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Rapid microfluidic sample preparation for mass spectrometric analysis of wild-type and mutant BRAF protein.

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Τίτλος: Rapid microfluidic sample preparation for mass spectrometric analysis of wild-type and mutant BRAF protein.
Συγγραφείς: Lin YH; Department of Biomedical Engineering, Chang Gung University, Taoyuan City, Taiwan; Department of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan City, Taiwan. Electronic address: yenheng@mail.cgu.edu.tw., Chang HY; Department of Biomedical Engineering, Chang Gung University, Taoyuan City, Taiwan., Wu CC; Molecular Medicine Research Center, Chang Gung University, Taoyuan City, Taiwan., Hsiao YC; Molecular Medicine Research Center, Chang Gung University, Taoyuan City, Taiwan; Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan City, Taiwan; Department of Otolaryngology-Head and Neck Surgery, Chang Gung Memorial Hospital at Linkou, Taoyuan City, Taiwan., Wen YH; Department of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan City, Taiwan; School of Medicine, National Tsing Hua University, Hsinchu City, Taiwan; School of Medicine, Chang Gung University, Taoyuan City, Taiwan., Yu JS; Molecular Medicine Research Center, Chang Gung University, Taoyuan City, Taiwan; Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan City, Taiwan; Department of Otolaryngology-Head and Neck Surgery, Chang Gung Memorial Hospital at Linkou, Taoyuan City, Taiwan; Research Center for Food and Cosmetic Safety, College of Human Ecology, Chang Gung University of Science and Technology, 33302, Taoyuan, Taiwan. Electronic address: yusong@mail.cgu.edu.tw.
Πηγή: Analytica chimica acta [Anal Chim Acta] 2026 Mar 15; Vol. 1391, pp. 345146. Date of Electronic Publication: 2026 Jan 23.
Τύπος έκδοσης: Journal Article; Research Support, Non-U.S. Gov't
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: Elsevier Country of Publication: Netherlands NLM ID: 0370534 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-4324 (Electronic) Linking ISSN: 00032670 NLM ISO Abbreviation: Anal Chim Acta Subsets: MEDLINE
Imprint Name(s): Publication: Amsterdam : Elsevier
Original Publication: Amsterdam.
Ιατρικοί όροι (MeSH): Proto-Oncogene Proteins B-raf*/genetics , Proto-Oncogene Proteins B-raf*/analysis , Microfluidic Analytical Techniques*/instrumentation , Mutant Proteins*/analysis , Mutant Proteins*/genetics, Humans ; Mass Spectrometry ; Mutation
Περίληψη: Background: The BRAF V600E mutant protein is a valuable biomarker for the diagnosis and prognosis of colorectal cancer. Quantitative detection by mass spectrometry (MS) requires purification from complex cell extracts and digestion into surrogate peptides, a process that traditionally takes at least ∼12 h, which is time-consuming and labor-intensive, limiting clinical applicability.
Results: We present an automated microfluidic sample-preparation chip that integrates immunoprecipitation, multistep washing, and on-bead tryptic digestion by integrating pneumatically driven micromixers, microvalves, and magnetically guided beads. On-bead digestion was employed to eliminate multiple buffer-exchange steps, simplifying fluidic control and producing more BRAF peptides than the conventional elution-digestion workflow, in which only ∼50 % of captured proteins are recovered. Starting from clarified cell lysate, the device produces peptide samples within approximately 2.5-3 h.
Significance and Novelty: Although individual components of the workflow have been reported previously, the present platform uniquely achieves end-to-end integration and automation of immuno-MS sample preparation. This work emphasizes operational simplicity and rapid turnaround time, providing a practical solution for MS-based mutant protein analysis in translational and precision medicine applications.
(Copyright © 2026 Elsevier B.V. All rights reserved.)
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Contributed Indexing: Keywords: Mass spectrometry; Microfluidics; Mutant BRAF; Proteomics; Sample preparation
Substance Nomenclature: EC 2.7.11.1 (Proto-Oncogene Proteins B-raf)
EC 2.7.11.1 (BRAF protein, human)
0 (Mutant Proteins)
Entry Date(s): Date Created: 20260209 Date Completed: 20260707 Latest Revision: 20260806
Update Code: 20260806
DOI: 10.1016/j.aca.2026.345146
PMID: 41663222
Βάση Δεδομένων: MEDLINE
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  Data: Rapid microfluidic sample preparation for mass spectrometric analysis of wild-type and mutant BRAF protein.
