Online matrix removal platform for coupling gel-based separations to whole protein electrospray ionization mass spectrometry

Ki Hun Kim, Philip D. Compton, John C. Tran, Neil L. Kelleher*

*Corresponding author for this work

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

A fractionation method called gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) has been used to dramatically increase the number of proteins identified in top-down proteomic workflows; however, the technique involves the use of sodium dodecyl sulfate (SDS), a surfactant that interferes with electrospray ionization. Therefore, an efficient removal of SDS is absolutely required prior to mass analysis. Traditionally, methanol/chloroform precipitation and spin columns have been used, but they lack reproducibility and are difficult to automate. Therefore, we developed an in-line matrix removal platform to enable the direct analysis of samples containing SDS and salts. Only small molecules like SDS permeate a porous membrane and are removed in a manner similar to cross-flow filtration. With this device, near-complete removal of SDS is accomplished within 5 min and proteins are subsequently mobilized into a mass spectrometer. The new platform was optimized for the analysis of GELFrEE fractions enriched for histones extracted from human HeLa cells. All four core histones and their proteoforms were detected in a single spectrum by high-resolution mass spectrometry. The new method versus protein precipitation/resuspension showed 2- to 10-fold improved signal intensities, offering a clear path forward to improve proteome coverage and the efficiency of top-down proteomics.

Original languageEnglish (US)
Pages (from-to)2199-2206
Number of pages8
JournalJournal of Proteome Research
Volume14
Issue number5
DOIs
StatePublished - May 1 2015

Keywords

  • SDS cleanup
  • cross-flow filtration
  • gel-based separation
  • online matrix removal device
  • top-down proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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