Journal
JOURNAL OF PROTEOME RESEARCH
Volume 19, Issue 9, Pages 3779-3791Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.0c00303
Keywords
polyacrylamide gel electrophoresis; Coomassie brilliant blue; fractionation; mass spectrometry; top-down proteomics; native mass spectrometry; 21 tesla FT-ICR
Categories
Funding
- Scientific Research on Innovative Areas Chemistry for Multimolecular Crowding Biosystems (JSPS KAKENHI) [18H04559]
- National Science Foundation Division of Materials Research [DMR-1644779]
- National Science Foundation Division of Chemistry [DMR-1644779]
- Biotechnology and Biological Sciences Research Council [BB/M025756/1, BB/R005311/1]
- U.S. National Institutes of Health [R01GM104610, R01GM103479]
- National Institute of General Medical Sciences, National Institutes of Health [P41 GM108569]
- Sherman Fairchild Foundation
- State of Florida
- JSPS KAKENHI [16K08937, 19K05526]
- Grants-in-Aid for Scientific Research [16K08937, 18H04559, 19K05526] Funding Source: KAKEN
- BBSRC [BB/M025756/1, BB/R005311/1] Funding Source: UKRI
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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.
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