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

## TL;DR

This study compares three methods for extracting proteins from olive leaves to find the most effective one for proteomics analysis.

## Contribution

The study evaluates and compares three protein extraction protocols for olive leaves with a focus on LC–MS/MS compatibility.

## Key findings

- Protocol A provided the highest protein yields and identified the most unique proteins and peptide sequences.
- Protocol A was more efficient in handling hydrophobic proteins based on RP-HPLC results.
- Protocol A showed greater sensitivity, efficiency, and reproducibility in LC–MS/MS analysis compared to other methods.

## Abstract

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 (Olea europaea), 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
(O. europaea 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.