Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Hydrophobic proteins, especially membrane proteins, are challenging to analyze due to low solubility.
• Existing protein digestion protocols often struggle with efficient solubilization and enzymatic accessibility of hydrophobic proteins.

Purpose of the Study:

• To develop and validate a novel protocol for enhanced digestion of hydrophobic proteins using phase-transfer surfactants (PTS).
• To improve the identification and recovery of hydrophobic peptides during proteomic analysis.

Main Methods:

• Development of a phase-transfer surfactant (PTS) protocol utilizing sodium deoxycholate (SDC) for protein digestion.
• Removal of SDC from acidified solutions using a water-immiscible organic solvent post-digestion.
• Application of the PTS protocol to membrane-enriched fractions from Escherichia coli and human cervical cancer HeLa cells.
• Analysis using two-dimensional strong cation exchange (SCX)-C18 LC-MS/MS.

Main Results:

• The PTS protocol significantly increased the number of identified proteins and hydrophobic peptide recovery compared to acid-labile surfactant methods.
• Analysis of HeLa cell membrane proteome identified 1450 proteins, with 53% being membrane proteins.
• The identified transmembrane domain distribution aligns with human databases, indicating unbiased membrane proteome digestion.

Conclusions:

• Phase-transfer surfactants (PTS), specifically SDC, offer a robust solution for digesting hydrophobic proteins.
• This PTS protocol enables more comprehensive and unbiased analysis of membrane proteomes.
• The method has broad applicability in proteomics for studying membrane-associated proteins.