Identification and Characterization of the Sulfolobus solfataricus P2 Proteome

Poh Kuan Chong and Phillip C. Wright*
Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom
J. Proteome Res., 2005, 4 (5), pp 1789–1798
DOI: 10.1021/pr0501214
Publication Date (Web): September 7, 2005
Copyright © 2005 American Chemical Society
*

 To whom correspondence should be addressed. Tel.:  +44(0)114 22 7577. Fax:  +44(0)114 2227501. E-mail:  p.c.wright@sheffield.ac.uk.

Abstract

Abstract Image

Via combined separation approaches, a total of 1399 proteins were identified, representing 47% of the Sulfolobus solfataricus P2 theoretical proteome. This includes 1323 proteins from the soluble fraction, 44 from the insoluble fraction and 32 from the extra-cellular or secreted fraction. We used conventional 2-dimensional gel electrophoresis (2-DE) for the soluble fraction, and shotgun proteomics for all three cell fractions (soluble, insoluble, and secreted). Two gel-based fractionation methods were explored for shotgun proteomics, namely:  (i) protein separation utilizing 1-dimensional gel electrophoresis (1-DE) followed by peptide fractionation by iso-electric focusing (IEF), and (ii) protein and peptide fractionation both employing IEF. Results indicate that a 1D-IEF fractionation workflow with three replicate mass spectrometric analyses gave the best overall result for soluble protein identification. A greater than 50% increment in protein identification was achieved with three injections using LC−ESI−MS/MS. Protein and peptide fractionation efficiency; together with the filtration criteria are also discussed.

Keywords: 2-DE • shotgun • LC−MS/MS • multiple injections • pre-fractionation • S. solfataricus

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History

  • Published In Issue October 10, 2005
  • Received April 27, 2005

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