and development of diseases such as tumors. To date, formalin fixed paraffin-embedded (FFPE) clinical tissues are believed to be one of the most valuable sample resources in the study of human diseases. Therefore, it is of great significance to reveal the molecular mechanism of cancer and discover the markers of tumor. Proteomics, based on high performance liquid chromatograph-tandem mass spectrometry (HPLC-MS/MS), has become a powerful tool for HPTM identification. However, HPTM analysis of FFPE samples is yet to be explored; it has not been reported in China to our best knowledge. In this study, a new method based on HPLC-MS/MS was developed for the extraction and separation of histone proteins and analysis and quantification of HPTMs in FFPE tissues. First, the strategy for the extraction and separation of histone proteins from FFPE samples were optimized. After comparing the extraction efficiency of two different methods, which include the acid extraction of histone and extraction of total protein, a novel method was developed for histone extraction, separation, and HPTMs analysis by integrating dewaxed hydration treatment of FFPE tissues with protein extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Furthermore, the effects of operation parameters on histone extraction and HPTM identification were investigated, including number of paraffin embedded sections and chemical derivation of histone proteins. Thereafter, the identification and quantification of HPTMs were performed using reversed-phase HPLC-MS/MS in data independent acquisition (DIA) mode. Finally, the optimized methods were applied to quantitative analysis of HPTMs in FFPE tissues. A variety of HPTMs were identified; they included lysine methylation, acetylation, crotonylation, etc. Therefore, the spectrum of HPTMs on global level was set for human breast cancer and paracancerous tissues from two cases of FFPE clinical tissues. The sites holding differential HPTM level in cancer and paracancerous tissues were further obtained by quantifying the individual HPTMs. Thus, the relationship between HPTM level and tumor was discussed. Abnormal HPTM sites were characterized using cluster analysis, thus their similar tendency was found. For example, abnormal HPTM sites such as H3K9me3, H3K9ac, and H3K27me3 in cancers are associated with epigenetic regulation. It indicated that different epigenetic events might occur in cancer and paracancerous tissues. Interestingly, we found that the level of H4K20me3 were both significantly down-regulated in the two cancer tissues. HPTM had been thought to be a potential biomarker in breast cancer; therefore, these positive results indicated that our method is effective for HPTMs of clinical FFPE samples. Our study provides a useful tool for the isolation and analysis of HPTMs in clinical FFPE samples, showing the potential for the detection of epigenetic biomarker in cancer.

Key words: high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), histone post-translational modifications (HPTMs), clinical tissues, paraffin-embedded, breast cancer

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