ObjectivesTo explore the molecular mechanisms underlying aggressive progression of papillary thyroid microcarcinoma and identify potential biomarkers.MethodsSamples were collected and sequenced using tandem mass tag-labeled liquid chromatography–tandem mass spectrometry. Differentially expressed proteins (DEPs) were identified and further analyzed using Mfuzz and protein–protein interaction analysis (PPI). Parallel reaction monitoring (PRM) and immunohistochemistry (IHC) were performed to validate the DEPs.ResultsFive thousand, two hundred and three DEPs were identified and quantified from the tumor/normal comparison group or the N1/N0 comparison group. Mfuzz analysis showed that clusters of DEPs were enriched according to progressive status, followed by normal tissue, tumors without lymphatic metastases, and tumors with lymphatic metastases. Analysis of PPI revealed that DEPs interacted with and were enriched in the following metabolic pathways: apoptosis, tricarboxylic acid cycle, PI3K-Akt pathway, cholesterol metabolism, pyruvate metabolism, and thyroid hormone synthesis. In addition, 18 of the 20 target proteins were successfully validated with PRM and IHC in another 20 paired validation samples. Based on machine learning, the five proteins that showed the best performance in discriminating between tumor and normal nodules were PDLIM4, ANXA1, PKM, NPC2, and LMNA. FN1 performed well in discriminating between patients with lymph node metastases (N1) and N0 with an AUC of 0.690. Finally, five validated DEPs showed a potential prognostic role after examining The Cancer Genome Atlas database: FN1, IDH2, VDAC1, FABP4, and TG. Accordingly, a nomogram was constructed whose concordance index was 0.685 (confidence interval: 0.645–0.726).ConclusionsPDLIM4, ANXA1, PKM, NPC2, LMNA, and FN1 are potential diagnostic biomarkers. The five-protein nomogram could be a prognostic biomarker.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40618-022-01960-x.