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BRAF protein immunoprecipitation, elution, and digestion from cell extract using a microfluidic mixer for mutant BRAF protein quantification by mass spectrometry

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Abstract

This study utilized a microfluidic mixer for the sample pretreatment of cell extracts for target protein quantification by mass spectrometers, including protein immunoprecipitation and protein enzymatic digestion. The time of sample pretreatment was reduced and thus the throughput of quantitative mutant proteins was increased by using the proposed method. Whole cell lysates of the cancer cell line HT-29 with gene mutations were used as the sample. The target protein BRAF was immunoprecipitated using magnetic beads in a pneumatic micromixer. Purified protein was then eluted and digested by trypsin in another two micromixers to yield peptide fragments in the solution. Using stable isotope-labeled standard as the internal control, wild-type and mutant BRAF proteins were quantified using mass spectrometry, which could be used for cancer screening. Compared with conventional methods in which protein immunoprecipitation lasts overnight, the micromixer procedure takes only 1 h, likely improving the throughput of mutant BRAF protein quantification by mass spectrometry.

Three micromixers were used to reduce the sample pretreatment time of cell extracts for target protein quantification by mass spectrometers, including protein immunoprecipitation, protein elution, and protein enzymatic digestion.

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Funding

The authors would like to thank the Ministry of Science and Technology of Taiwan and the Chang Gung Memorial Hospital for their funding support (105-2221-E-182-036-MY3, CMRPD2F0241, BMRPC01, CLRPD190018). This study was also supported by the “Molecular Medicine Research Center, Chang Gung University” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.

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Authors and Affiliations

  1. Department of Electronic Engineering, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Taoyuan, 333, Taiwan

    Yen-Heng Lin

  2. Graduate Institute of Biomedical Engineering, Chang Gung University, Kwei-Shan, Taoyuan, 333, Taiwan

    Yen-Heng Lin, Heng-Yun Chang & Chia-Wei Wu

  3. Department of Otolaryngology-Head & Neck Surgery, Chang Gung Memorial Hospital, Linkou, 333, Taiwan

    Yen-Heng Lin & Kai-Ping Chang

  4. Graduate Institute of Biomedical Sciences, Chang Gung University, 259 Wen-Hua 1st Road, Kwei-Shan, Taoyuan, 333, Taiwan

    Chia-Chun Wu & Jau-Song Yu

  5. Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan

    Kai-Ping Chang & Jau-Song Yu

  6. Liver Research Center, Chang Gung Memorial Hospital, Linkou, 333, Taiwan

    Jau-Song Yu

  7. Research Center for Food and Cosmetic Safety, Research Center for Chinese Herbal Medicine, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, 333, Taiwan

    Jau-Song Yu

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  1. Yen-Heng Lin
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Lin, YH., Chang, HY., Wu, CC. et al. BRAF protein immunoprecipitation, elution, and digestion from cell extract using a microfluidic mixer for mutant BRAF protein quantification by mass spectrometry. Anal Bioanal Chem 411, 1085–1094 (2019). https://doi.org/10.1007/s00216-018-1536-2

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