...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Analytica chimica acta >Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry
【24h】

Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry

机译：免疫富集和靶向质谱法定量分析大肠癌中野生型和V600E突变型BRAF蛋白

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The BRAF V600E mutation is one of the most common mutations implicated in the development of several types of cancer including colorectal cancer (CRC), where it is associated with aggressive disease phenotypes and poor outcomes. The status of the BRAF V600E mutation is frequently determined by direct DNA sequencing. However, no previous study has sought to quantify the BRAF V600E protein in cancer specimens. Here, we evaluated immunoenrichment coupled with two MS-based quantitative techniques, namely multiple reaction monitoring (MRM) and single ion monitoring conjugated accurate inclusion mass screening (SIM-AIMS), to detect and precisely quantify wild-type (WT) and V600E mutant BRAF proteins in DNA sequence-confirmed CRC tissue specimens. WT and V600E BRAF proteins were immunoprecipitated from a CRC cell line (HT-29), and their representative peptides ((592)IGDFGLATVK(601) and (592)IGDFGLATEK(601), respectively) were confirmed by LC-MS/MS analysis and then quantified by MRM or SIM-AIMS with spiked stable isotope-labeled peptide standards. Both assays worked well for measuringWT BRAF from different amounts of HT-29 cell lysates, but the MRM assay was more sensitive than SIM-AIMS assay for quantifying lower levels of V600E BRAF. In protein extracts (2 mg) from 11 CRC tissue specimens, the MRM assay could measure WT BRAF in all 11 cases (0.32-1.66 ng) and the V600E BRAF in two cases (0.1-0.13 ng; mutant-to-WT ratio, 0.16-0.17). The SIM-AIMS assay could also detect WT and V600E BRAF in CRC specimens, but the measured levels of both targets were lower than those determined by MRM assay. Collectively, this study provides an effective method to precisely quantify WT and V600E BRAF proteins in complex biological samples using immunoenrichment-coupled targeted MS. Since the V600E BRAF protein has emerged as an important therapeutic target for cancer, the developed assay should facilitate future BRAF-related basic and clinical studies. (C) 2016 Elsevier B.V. All rights reserved.
机译：BRAF V600E突变是与几种类型的癌症（包括结直肠癌（CRC））相关的最常见突变之一，在这种情况下，它与侵袭性疾病表型和不良预后相关。 BRAF V600E突变的状态通常通过直接DNA测序来确定。然而，以前没有研究试图量化癌症标本中的BRAF V600E蛋白。在这里，我们评估了免疫富集结合两种基于MS的定量技术，即多反应监测（MRM）和单离子监测共轭精确包合质量筛选（SIM-AIMS），以检测和精确定量野生型（WT）和V600E突变体DNA序列确认的CRC组织标本中的BRAF蛋白。从CRC细胞系（HT-29）中免疫沉淀WT和V600E BRAF蛋白，并通过LC-MS / MS分析确认了它们的代表肽（分别为（592）IGDFGLATVK（601）和（592）IGDFGLATEK（601））。然后通过MRM或SIM-AIMS用加标的稳定同位素标记的肽标样进行定量。两种测定法都能很好地测量不同量的HT-29细胞裂解液中的WT BRAF，但是MRM测定法比SIM-AIMS测定法更灵敏，可定量测定较低水平的V600E BRAF。在11个CRC组织标本中的蛋白质提取物（2毫克）中，MRM分析可以测量所有11例（0.32-1.66 ng）的WT BRAF和两个案例（0.1-0.13 ng;突变体与WT的比率， 0.16-0.17）。 SIM-AIMS分析还可以检测CRC标本中的WT和V600E BRAF，但是两个靶标的测定水平均低于通过MRM测定法测定的水平。总而言之，这项研究提供了一种有效的方法，可以使用免疫富集偶联的靶向MS精确定量复杂生物样品中的WT和V600E BRAF蛋白。由于V600E BRAF蛋白已成为癌症的重要治疗靶标，因此开发的测定方法应有助于将来进行BRAF相关的基础和临床研究。 （C）2016 Elsevier B.V.保留所有权利。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号