Intact protein mass spectrometry imaging at isotopic resolution enables assessment of covalent drug candidates

Covalent drugs form permanent bonds with their target proteins, offering distinct pharmacological advantages but presenting unique analytical challenges for in vivo assessment. We apply matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to detect covalent drug-intact target protein complexes. We optimize instrumentation parameters to increase the upper mass limit of MSI ( < 17 kDa) on a Fourier-transform ion cyclotron resonance (FT-ICR) MS, demonstrating that isotopically resolved spectra obtained by FT-ICR MS better enable removal of interfering ion’s signals than time of flight (TOF) MS. We generate a spatial map of in vivo percent target engagement [%TE = modified target protein/ (unmodified + modified) target protein] for a familial amyotrophic lateral sclerosis (fALS) drug candidate. Ratiometric imaging with high resolution deisotoping improves MS image quality, while spatially resolved %TE and intact hemoglobin signal enables blood-brain barrier penetration assessment. These techniques fill an important gap by enabling MSI studies of covalent drugs and provide in situ tools for early-stage pharmacological evaluation. Mass spectrometry plays a crucial role in drug development, yet traditional methods struggle with covalent drug pharmacokinetics and pharmacodynamics. Here, the authors demonstrate that direct measurement of drug–protein conjugates offers a more effective approach, with imaging providing spatially resolved insight that enhances the understanding of covalent drug behavior.