Comparative proteome analysis of subcellular fractions from Borrelia burgdorferi by NEPHGE and IPG.
Borrelia burgdorferi, the cause of Lyme disease, produces excessive amounts of membrane lipoproteins such as outer surface protein A (OspA) when grown in vitro, and consequently many low or moderately abundant proteins are underrepresented when cell lysates are examined by 2-DE. We analyzed the B. burgdorferi B31 proteome computationally and by IPG or modified NEPHGE after subcellular fractionation into membrane-associated and soluble proteins. The B. burgdorferi B31 theoretical proteome is comprised of 1623 proteins and has a mean pI of 8.36 and a median pI of 9.03 with 68% of the proteome possessing a pI >/=7.5. Separation of soluble proteins by IPG resulted in 205 individual spots and identification of 78 protein spots by MALDI-TOF MS. Separation by modified NEPHGE routinely resulted in approximately 185 soluble and 160 membrane protein spots with the identification of 88 individual protein spots combined by MALDI-TOF MS. Homologues to GroEL and aminopeptidase I were present in greater amounts in the membrane faction, with enolase at nearly equivalent amounts in the soluble and membrane fractions. Identification of proteins isolated and separated by such methods will enable future determination of proteome changes in membrane and soluble protein fractions as spirochetes adapt to their changing environments.
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Andrew J
AJ
Nowalk
Christi
C
Nolder
Dawn R
DR
Clifton
James A
JA
Carroll
D001426:Bacterial Proteins / Q000032:analysis* / Q000302:isolation & purification; D025065:Borrelia burgdorferi / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D007525:Isoelectric Focusing / Q000379:methods; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013347:Subcellular Fractions / Q000737:chemistry
D001426:Bacterial Proteins