Optimizing capillary electrophoresis for top-down proteomics of 30-80 kDa proteins

Yihan Li, Philip D. Compton, John C. Tran, Ioanna Ntai, Neil L. Kelleher*

*Corresponding author for this work

Research output: Contribution to journalArticle

59 Scopus citations

Abstract

The direct analysis of intact proteins via MS offers compelling advantages in comparison to alternative methods due to the direct and unambiguous identification and characterization of protein sequences it provides. The inability to efficiently analyze proteins in the "middle mass range," defined here as proteins from 30 to 80 kDa, in a robust fashion has limited the adoption of these "top-down" methods. Largely, a result of poor liquid chromatographic performance, the limitations in this mass range may be addressed by alternative separations that replace chromatography. Herein, the short migration times of CZE-ESI-MS/MS have been extended to size-sorted whole proteins in complex mixtures from Pseudomonas aeruginosa PA01. An electrokinetically pumped nanospray interface, a coated capillary, and a stacking method for on-column sample concentration were developed to achieve high-loading capacity and separation resolution. We achieved full width at half maximum of 8-16 s for model proteins up to 29 kDa and identified 30 proteins in the mass range of 30-80 kDa from P. aeruginosa PA01 whole cell lysate. These results suggest that CZE-ESI-MS/MS is capable of identifying proteins in the middle mass range in top-down proteomics.

Original languageEnglish (US)
Pages (from-to)1158-1164
Number of pages7
JournalProteomics
Volume14
Issue number10
DOIs
StatePublished - May 2014

Keywords

  • CZE-ESI-MS/MS
  • Pseudomonas aeruginosa PA01
  • Technology
  • Top-down proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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