Native GELFrEE: A New Separation Technique for Biomolecular Assemblies

Abstract

The cadre of protein complexes in cells performs an array of functions necessary for life. Their varied structures are foundational to their ability to perform biological functions, lending great import to the elucidation of complex composition and dynamics. Native separation techniques that are operative on low sample amounts and provide high resolution are necessary to gain valuable data on endogenous complexes. Here, we detail and optimize the use of tube gel separations to produce samples proven compatible with native, multistage mass spectrometry (nMS/MS). We find that a continuous system (i.e., no stacking gel) with a gradient in its extent of cross-linking and use of the clear native buffer system performs well for both fractionation and native mass spectrometry of heart extracts and a fungal secretome. This integrated advance in separations and nMS/MS offers the prospect of untargeted proteomics at the next hierarchical level of protein organization in biology.

Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics

“…hannah venom was fractionated by NativeGELFrEE as described previously (28). Briefly, 1 mg of venom was suspended in 200 l of low salt solubilization buffer (50 mM imidazole, 500 mM aminocaproic acid, 1 mM EDTA, pH 7.0) and mixed with 30 l of 70% glycerol and 0.1% Ponceau S. Fractionation was performed via imidazole-based clear native GELFrEE carried out using 1-12% T gradient tube gel.…”

“…This was followed by gas-phase ejection of subunits (27), with the fragmentation of these by MS/MS and their identification being a more recent advance (23). The development of a separation technique termed native gel-eluted liquid fraction entrapment electrophoresis (native GELFrEE), allowed its linked use with native MS (23) to fully characterize intact complexes from endogenous systems (28). With many proteins performing their functions as members of protein assemblies, their direct observation could provide a highly informative view of the molecular composition and proteinprotein interactions in snake venom.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…Conversely, gel elution liquid fraction entrapment electrophoresis (GELFrEE) enables separation of intact proteins in a gel-based format but allows for liquid phase recovery of separated species, thereby bypassing the requirement for further sample pretreatment prior to MS analysis [167]. A slightly modified format of the GELFrEE system (native GELFrEE) has also been shown to be a viable technique for intact protein separations under native conditions [168].…”

“…In native MS, where physiological or near-physiological conditions are required, the task of separating non-denatured proteins and protein complexes at high resolution is further complicated. Conventional separation techniques compatible with native MS include size exclusion chromatography (SEC) [302], ion exchange chromatography (IEX) [163], hydrophobic interaction chromatography (HIC) [303], affinity chromatography [304], capillary isoelectric focusing (cIEF) [305,306], native gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) [167,307], native polyacrylamide gel electrophoresis [308], capillary zone electrophoresis (CZE) [67,309] and flow field-flow fractionation (F4) [310,311]. However, for many of these methods, online interfacing to a mass spectrometer is challenging or yet impossible.…”

“…cIEF may lead to unfolding of proteins and dissociation of protein complexes at their pIs and require the use of denaturing sheath flow buffers to maintain stable electrospray, which may limit native MS applications to relatively stable complexes. The native GELFrEE approach, although viable for the separation of native multimeric complexes and proteins in mixtures, has not yet been directly interfaced with MS or demonstrated the capability of high-resolution separations of charge-based proteoforms and species with very subtle structural differences [167,307].…”