Continuous flow-through multi-phase electromembrane extraction for protein desalting

Micro-electromembrane extraction (μ-EME) enables efficient protein desalting prior to mass spectrometry by applying an electric field to actively drive interfering salt ions across a free liquid membrane. While this technique provides excellent sample cleanup, conventional static micropipette tip formats face severe physical constraints, including intricate manual phase assembly, prolonged processing times per droplet, and fundamentally low sample throughput. Therefore, the critical problem addressed in this study is overcoming the operational limitations of static extraction by engineering a continuous flow-through EME architecture that automates liquid handling and maximizes protein desalting throughput.

A continuous flow-through EME platform was developed, processing samples at 2-6 μL/min. The baseline three-phase configuration, utilizing 1-pentanol organic phases and dialysis membranes, achieved 88.8-96.8% salt removal and 92.3-98.7% lysozyme recovery for 92.4-123 mM NaCl samples. To resolve electrolysis-induced acidification, an advanced five-phase configuration was engineered with outermost aqueous acceptor solutions. For 110 mM NaCl, this setup removed 98.2% Na⁺ and 99.6% Cl^- with 80.2% lysozyme recovery. Crucially, to tackle the challenging removal of weak-acid phosphate buffers, a concentration-matched ammonia solution was introduced into the anodic acceptor. This dynamically neutralized electrolysis-generated protons, stabilized the sample pH at ∼7.4, and prevented phosphate protonation, enabling >85.8% removal of 1-10 mM phosphate. Operating near the analytes' isoelectric points minimized electromigration, ensuring high recoveries of cytochrome C (>67.6%) and myoglobin (>91.9%).

The novelty of this study involves integrating a continuous flow-through architecture with an active, ammonia-based pH modulation strategy to facilitate the removal of complex polyprotic salts. Findings indicate that aligning the environmental pH with the protein's isoelectric point helps minimize adsorptive loss. Ultimately, this structurally optimized platform serves as a fundamental proof-of-concept for an automated sample pretreatment approach that enhances post-desalination mass spectrometry signal-to-noise ratios.