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

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

PEPPI-MS: Strategies to Enhance the Extraction of Electrophoretically Separated Proteins from Polyacrylamide Gels and Their Application to Top-Down/native Mass Spectrometry

Polyacrylamide gel electrophoresis (PAGE) is a powerful technique for separating proteins from complex biological samples. However, the difficulty in recovering proteins with high yields from polyacrylamide matrices often precludes further analyses of intact proteins. Here, we propose a novel experimental workflow named Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS (‘PEPPI-MS’), which allows intact mass spectrometry (MS) of PAGE separated proteins. We discovered that staining proteins with certain Coomassie brilliant blue formulations immediately after PAGE improves the efficiency of extraction in a medium with pH 7–11. Post-staining, proteins spanning a broad range of molecular weights were recovered efficiently in a 10-minute procedure. High recovery yields were also obtained from dried and archived gels. This workflow is effective for top-down proteomics analysis of the target molecular region in the gel. An alternative procedure was developed for the extraction of protein complexes exceeding 400 kDa, which were separated using native PAGE, from unstained gels. Non-covalent hemoglobin tetramer, purified from cell lysate with two-dimensional native PAGE and extracted with the mild detergent octyl-β-Dglucopyranoside, was amenable for native MS analysis. We anticipate that the established workflow will facilitate the purification, storage, and transport of proteins destined for detailed characterization by MS

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

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