Protein Extraction Protocol for Legumes Compatible with Detergent: Gel Fractionation and Mass Spectrometry

Liquid chromatography coupled with mass spectrometry is a vital tool for proteomic analyses. While significant progress has been made in decoding the genomes of crops in recent decades, the composition of their proteomes-the complete set of expressed proteins in a species-remains largely unknown. The success of this technique heavily relies on efficient and optimized sample preparation, which is one of the most critical steps for maximizing the recovery of reliable information. In this chapter, we present a universal protein extraction protocol originally developed for a gel-based approach combined with an initial extraction step using methanol: chloroform: water (MCW) to remove high concentrations of secondary metabolites, such as pigments, phenolic compounds, lipids, carbohydrates, and terpenes. This enhanced protocol was specifically designed for extracting proteins from the phenolic-rich tissues of legumes. Our workflow allows the use of small amounts (less than 20 mg) of fresh-weight tissue and can identify over 2000 proteins per sample. Additionally, this approach is cost-effective compared to commercial kits, and its broad applicability across various plant tissues makes it particularly effective for challenging leguminous samples.