Nucleotide-enrichment-based MALDI-TOF MS assay
Multiple mutation detection is becoming necessary for clinical applications interested in the diagnosis, treatment decision, 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 for simultaneously achieving high sensitivity and high throughput. Herein, we present a novel multiple mutation 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 thyroid cancer somatic hotspot mutations. This method enhances sensitivity by removing the wild-type matched mass-modified dideoxynucleotide (ddNTP) from the nucleotide mixture in the single base extension reaction, leaving the mutant alleles available for analysis. The performance of the NE-MS assay was determined with an order of 1/8 fold lower limit of detection (LOD) than the regular MS method. This NE-MS strategy provided an excellent diagnostic performance for thyroid cancer, revealing that multiple mutations were associated with a poor prognosis in thyroid cancer. Accordingly, this study demonstrated that the NE-MS assay was a highly sensitive and reliable initial screening method for detecting somatic mutations and as a diagnostic and prognostic tool for thyroid nodules.