Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements

George M. Janini, Thomas P. Conrads, Timothy D. Veenstra, Haleem J. Issaq*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

27 Scopus citations


We have developed an effective two-dimensional fractionation protocol of complex proteome mixtures that extends the ability to conduct more comprehensive proteome measurements. A sample containing intact proteins extracted from Saccharomyces cerevisiae was fractionated by liquid phase isoelectric focusing, followed by tryptic digestion and solid-phase extraction (SPE) clean-up and reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS-MS) of the resultant peptides. The clean-up step is designed to desalt the fractions and rid them of urea and ampholytes prior to analysis by LC-MS-MS. Fifty milligrams of protein were separated into 20 fractions by liquid-phase isoelectric focusing, spanning a pH range of 3-10. The effectiveness of the removal of ampholytes was monitored by capillary zone electrophoresis and LC-MS-MS. The ability to analyze all of the 20 fractions without any noticeable decrease in the separation efficiency demonstrates the overall effectiveness of the SPE clean-up step. The results show that the separation strategy is effective for high throughput characterization of proteins from complex proteomic mixtures.

Original languageEnglish
Pages (from-to)43-51
Number of pages9
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Issue number1
StatePublished - 5 Apr 2003
Externally publishedYes


  • Peptides
  • Proteins
  • Proteomes


Dive into the research topics of 'Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements'. Together they form a unique fingerprint.

Cite this