An improved strategy for selective and efficient enrichment of integral plasma membrane proteins of mycobacteria.
Mycobacterial plasma membrane proteins play essential roles in many cellular processes, yet their comprehensive proteomic profiling remains challenging. This is mainly due to obstacles related to their extraction and solubilization. To tackle this problem, we have developed a novel procedure to selectively enrich mycobacterial plasma membrane proteins based on alkaline sodium carbonate washing of crude membranes followed by Triton X-114 phase partitioning. The present study assesses the efficiency of this method by proteome analysis of plasma membrane proteins from Mycobacterium bovis BCG. Extracted proteins were separated in parallel by 1-D SDS-PAGE and 2-DE and then analyzed by LC-MS/MS and MALDI-MS/MS. Our study revealed 125 proteins, of which 54 contained 1-14 predicted transmembrane domains (TMD) including nine novel proteins. The 1-D SDS-PAGE-based proteome analysis identified 81 proteins, of which 49 (60.5%) harbored TMD. This approach also revealed many hydrophobic membrane-associated/periplasmic proteins lacking TMD, but only few soluble proteins. The identified proteins were characterized with regard to biological functions and physicochemical properties providing further evidence for the high efficiency of the prefractionation method described herein.
Mattow
Jens
J
Max Planck Institute for Infection Biology, Department of Immunology, Berlin, Germany. mattow@mpiib-berlin.mpg.de
Siejak
Frank
F
Hagens
Kristine
K
Schmidt
Frank
F
Koehler
Christian
C
Treumann
Achim
A
Schaible
Ulrich E
UE
Kaufmann
Stefan H E
SH
D001426:Bacterial Proteins / Q000032:analysis / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification*; D009163:Mycobacterium bovis / Q000737:chemistry* / Q000166:cytology; D020543:Proteome / Q000032:analysis
D001426:Bacterial Proteins; D008565:Membrane Proteins; D020543:Proteome