Comparative Analysis of Protein Extraction Protocols for Olive Leaf Proteomics: Insights into Differential Protein Abundance and Isoelectric Point Distribution.

Plant proteomics studies face two major challenges: limited databases due to the need for sequenced genomes and the difficulty in obtaining high-quality protein extracts. Olive (), a key species in Mediterranean flora known for its rich biochemical content, presents additional complexity due to its lipidic structure and high levels of inhibitory compounds that hinder protein extraction. Consequently, various studies have focused on optimizing the protein extraction methods for olives. While different extraction protocols exist for leaf proteome analysis, their compatibility with LC-MS/MS has been scarcely studied. This work was carried out to compare three protein extraction protocols for LC-MS/MS analysis using olive ( L) leaf tissue. Denaturing SDS (Method A), physiological CHAPS (Method B), and phenolic TCA/acetone (Method C) were evaluated with LC-MS/MS data. The quantitative comparisons of the three extraction methods revealed that Protocol A gave the greatest yields. According to the results obtained, Protocol A uniquely identified 77 proteins, Protocol B identified 10 unique proteins, and Protocol C identified 19 unique proteins. Similarly, the peptide sequence analysis showed that Protocol A uniquely identified 208 peptide sequences, Protocol B identified 29, and Protocol C identified 36. Moreover, reversed-phase high-performance liquid chromatography (RP-HPLC) results suggest that Method A may be more efficient in removing and retaining hydrophobic proteins. Overall, Protocol A demonstrated greater sensitivity, efficiency, and reproducibility in LC-MS/MS analysis.