Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry |
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| Institution: | 1. Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan;2. 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;3. Department of Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan;4. Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan;5. Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan;6. Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan;7. Experimental Center of Functional Subjects, College of Basic Medicine, China Medical University, Shenyang, Liaoning 110122, China;8. Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan, Taiwan;9. Department of Otolaryngology, Chang Gung Memorial Hospital, Linkou, Taiwan;10. Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan;1. Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan;2. Global Research Cluster, RIKEN, Wako 351-0198, Japan;1. INRA, UR1268 Biopolymers Interactions Assemblies F-44316 Nantes, France;2. Synchrotron SOLEIL, L''Orme des Merisiers, F-91190 Gif-sur-Yvette, France;3. UAR 1008 CEPIA, INRA, F-44316 Nantes, France;4. Sorbonne Universités, Université Pierre et Marie Curie, Paris VI, CNRS, Integrative Biology of Marine Models, UMR 8227, Station Biologique, Place George Teissier, F29688 Roscoff Cedex, France;1. Department of Chemical Engineering, Hanyang University, Seoul 04763, South Korea;2. Department of Chemical and Biological Engineering, Korea University, Seoul 02841, South Korea;1. State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China;2. School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China;1. Departamento de Química Analítica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Instituto de Química de Rosario (IQUIR-CONICET), Suipacha 531, Rosario S2002LRK, Argentina;2. Universidade Federal da Paraíba (UFPB), Centro de Ciências Exatas e da Natureza, Departamento de Química, Castelo Branco, João Pessoa, PB, Brazil;3. Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17 177 Stockholm, Sweden;;4. SciLifeLab, 17165 Stockholm, Sweden |
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| 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 (592IGDFGLATVK601 and 592IGDFGLATEK601, 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. |
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| Keywords: | Oncogene BRAF V600E mutation Protein quantification Immunoenrichment Targeted mass spectrometry |
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