Quantitative Mass Spectrometry Imaging of Amino Acids Enabled by Pyridinium Salt MALDI Probe

Amino acids (AAs) are closely linked to various diseases. Investigating their spatial distribution and content differences can provide deeper insights into specific disease mechanisms. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) enables spatial visualization of biomolecules, but conventional matrices introduce significant background interference that limits the detection of small molecules such as AAs. On-tissue chemical derivatization (OTCD) using permanently charged pyridinium probes significantly enhances the detection sensitivity of poorly ionizable compounds like AAs, allowing for their spatial mapping. However, current quantitative mass spectrometry imaging (QMSI) strategies for AAs using conventional matrix remain limited, highlighting the urgent need for the development of a widely applicable absolute quantification method for AAs that integrates OTCD. In this study, a series of pyridinium salt-based MALDI-MS probes were designed and characterized, leading to the identification of an efficient candidate, 1-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)-2-methylphenyl)-2,4,6-triphenylpyridin-1-ium tetrafluoroborate (DCMT-4FB). This probe was then combined with deuterium-labeled internal standard to establish calibration curve, and its linear correction capability was validated, demonstrating strong correlation coefficients. Furthermore, a novel quantitative endogenous substance spraying approach was employed to perform absolute quantification MSI analysis of AAs (leucine and isoleucine) in different regions of human hepatocellular carcinoma (HCC) tissue sections. Finally, by cospraying the DCMT-4FB probe with its deuterium-labeled isotope analog, DCMT-d₂-4FB, the spatial distribution of AAs and other metabolites within HCC tissues was rapidly obtained, providing valuable insights for clinical research. This study highlights the superior AAs quantification capability of the DCMT-4FB probe and offers new perspectives for probe development and quantitative analysis of endogenous metabolites.