Membrane and Monolith Enzyme Reactors for Rapid Digestion of Proteins Prior to Mass Spectrometry Analysis

Using layer-by-layer adsorption of polyelectrolytes and enzymes in nylon membranes and polymer monoliths, this dissertation develops enzymatic reactors to rapidly and simply digest proteins for their analysis using mass spectrometry (MS). The research first develops modified membranes for electrodigestion of proteins separated in sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) gels. Electrophoresis remains a popular method for protein separation prior to MS analysis. Additionally, IEF separates protein charge variants for analysis of their post-translational modifications (PTMs) such as deamidations or glycosylations, which are important in understanding disease and creating effective protein therapeutics. However, MS analysis of gel-separated proteins requires laborious in-gel digestion procedures to create peptides and remove them from the gels. This dissertation describes direct electroblotting through enzyme-containing membranes to simplify extraction and digestion of proteins separated by SDS-PAGE and IEF.

MS analysis of extracted peptides showed that electrodigestion of an Escherichia coli (E. coli) cell lysate separated by SDS-PAGE results in only 13% fewer protein identifications than in-gel digestion, and in far less time. Both tryptic and peptic electrodigestion of a standard set of IEF-separated proteins led to identification of proteins with high % sequence coverages. Moreover, identified proteins show their strongest signals in the expected band, demonstrating retention of spatial resolution during electrodigestion. However, electrodigestion after IEF has not yet allowed identification of protein PTMs.

Next, this research aimed to separate intact proteins using capillary electrophoresis (CE) and rapidly digest these proteins in a monolith enzyme reactor (1-3 mm long) just prior to MS. Layer-by-layer adsorption of polyelectrolyte films and enzymes inside the monolith pores created highly active proteolytic reactors. Preliminary results confirm that compared to covalent immobilization in a monolith, adsorbed polyelectrolyte layers increase the enzyme immobilization several fold. An enzyme-modified monolith in an emitter tip for online CE analysis performed well offline and led to LC-MS/MS identification of bovine serum albumin (BSA) peptides with 96% sequence coverage. Future work with the monolith-containing emitter tip may yield successful digestion after CE protein separations.