Chen Hang, Hsiao Yung-Chin, Chiang Sum-Fu, Wu Chia-Chun, Lin Yu-Tsun, Liu Hsuan, Zhao Hong, Chen Jinn-Shiun, Chang Yu-Sun, Yu Jau-Song
Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110122, China.
Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; Department of Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan.
Anal Chim Acta. 2016 Aug 24;933:144-55. doi: 10.1016/j.aca.2016.05.037. Epub 2016 Jun 2.
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 measuring WT 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.
BRAF V600E突变是与包括结直肠癌（CRC）在内的几种癌症发生相关的最常见突变之一，在结直肠癌中，它与侵袭性疾病表型和不良预后相关。BRAF V600E突变状态通常通过直接DNA测序来确定。然而，以前没有研究试图对癌症标本中的BRAF V600E蛋白进行定量。在这里，我们评估了免疫富集结合两种基于质谱的定量技术，即多反应监测（MRM）和单离子监测共轭精确包含质量筛选（SIM-AIMS），以检测并精确量化DNA序列确认的CRC组织标本中的野生型（WT）和V600E突变型BRAF蛋白。从CRC细胞系（HT-29）中免疫沉淀WT和V600E BRAF蛋白，通过液相色谱-串联质谱（LC-MS/MS）分析确认其代表性肽段（分别为(592)IGDFGLATVK(601)和(592)IGDFGLATEK(601)），然后用加标的稳定同位素标记肽标准品通过MRM或SIM-AIMS进行定量。两种测定方法在测量不同量的HT-29细胞裂解物中的WT BRAF时都表现良好，但在定量较低水平的V600E BRAF时，MRM测定比SIM-AIMS测定更灵敏。在11个CRC组织标本的蛋白质提取物（2mg）中，MRM测定能够在所有11例中检测到WT BRAF（0.32 - 1.66ng），在2例中检测到V600E BRAF（0.1 - 0.13ng；突变型与野生型比例为0.16 - 0.17）。SIM-AIMS测定也能在CRC标本中检测到WT和V600E BRAF，但两个靶点的测量水平均低于MRM测定所确定的水平。总体而言，本研究提供了一种使用免疫富集耦合靶向质谱精确量化复杂生物样品中WT和V600E BRAF蛋白的有效方法。由于V600E BRAF蛋白已成为癌症的重要治疗靶点，所开发的测定方法应有助于未来与BRAF相关的基础和临床研究。