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  Data: <searchLink fieldCode="AU" term="%22Lin+YH%22">Lin YH</searchLink>; Department of Biomedical Engineering, Chang Gung University, Taoyuan City, Taiwan; Department of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan City, Taiwan. Electronic address: yenheng@mail.cgu.edu.tw.<br /><searchLink fieldCode="AU" term="%22Chang+HY%22">Chang HY</searchLink>; Department of Biomedical Engineering, Chang Gung University, Taoyuan City, Taiwan.<br /><searchLink fieldCode="AU" term="%22Wu+CC%22">Wu CC</searchLink>; Molecular Medicine Research Center, Chang Gung University, Taoyuan City, Taiwan.<br /><searchLink fieldCode="AU" term="%22Hsiao+YC%22">Hsiao YC</searchLink>; Molecular Medicine Research Center, Chang Gung University, Taoyuan City, Taiwan; Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan City, Taiwan; Department of Otolaryngology-Head and Neck Surgery, Chang Gung Memorial Hospital at Linkou, Taoyuan City, Taiwan.<br /><searchLink fieldCode="AU" term="%22Wen+YH%22">Wen YH</searchLink>; Department of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan City, Taiwan; School of Medicine, National Tsing Hua University, Hsinchu City, Taiwan; School of Medicine, Chang Gung University, Taoyuan City, Taiwan.<br /><searchLink fieldCode="AU" term="%22Yu+JS%22">Yu JS</searchLink>; Molecular Medicine Research Center, Chang Gung University, Taoyuan City, Taiwan; Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan City, Taiwan; Department of Otolaryngology-Head and Neck Surgery, Chang Gung Memorial Hospital at Linkou, Taoyuan City, Taiwan; Research Center for Food and Cosmetic Safety, College of Human Ecology, Chang Gung University of Science and Technology, 33302, Taoyuan, Taiwan. Electronic address: yusong@mail.cgu.edu.tw.
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  Data: <searchLink fieldCode="MM" term="%22Proto-Oncogene+Proteins+B-raf%22">Proto-Oncogene Proteins B-raf*</searchLink>/<searchLink fieldCode="MM" term="%22Proto-Oncogene+Proteins+B-raf+genetics%22">genetics</searchLink> <br /><searchLink fieldCode="MM" term="%22Proto-Oncogene+Proteins+B-raf%22">Proto-Oncogene Proteins B-raf*</searchLink>/<searchLink fieldCode="MM" term="%22Proto-Oncogene+Proteins+B-raf+analysis%22">analysis</searchLink> <br /><searchLink fieldCode="MM" term="%22Microfluidic+Analytical+Techniques%22">Microfluidic Analytical Techniques*</searchLink>/<searchLink fieldCode="MM" term="%22Microfluidic+Analytical+Techniques+instrumentation%22">instrumentation</searchLink> <br /><searchLink fieldCode="MM" term="%22Mutant+Proteins%22">Mutant Proteins*</searchLink>/<searchLink fieldCode="MM" term="%22Mutant+Proteins+analysis%22">analysis</searchLink> <br /><searchLink fieldCode="MM" term="%22Mutant+Proteins%22">Mutant Proteins*</searchLink>/<searchLink fieldCode="MM" term="%22Mutant+Proteins+genetics%22">genetics</searchLink><br /><searchLink fieldCode="MH" term="%22Humans%22">Humans</searchLink> ; <searchLink fieldCode="MH" term="%22Mass+Spectrometry%22">Mass Spectrometry</searchLink> ; <searchLink fieldCode="MH" term="%22Mutation%22">Mutation</searchLink>
– Name: Abstract
  Label: Abstract
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  Data: Background: The BRAF V600E mutant protein is a valuable biomarker for the diagnosis and prognosis of colorectal cancer. Quantitative detection by mass spectrometry (MS) requires purification from complex cell extracts and digestion into surrogate peptides, a process that traditionally takes at least ∼12 h, which is time-consuming and labor-intensive, limiting clinical applicability.<br />Results: We present an automated microfluidic sample-preparation chip that integrates immunoprecipitation, multistep washing, and on-bead tryptic digestion by integrating pneumatically driven micromixers, microvalves, and magnetically guided beads. On-bead digestion was employed to eliminate multiple buffer-exchange steps, simplifying fluidic control and producing more BRAF peptides than the conventional elution-digestion workflow, in which only ∼50 % of captured proteins are recovered. Starting from clarified cell lysate, the device produces peptide samples within approximately 2.5-3 h.<br />Significance and Novelty: Although individual components of the workflow have been reported previously, the present platform uniquely achieves end-to-end integration and automation of immuno-MS sample preparation. This work emphasizes operational simplicity and rapid turnaround time, providing a practical solution for MS-based mutant protein analysis in translational and precision medicine applications.<br /> (Copyright © 2026 Elsevier B.V. All rights reserved.)
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  Data: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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  Data: <i>Keywords: </i>Mass spectrometry; Microfluidics; Mutant BRAF; Proteomics; Sample preparation
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