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

[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.