Probing proteomes using capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.
Unlike the genome, the proteome is exquisitely sensitive to cellular conditions and will consist of proteins having abundances dependent upon stage in the cell cycle, cell differentiation, response to environmental conditions (nutrients, temperature, stress etc.), or disease state(s). Therefore, the study of proteomes under well-defined conditions can provide a better understanding of complex biological processes and inference of protein function. Thus, much faster, more sensitive, and precise capabilities for the characterization of cellular constituents are desired. We describe progress in the development and initial application of the powerful combination of capillary isoelectric focusing (CIEF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for measurements of the proteome of the model system Escherichia coli. Isotope depletion of the growth media has been used to improve mass measurement accuracy, and the comparison of CIEF-FTICR results for the analysis of cell lysates harvested from E. coli cultured in normal and isotopically depleted media are presented. The initial studies have revealed 400-1000 putative proteins in the mass range 2-100 kDa from total injections of approximately 300 ng of E. coli proteins in a single CIEF-FTICR analysis.
Jensen
P K
PK
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Pasa-Tolić
L
L
Anderson
G A
GA
Horner
J A
JA
Lipton
M S
MS
Bruce
J E
JE
Smith
R D
RD
D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000254:growth & development; D005583:Fourier Analysis; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007525:Isoelectric Focusing / Q000379:methods; D007554:Isotopes; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D008970:Molecular Weight; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D003470:Culture Media; D007554:Isotopes