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王莽原, 霍强, 杨毅宁. 人类心房组织转录因子新一代蛋白质组学高通量筛查TFRE实验方法的建立[J]. 四川大学学报(医学版), 2021, 52(2): 274-278. DOI: 10.12182/20210360208
引用本文: 王莽原, 霍强, 杨毅宁. 人类心房组织转录因子新一代蛋白质组学高通量筛查TFRE实验方法的建立[J]. 四川大学学报(医学版), 2021, 52(2): 274-278. DOI: 10.12182/20210360208
WANG Mang-yuan, HUO Qiang, YANG Yi-ning. Establishment of a New-generation High-throughput Proteomic Profiling of Transcription Factor in Human Atrial Tissue[J]. Journal of Sichuan University (Medical Sciences), 2021, 52(2): 274-278. DOI: 10.12182/20210360208
Citation: WANG Mang-yuan, HUO Qiang, YANG Yi-ning. Establishment of a New-generation High-throughput Proteomic Profiling of Transcription Factor in Human Atrial Tissue[J]. Journal of Sichuan University (Medical Sciences), 2021, 52(2): 274-278. DOI: 10.12182/20210360208

人类心房组织转录因子新一代蛋白质组学高通量筛查TFRE实验方法的建立

Establishment of a New-generation High-throughput Proteomic Profiling of Transcription Factor in Human Atrial Tissue

  • 摘要:
      目的  探究利用人类心房组织建立高通量高效筛查转录因子的实验技术,即转录因子反应元件(transcription factor response elements,TFRE)的方法。
      方法  本研究纳入2例术后患者的右心房组织(1例术前无房颤,1例术前有房颤)。提取人类心房组织细胞核蛋白质,测定其蛋白质浓度,制备磁珠-串联双拷贝转录因子反应元件序列(beads-catTFRE)复合体,利用beads-catTFRE复合体在核蛋白提取液中富集转录因子,高温高盐使核蛋白与beads-catTFRE复合体去结合后进行蛋白凝胶电泳,切胶、褪色后利用胰酶溶液进行胶内酶解,利用乙腈收缩凝胶条挤出其中的肽段并得到肽段溶液,干燥、溶解后上样进行质谱检测。利用Firmiana进行一站式数据分析和处理。
      结果  本次实验在正常右心房组织和房颤患者右心房组织两个实验组中分别鉴定到了220种、181种转录因子,两组一共鉴定到了241种转录因子,其中正常右心房组织中表达量高于中位数的转录因子前10%的有12个;房颤右心房组织中表达量高于中位数的转录因子前10%的有12个。
      结论  本研究建立高通量筛查方法覆盖度高,其检出数据可以用于支持进一步的实验验证研究。

     

    Abstract:
      Objective  To explore for the establishment of an experimental technique for profiling transcription factors, namely transcription factor response elements (TFRE), with high throughput and efficiency using human atrial tissue.
      Methods  Postoperative right atrial tissues from 2 patients, one with preoperative atrial fibrillation and the one with no preoperative atrial fibrillation, were included in the study. The nucleus protein was extracted from the human atrial tissue, and the protein concentration was then measured. A solution with a complex formed through combining magnetic beads with concatenated tandem array of the consensus transcription factor response element DNA sequence (beads-catTFRE) was prepared, and the beads-catTFREs were then used to enrich transcription factors in the nucleoprotein extraction. SDS-PAGE electrophoresis was performed after dissociating beads-catTFRE from nucleoprotein with high temperature and high salt. The gel was then cut and faded before enzymolysis by trypsin in the gels was performed. Acetonitrile was used to extract the peptides from the gels, and the peptide solution was then dried. After that, we dissolved the peptides and performed mass spectrum tests, and the data were analyzed and processed with Firmiana one-stop proteomic analysis platform.
      Results  In this study, 220 and 181 transcription factors were identified in the normal right atrial tissue and the right atrial tissue with atrial fibrillation, respectively. A total of 241 transcription factors were identified in the two groups. Among the 241 transcription factors, 12 were in the top 10% of those transcription factors that were above the median expression level of the normal right atrial tissue, and 12 transcription factors were in the top 10% of those above the median expression level of the right atrial tissue with atrial fibrillation.
      Conclusion  The high-throughput profiling method established in this study has high coverage, and the data collected can be used to support further validation studies.

     

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