一种用于精确定量致癌蛋白的稳健自动化工作流程，以加速靶向降解和肿瘤学研究

Quantitative and reproducible measurement of oncoproteins is essential for evaluating signaling biology, validating biomarkers, and advancing targeted degradation strategies such as Proteolysis-Targeting Chimeras PROTACs. Traditional Western blotting is widely used but is limited by variable assay performance and poor reproducibility, hindering its ability to support rigorous protein quantification across diverse sample types. 1 Similarly, ELISA detection methods are often not sufficiently sensitive and do not confirm the molecular weight of the detected protein. We developed a fully automated, calibration-based workflow using the Jess™ automated Western system to enable sensitive and absolute quantification of endogenous and overexpressed proteins. KRAS was used as a representative oncogenic target. While historically KRAS has been regarded as an undruggable target, recent advances in the field have demonstrated that PROTAC strategies can effectively degrade this oncogenic protein, increasing the important of precise KRAS quantification tools. 2 A recombinant KRAS standard curve (0.012-1.5 ng/µL) demonstrated excellent linearity (R² > 0.99), broad dynamic range, and high sensitivity. Precision testing showed robust peak area agreement across capillaries and days, confirming stable performance suitable for quantitative protein profiling. Application of this workflow to HeLa lysates, transiently transfected HEK293 cells, and extracellular vesicle samples enabled reliable detection of both high- and low-abundance KRAS populations. Quantitative assessment of the constitutively active KRAS mutant G12D antibody further highlighted the platform's ability to resolve clinically relevant variants with high specificity and sensitivity. 3 Although demonstrated using KRAS, this automated workflow is broadly applicable to additional oncoproteins, signaling molecules, and degradation substrates. Its integration of automated capillary electrophoresis, defined calibration standards, and digital quantification supports key applications including PROTAC efficacy assessment, variant-specific analysis, and biomarker development. By coupling reproducible assay performance with absolute quantification, this method provides a powerful platform to accelerate oncology research and therapeutic discovery across multiple protein targets. 1. S. C. Taylor, L. K. Rosselli-Murai, B. Crobeddu, I. Plante, A critical path to producing high quality, reproducible data from quantitative western blot experiments. Scientific Reports . 12 (2022) 2. T. Kos, D. Saur, Breaking down KRAS: Small-molecule degraders for cancer therapy. Signal Transduction and Targeted Therapy . 10 (2025) 3. Y. Tang et al. , Targeting KRASG12D mutation in non-small cell lung cancer: Molecular mechanisms and therapeutic potential. Cancer Gene Therapy . 31, 961-969 (2024)