Abstract
Multiple mutation detection is increasingly essential for clinical applications targeting the diagnosis, treatment decision-making, and prognosis assessment of thyroid cancer, especially for the limited amount of thyroid fine-needle aspiration (FNA) samples. However, there is a lack of cost-effective methods that can simultaneously achieve high sensitivity and high throughput for thyroid cancer. Herein, we present a novel multiplex mutation detection technology that integrates nucleotide enrichment (NE)-assisted specific identification of variant alleles with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), termed NE-MS, enabling the simultaneous identification of 26 somatic hotspot mutations in thyroid cancer. This method enhances sensitivity by removing the mass-modified dideoxynucleotide (ddNTP) matching the wild-type from the nucleotide mixture during the single-base extension reaction, leaving the mutant alleles available for analysis. NE-MS exhibits an 8-fold lower LOD compared to the regular MS method. This strategy provided an excellent diagnostic performance for thyroid cancer, revealing that multiple mutations are associated with poor prognosis in thyroid cancer patients. Accordingly, this study demonstrated that the NE-MS assay is a highly sensitive and reliable initial screening method for somatic mutation detection, as well as a diagnostic and prognostic tool for thyroid nodules. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-39755-2.