SDS Depletion from Intact Membrane Proteins by KCl Precipitation Ahead of Mass Spectrometry Analysis

TL;DR

This paper introduces a new method to remove SDS from membrane proteins before mass spectrometry, improving analysis of intact proteins.

Contribution

A novel KCl precipitation workflow is developed for SDS removal, enabling high-quality proteoform analysis of intact membrane proteins.

Findings

KCl precipitation at pH 12 with urea maximized membrane protein recovery and purity.

Bottom-up analysis identified 1826 distinct liver protein groups using the optimized protocol.

The workflow achieved 69.3% identification of membrane proteins under optimal conditions.

Abstract

Background: Membrane proteins are preferentially solubilized with sodium dodecyl sulfate (SDS), which necessitates a purification protocol to deplete the surfactant prior to mass spectrometry analysis. However, maintaining solubility of intact membrane proteins is challenged in an SDS-free environment. SDS precipitation with potassium salts (KCl) offers a potentially viable workflow to deplete SDS and permit proteoform analysis. The purpose of this study is to devise a robust detergent-based protocol applicable for processing and analysis of intact membrane-associated proteoforms. Methods: The precipitation conditions impacting SDS removal from spinach chloroplasts and liver membrane proteome preparations were evaluated, capitalizing on optimization of pH (highly basic), addition of MS-compatible solubilizing additives (urea) and adjustment of the KCl to SDS ratio to maximize recovery and purity. Results: Characterization of the SDS-solubilized, KCl-precipitated spinach membrane preparation revealed multiple charge envelope MS spectra displaying high signal to noise, free of SDS adducts. Precipitation at pH 12 or with urea improved protein recovery and purity. Bottom-up analysis identified 1826 distinct liver protein groups from four independent SDS precipitation conditions. While precipitation at pH 8 without urea revealed a greater number of protein identifications by mass spectrometry, precipitation under highly basic conditions (pH 12) with urea provided higher membrane protein recovery and achieved the greatest number (732 of 1056) and largest percentage (69.3%) of membrane proteins identified in the SDS removal workflow. Conclusion: This workflow provides new opportunities for MS-based proteoform analysis by capitalizing on the benefits of SDS for protein extraction while maintaining high solubility and purity of intact proteins though KCl precipitation of the surfactant.

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