Research

Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

Rembert Pieper¹ , Shih-Ting Huang¹ , David J Clark¹ , Jeffrey M Robinson¹ , Hamid Alami¹ , Prashanth P Parmar¹ , Moo-Jin Suh¹ , Srilatha Kuntumalla¹ , Christine L Bunai¹ , Robert D Perry² , Robert D Fleischmann¹ and Scott N Peterson¹

¹  J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland, USA

²  Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky, USA

author email corresponding author email

Proteome Science 2009, 7:5doi:10.1186/1477-5956-7-5

Published:	19 February 2009

Abstract

Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr), an alkaline solution (180 mM Na₂CO₃, pH 11.3) and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14). Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries). Thirty-one proteins were experimentally associated with the outer membrane (OM). Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high M_r fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM) proteins with two or more predicted transmembrane domains (TMDs) were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.