Analysis of Proteins and Peptides by Electrokinetic Stacking Coupled with Paper Spray Mass Spectrometry

Less expensive, faster, and laborious protocols are needed for protein and peptide mass spectrometry (MS) for therapeutic protein characterization. This work describes a simple paper-based device that couples electrokinetic (EK) stacking via faradaic ion concentration polarization (f-ICP) with paper spray (PS) MS to enrich proteins and peptides on paper in minutes. The EK-PS device consists of a three-dimensional (3D)-printed holder with two polytetrafluoroethylene (PTFE) filter papers that act both as the media for electrokinetic stacking by f-ICP and as the supports for paper spray ionization. Modification of the PTFE filters via silanization improves MS ionization and imparts a positive surface charge to enable electro-osmotic flow. A potential of ∼280 V along the length of the papers induces electrokinetic stacking, whereas simultaneously floating the device at 4000 V generates online ionization via paper spray. Stacking and elution of proteins and peptides, which occurs by a combination of electro-osmotic flow and electrophoresis, takes ∼5-10 min and improves detection sensitivity by over 10-fold. In the analysis of tryptic protein digests and glycosylation profiling of monoclonal antibodies, electrokinetic stacking increases peptide identifications ∼9-fold and enables the detection of glycoforms above 3% relative abundance. The method is relatively simple and rapid, which may be useful for monitoring protein manufacturing.