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Analytical and Bioanalytical Chemistry

, Volume 411, Issue 5, pp 1085–1094 | Cite as

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

  • Yen-Heng LinEmail author
  • Heng-Yun Chang
  • Chia-Chun Wu
  • Chia-Wei Wu
  • Kai-Ping Chang
  • Jau-Song YuEmail author
Research Paper

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.

Graphical abstract

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.

Keywords

Micromixer Immunoprecipitation Enzymatic digestion Mutant BRAF Mass spectrometry Protein quantification 

Notes

Funding information

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.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

216_2018_1536_MOESM1_ESM.pdf (486 kb)
ESM 1 (PDF 486 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Electronic EngineeringChang Gung UniversityTaoyuanTaiwan
  2. 2.Graduate Institute of Biomedical EngineeringChang Gung UniversityTaoyuanTaiwan
  3. 3.Department of Otolaryngology-Head & Neck SurgeryChang Gung Memorial HospitalLinkouTaiwan
  4. 4.Graduate Institute of Biomedical SciencesChang Gung UniversityTaoyuanTaiwan
  5. 5.Molecular Medicine Research CenterChang Gung UniversityTaoyuanTaiwan
  6. 6.Liver Research CenterChang Gung Memorial HospitalLinkouTaiwan
  7. 7.Research Center for Food and Cosmetic Safety, Research Center for Chinese Herbal Medicine, College of Human EcologyChang Gung University of Science and TechnologyTaoyuanTaiwan

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