Intact protein profiling of Chlorobium tepidum by capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry.
Capillary isoelectric focusing (CIEF) coupled with reversed-phase liquid chromatography (RPLC) and electrospray ionization (ESI) mass spectrometry (MS) is shown to provide a liquid-based alternative to 2D-PAGE for intact protein profiling. This combination exhibits high resolution, sensitivity and throughput for protein profiling based on pI vs MW. The CIEF-RPLC-MS system described here facilitates the use of IEF markers for internal calibration of pI. It also provides a high dynamic range as evidenced by the detection of 100 pg (3 fmol) of a test protein spiked into 1 microg of a complex protein mixture. About 1200 individual proteins/polypeptides were detected from lysates of the green sulfur bacterium Chlorobium tepidum in a single <8 h run. The pI vs MW profile obtained from CIEF-RPLC-MS compares favorably with theoretical data derived from the C. tepidum genome and experimental data obtained from 2D-PAGE.
Zhou
Feng
F
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, 19716, USA.
Hanson
Thomas E
TE
Johnston
Murray V
MV
D041883:Chlorobium / Q000737:chemistry*; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D036103:Nanotechnology; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012680:Sensitivity and Specificity
D011506:Proteins