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



Takemori, Ayako, Butcher, David S, Harman, Victoria M ORCID: 0000-0002-0990-153X, Brownridge, Philip, Shima, Keisuke, Higo, Daisuke, Ishizaki, Jun, Hasegawa, Hitoshi, Suzuki, Junpei, Yamashita, Masakatsu
et al (show 5 more authors) (2020) PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry. JOURNAL OF PROTEOME RESEARCH, 19 (9). pp. 3779-3791.

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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.

Item Type: Article
Uncontrolled Keywords: polyacrylamide gel electrophoresis, Coomassie brilliant blue, fractionation, mass spectrometry, top-down proteomics, native mass spectrometry, 21 tesla FT-ICR
Depositing User: Symplectic Admin
Date Deposited: 21 Aug 2020 07:11
Last Modified: 18 Jan 2023 23:36
DOI: 10.1021/acs.jproteome.0c00303
Related URLs:
URI: https://livrepository.liverpool.ac.uk/id/eprint/3098283