title: Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry.
abstract: 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.
authors: 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.
authors: Hang
authors: H
authors: Chen
authors: Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; Department of Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan.
authors: Yung-Chin
authors: YC
authors: Hsiao
authors: Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan; Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors: Sum-Fu
authors: SF
authors: Chiang
authors: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors: Chia-Chun
authors: CC
authors: Wu
authors: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors: Yu-Tsun
authors: YT
authors: Lin
authors: Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan; Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors: Hsuan
authors: H
authors: Liu
authors: Experimental Center of Functional Subjects, College of Basic Medicine, China Medical University, Shenyang, Liaoning 110122, China.
authors: Hong
authors: H
authors: Zhao
authors: Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan.
authors: Jinn-Shiun
authors: JS
authors: Chen
authors: Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; Department of Otolaryngology, Chang Gung Memorial Hospital, Linkou, Taiwan.
authors: Yu-Sun
authors: YS
authors: Chang
authors: Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan. Electronic address: yusong@mail.cgu.edu.tw.
authors: Jau-Song
authors: JS
authors: Yu
mesh_terms: D015179:Colorectal Neoplasms / Q000175:diagnosis*; D006801:Humans; D013058:Mass Spectrometry; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000032:analysis* / Q000235:genetics / Q000276:immunology*
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
keywords: BRAF V600E mutation; Immunoenrichment; Oncogene; Protein quantification; Targeted mass spectrometry