Improving the Annotation for Spatial Proteomics: A Computational Approach to Enhance Molecular Characterization of Thyroid Nodules.

The present work proposes a reproducible and automated workflow for integrating digital pathology in matrix-assisted laser-desorption ionization mass spectrometry imaging (MALDI-MSI) data analysis, using thyroid tissue as a proof-of-concept application. MALDI-MSI has shown promise in the molecular characterization of thyroid neoplasms. Yet challenges remain in minimizing signal interferents and improving diagnostic discrimination. In this study, we propose an interdisciplinary approach integrating digital pathology with spatial proteomics to enhance MALDI-MSI analysis of thyroid lesions from formalin-fixed paraffin-embedded tissue sections. We trained a pixel classifier to automatically select cell-rich regions of interest (ROIs) from hematoxylin and eosin-stained tissue microarrays, reducing interference from colloid-rich areas. The proteomics signals obtained with the pixel classifier (PC) were compared with those obtained from the full core (FC) and those manually annotated by the pathologist (PAT). The results showed that PC ROIs significantly decreased interfering signals (15%) while increasing the signal-to-noise ratio of tryptic peptides (+37%). Indeed, we detected a greater number of m/z signals (between 9 and 24%), improving the spectral clustering by means of principal component analysis to distinguish different histopathological regions. Receiver operating characteristic (ROC) analysis further confirmed the improved classification power, with a 50% increase in discriminatory m/z features across different thyroid nodules diagnosis compared to conventional FC and PAT data. Using a PC to select cell-specific regions globally enhances reproducibility, reduces operator workload, and optimizes MALDI-MSI workflows. Altogether, the proposed approach paves the way for more accurate molecular characterization of thyroid neoplasms and holds potential for improving biomarker discovery and diagnostic precision in clinical pathology.