Quantitative Mass Spectrometry Imaging of Intact Proteins in Tissues.

Quantitative imaging of endogenous proteins in biological tissues is essential for understanding their roles in cellular signaling and metabolism. Mass spectrometry imaging (MSI) using nanospray desorption electrospray ionization (nano-DESI) is a label-free approach for mapping intact proteins in biological tissues with minimal sample preparation. However, signal suppression during ionization presents a challenge for quantification. In this study, we introduce a quantitative nano-DESI MSI approach by incorporating protein internal standards (IS) into the extraction solvent. Ion images were generated using iFAMS deconvolution software, which efficiently isolates protein-specific signals from a complex background. A systematic evaluation of normalization strategies indicates that accurate quantification in each pixel is achieved using charge-weighted signal normalization to the IS signal. We demonstrate quantitative imaging of endogenous proteins using only one IS and validate the approach using immunofluorescence imaging and bulk analysis. This robust approach enables accurate protein mapping and quantification in MSI experiments, providing deeper insights into protein function in complex biological systems.