Formic Acid Enhanced Single-Pot Sample Preparation to Improve Identification Coverage of Membrane Proteome of Laser Capture Microdissected Tissue Sections

-Dodecyl-d-maltoside (DDM) is commonly used for protein extraction from small tissue sections in spatially resolved proteomics workflows, improving low-input sample recovery without surfactant removal prior to MS analysis. However, extracting and solubilizing highly hydrophobic membrane proteins using mild, nondenaturing detergents remains challenging. Herein, we propose a formic acid (FA)-enhanced single-pot sample preparation (FAESP) method to improve protein solubility and extraction from tissue samples, enhancing membrane proteome identification coverage in laser capture microdissected tissue sections. Due to its high volatility, FA is rapidly removed by vacuum drying without cleaning steps. Combination with DDM ensures compatibility with subsequent digestion and LC-MS analysis. Compared with the FA-free DDM-based method, FAESP achieves a 38% increase in proteome coverage, with 40% of identified proteins annotated as membrane proteins. The FAESP workflow was applied to neuron type-specific proteome profiling in the medial habenula (MHb) of mouse brain, revealing regional specificity of synaptic proteins and solute carrier proteins in aging MHb subregions. These results demonstrate that FAESP provides a superior tool for discovering membrane protein candidates that distinguish neuronal types, facilitating biological and clinical applications.