Enhancing MALDI-MSI spatial proteomics analysis through matrix solution acidification

Mass spectrometry imaging (MSI) is a powerful technique for spatially resolved proteomics analysis, offering rapid data acquisition and high lateral resolution. However, its utility is often constrained by limited proteomics coverage. In this study, we introduce a simple yet effective method to enhance the detection efficiency of matrix-assisted laser desorption/ionization (MALDI)-MSI for both intact proteins and high-mass tryptic peptides in tissues. By reformulating conventional matrices with elevated concentrations of formic acid (FA), we observed significant improvements in imaging performance, including enhanced peak detection and superior image quality. The efficacy of this approach was first validated using sinapinic acid (SA), a widely adopted matrix for intact protein imaging. Notably, the matrix acidification approach exhibited complementary benefits in tryptic peptide MSI. When using matrix α-cyano-4-hydroxycinnamic acid (CHCA) with 5% FA, we observed selective enhancement of high-mass peptides (>1500 <i>m</i>/<i>z</i>) in MSI, along with improved image quality. Collectively, our results demonstrate the potential of this approach to improve the proteomic coverage in MSI-based spatial proteomics analysis.