Integrating 'top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis.
Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.
VerBerkmoes
Nathan C
NC
Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6365, USA.
Bundy
Jonathan L
JL
Hauser
Loren
L
Asano
Keiji G
KG
Razumovskaya
Jane
J
Larimer
Frank
F
Hettich
Robert L
RL
Stephenson
James L
JL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis*; D002458:Cell Fractionation; D002845:Chromatography / Q000379:methods; D030562:Databases, Protein; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis*; D016415:Sequence Alignment; D020592:Shewanella / Q000737:chemistry*
D001426:Bacterial Proteins; D020543:Proteome