Dual Enhancement of Protein Imaging in Complex Tissues Using HydroWash: A Sequential Washing and Hydrogel Conditioning Strategy for DESI-MS.

Protein mass spectrometry imaging (MSI) is a promising tool for spatial proteomics but remains limited by low desorption efficiency and chemical interference from complex tissue matrices. To address these challenges, we developed HydroWash, a two-step strategy that combines conventional tissue washing with hydrogel conditioning to enhance DESI-MS protein imaging. A brief 5 min treatment with 5% gelatin hydrogel enhanced the DESI-MS signals of α-globin and β-globin in kidney tissues 3.0-3.6-fold and 1.7-2.5-fold, respectively. Mechanistic studies indicated that the hydrogel modulates the residual salt content following washing, thereby contributing to improved ionization efficiency. In total, two and five proteins showed significant signal enhancement in kidney and brain tissues, respectively. At a spatial resolution of 200 μm, the most pronounced improvement in brain tissue was observed in the white matter, where two isoforms of myelin basic protein (MBP) exhibited markedly elevated signals. Subsequent high-resolution imaging at 40 μm in cerebellar tissue revealed spatially coherent distributions of MBP isoforms in both the white matter and the granular layer using a standard DESI platform. Overall, HydroWash achieves significant improvement in protein MSI signal intensity while maintaining the appropriate spatial resolution. This suggests that HydroWash could serve as a viable method for improving the quality of protein imaging in complex biological tissues.