PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry

Abstract

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS (“PEPPI-MS”), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry

“…These instruments can transmit large protein complexes without providing excessive activation that could compromise protein complex structure. In recent years, important progress in native purication methods such as native gel-eluted liquid fraction entrapment electrophoresis (GeLFrEE) separation, 36 native gel electrophoresis, 37 ion exchange chromatography (IEX), 38 hydrophobic interaction chromatography (HIC), 39,40 and online buffer exchange, has made these methods more applicable for complex mixtures and for characterisation of endogenous ligands. 36,41 By using appropriate sample preparation, ionisation conditions, and instrumentation, many challenging analytes such as membrane proteins, 18,42 intrinsically disordered proteins, 43 highly dynamic or heterogeneous complexes, 19 or very large systems such as intact virus capsids 44 can all be investigated using native MS, as well as their associated proteoforms.…”

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis

“…In a very recent study by Takemori et al. [141], the authors showed increased recovery (∼75%) when using a novel approach based on a passive elution of intact proteins from polyacrylamide gels and the use of aqueous solvents. The intrinsic weakness of these approaches is also their poor resolving power for certain classes of proteins (i.e.…”

“…Takemori and co‐workers [141] present in their recent study a novel workflow called Passively Eluting Proteins from Polyacrylamide gels as Intact species (PEPPI), which allows further top‐down MS identification of separated proteins. An illustration of this workflow is given in Figure 7.…”

“…Distribution of recovery efficiency with the theoretical molecular weight of recovered proteins (C). Reprinted with permission from the American Chemical Society (Copyright, 2020) [141]…”

Proteomic Profiling of Emiliania huxleyi Using a Three-Dimensional Separation Method Combined with Tandem Mass Spectrometry

“…The classic approach starts with the separation of proteins by 2-DE or LC prior to enzymatic digestion and LC-MS/MS analysis of the peptide digest [37]. The second strategy is applied in top-down proteomics, which allows the multi-dimensional separation in protein level, followed by MS/MS analysis [38]. The third method is widely used in bottom-up proteomics studies, which first digests the proteins into peptides prior to multi-dimensional separation and MS/MS analysis [39].…”