Updated publication reference for DOI(s): 10.4172/2165-7920.1000766. Purpose: To clinically and proteomically profile a fine-needle aspirate biopsy (FNAB) from a single in situ cold thyroid nodule (CTN). Experimental Design: The FNAB lysate was digested with trypsin, and analysed by LC-MSMS on an LTQ Orbitrap Velos. Remaining peptides were separated by reversed-phase 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 CTN FNAB. Results: Sixty seven and 2,595 non-redundant protein groups (2 unique peptides) were identified from unfractionated and fractionated CTN FNAB, 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. GO analysis revealed high-enrichment for extracellular vesicular exosome and vesicle (cellular component); regulation of biological quality (biological processes); and structural molecule activity (molecular function). Conclusions and Clinical Significance: The CTN was clinically-classified as benign. Proteomic data from FNAB can provide additional diagnostic candidates indicative of benign or cancerous CTN without the need for invasive surgical intervention.