Proteomic and Clinical Analysis of a Fine-Needle Aspirate Biopsy from a Single Cold Thyroid Nodule: A Case Study

Abstract

Background: For cases where clinical and cytological data from cold thyroid nodules are ambiguous; presurgical proteomic profiling of fine-needle aspirate biopsies of cold thyroid nodules in situ can provide additional diagnostics to avoid invasive surgical intervention and thyroidectomy of benign or non-cancerous tissue. Methods: The fine-needle aspirate biopsy lysate was digested with trypsin, and analysed by liquid chromatography mass spectrometry on a linear trap quadrupole Orbitrap Velos. Remaining peptides were separated by reversedphase chromatography and fractions analysed as technical duplicates. Identified proteins were analysed by Gene Ontology and protein abundance were calculated using the Top3 label-free method. The proteomic data was complemented with ultrasonography and scintigraphy of the thyroid gland; and cytology of the cold thyroid nodule fine-needle aspirate biopsy. Results: Sixty seven and 2,595 non-redundant protein groups (≥2 unique peptides) were identified from unfractionated and fractionated cold thyroid nodule fine-needle aspirate biopsy, respectively. Label-free protein abundance ranged over 6 orders of magnitude from the most abundant proteins, haemoglobin and thyroglobulin; to the low-abundance protein SON. Many previously-reported markers of thyroid cancer were in the top 23% of the identified proteins. Gene Ontology analysis revealed high-enrichment for cytoplasmic and membrane-bound organelle (cellular component); single-organism and small molecular processes (biological processes); and poly(A) ribonucleic acid, ribonucleic acid and protein-binding (molecular function). Conclusions: The cold thyroid nodule was clinically-classified as benign. Proteomic data from fine-needle aspirate biopsies can provide additional diagnostic candidates indicative of a benign or cancerous cold thyroid nodule without the need for invasive surgical intervention.