In contemporary medical practice, human thyroglobulin (Tg) stands out as the primary serum biomarker for detecting recurrence or persistence (presence of residual tumor) of differentiated thyroid carcinoma (DTC) in patients post-thyroidectomy. Immunoassays (IMAs) and radioimmunoassays (RIAs) have been implemented in clinical settings to gauge Tg levels. However, these methods can be unreliable because anti-thyroglobulin antibodies (Tg-Abs) and heterophile antibodies (HAs) interfere with assay binding, leading to either under- or overestimation of true Tg concentrations. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has emerged as a distinctive alternative tool for Tg measurements. Despite its potential, the effectiveness of LC-MS/MS is under ongoing investigation. This review aims to provide a clear overview of existing follow-up procedures for Tg quantification and evaluate the potential of mass spectrometry (MS) in Tg analysis. The distinctive contribution of this review is the introduction of an emerging approach combining dried blood spots (DBSs) with LC-MS/MS for Tg measurement, emphasizing their translational potential for clinical follow-up of DTC patients.

Background/Objectives: In recent years, lipids have emerged as critical regulators of different disease processes, being involved in cancer pathogenesis, progression, and outcome. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) has significantly expanded the technology’s reach, enabling spatially resolved profiling of lipids directly from tissue, including formalin-fixed paraffin-embedded (FFPE) specimens. In this context, MALDI matrix selection is crucial for lipid extraction and ionization, influencing key aspects such as molecular coverage and sensitivity, especially in such specimens with already depleted lipid content. Thus, in this work, we aim to explore the feasibility of mapping lipid species in FFPE clinical samples with MALDI-MSI using 6-aza-2-thiothymine (ATT) as a matrix of choice. Methods: To do so, ATT performances were first compared to those two other matrices commonly used for lipidomic analyses, 2′,5′-dihydroxybenzoic acid (DHB) and Norharmane (NOR), on lipid standards. Results: As a proof-of-concept, we then assessed ATT’s performance for the MALDI-MSI analysis of lipids in FFPE brain sections, both in positive and negative ion modes, comparing results with those obtained from other commonly used dual-polarity matrices. In this context, ATT enabled the putative annotation of 98 lipids while maintaining a well-balanced detection of glycerophospholipids (60.2%) and sphingolipids (32.7%) in positive ion mode. It outperformed both DHB and NOR in the identification of glycolipids (3%) and fatty acids (4%). Additionally, ATT exceeded DHB in terms of total lipid count (62 vs. 21) and class diversity and demonstrated performance comparable to NOR in negative ion mode. Moreover, ATT was applied to a FFPE glioblastoma tissue microarray (TMA) evaluating the ability of this matrix to reveal biologically relevant lipid features capable of distinguishing normal brain tissue from glioblastoma regions. Conclusions: Altogether, the results presented in this work suggest that ATT is a suitable matrix for pathology imaging applications, even at higher lateral resolutions of 20 μm, not only for proteomic but also for lipidomic analysis. This could enable the use of the same matrix type for the analysis of both lipids and peptides on the same tissue section, offering a unique strategic advantage for multi-omics studies, while also supporting acquisition in both positive and negative ionization modes.

Matrix Assisted Laser Desorption/Ionisation-Mass Spectrometry Imaging (MALDI-MSI) is a well-established spatial omic technique which enables the untargeted mapping of various classes of biomolecules, including tryptic peptides, directly on tissue. This method relies on the use of matrices for the ionisation and volatilisation of analytes, and α-Cyano-4-hydroxycinnamic acid (CHCA) represents the most widespread matrix for tryptic peptides analysis. However, CHCA also presents certain limitations that foster the quest for novel matrix compounds. 6-aza-2-thiothymine (ATT), traditionally used in MALDI mass spectrometry (MS) for oligonucleotides, small molecules and oxidised phospholipids, has not been thoroughly investigated as a potential matrix for tryptic peptide analysis in MALDI-MS or MALDI-MSI. Therefore, this study addresses this gap by evaluating the capability of ATT to ionise tryptic peptides from Bovine Serum Albumin (BSA) and map in situ-digested peptides from formalin-fixed paraffin-embedded (FFPE) tissue sections in these respective applications. Comparative analysis with CHCA demonstrated the complementary strengths of these matrices for detecting tryptic peptides, establishing ATT as a feasible alternative to CHCA in the MALDI-MSI field and paving the way for future advancements in spatial proteomics.