High-throughput liquid-liquid fractionation of multiple protein post-translational modifications

James H. Deford, Jonathan E. Nuss, James Amaning, Robert D. English, Don Tjernlund, John Papaconstantinou

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Post-translational protein modifications have contributed significantly to the identification of macro-molecular biomarkers of biological processes. We have modified a two-dimensional HPLC system (Beckman Coulter PF2D ProteomeLab) to create proteome maps of post-translational protein modifications. This system resolves complex protein mixtures by anion exchange chromatofocusing in the first dimension and hydrophobicity (reverse phase chromatography) in the second dimension. The simultaneous identification of multiple protein modifications, accomplished by incorporating a photo diode array (PDA) detector into the PF2D system, facilitates the simultaneous production of three-dimensional proteome maps and visualization of both unmodified and post-translationally modified (PTM) proteins at their signature wavelengths within the proteome. We describe procedures for the simultaneous resolution of proteome maps, the identification of proteins modified by nitration, carbonylation, and phosphorylation, and proteins with unique spectra such as the heme containing proteins.

Original languageEnglish (US)
Pages (from-to)907-916
Number of pages10
JournalJournal of Proteome Research
Volume8
Issue number2
DOIs
StatePublished - Feb 2009

Keywords

  • Carbonylation
  • Nitration
  • Phosphorylation
  • Post-translational modification

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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