Analysis of whole cell lysate from the intercellular bacterium Coxiella burnetii using two gel-based protein separation techniques.
Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular gamma-proteobacterium, which replicates within large phagolysosome-like compartments formed in the host cell. The global protein profile of intracellular C. burnetii strain Nine Mile phase II was analyzed by two gel-based approaches coupled to MALDI-TOF MS. Colloidal Coomassie brilliant blue-stained 2-DE gels at the pH range 3-10 resolved over 600 protein spots and 125 spots in doubled-SDS-PAGE gels. Mass spectra obtained for each trypsin-digested protein-spot were compared to the C. burnetii genome database, and a total number of 185 different C. burnetii proteins were identified by both techniques. 2-DE in combination with MALDI-TOF MS, as a high-throughput method, allowed the identification of 172 proteins. On the other hand, the application of doubled-SDS-PAGE allowed the identification of 38 proteins, with some of them being very alkaline and membrane proteins not identified in the 2-DE approach. Most identified proteins were predicted to be involved in metabolism and biosynthesis. Several identified proteins are speculated to have a distinct and vital role in the pathogenesis and survival of C. burnetii within the harsh phagolysosomal environment.
Samoilis
Georgios
G
Division of Biochemistry, Department of Chemistry, University of Crete, P. O. Box 2208, GR-71003 Voutes, Greece.
Psaroulaki
Anna
A
Vougas
Konstantinos
K
Tselentis
Yannis
Y
Tsiotis
Georgios
G
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis*; D002522:Chlorocebus aethiops; D016997:Coxiella burnetii / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D010449:Peptide Mapping / Q000379:methods; D020543:Proteome / Q000032:analysis*; D011778:Q Fever / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014709:Vero Cells
D001426:Bacterial Proteins; D020543:Proteome