Profiling the alkaline membrane proteome of Caulobacter crescentus with two-dimensional electrophoresis and mass spectrometry

Mark P. Molloy, Nikhil D. Phadke, Hong Chen, Richard Tyldesley, David E. Garfin, Janine R. Maddock, Philip C. Andrews*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

49 Citations (Scopus)

Abstract

Attempts at protein profiling in the alkaline pH region using isoelectric focusing have often proved difficult, greatly limiting the scope of proteome analysis. We investigated several parameters using custom pH 8-11 immobilized pH gradients to separate a Caulobacter crescentus membrane preparation. These included sample application, quenching endoosomotic flow and gel matrix composition. Among these factors, the sample application position was the predominant parameter to affect two-dimensional gel quality. Separated proteins were silver stained and profiled using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The use of a prototype MALDI-Q-Tof mass spectrometer assisted identification of several proteins by providing highly informative peptide fragmentation data from the sample digests. Thirty-two unique alkaline proteins were identified in this study, which complements our previously described C. crescentus membrane proteome. Our experiments point towards new options for proteomic researchers aiming to both extend the scope of analysis, and simplify methods of identifying proteins with high confidence.

Original languageEnglish
Pages (from-to)899-910
Number of pages12
JournalProteomics
Volume2
Issue number7
DOIs
Publication statusPublished - 2002
Externally publishedYes

Keywords

  • Alkaline proteins
  • Caulobacter crescentus
  • Immobilized pH gradient
  • Mass spectrometry
  • Outer membrane proteins
  • Proteome

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