Integration of multidimensional chromatographic protein separations with a combined "top-down" and "bottom-up" proteomic strategy.
In this paper, we present a combined top-down/bottom-up proteomic analysis workflow for the characterization of proteomic samples. This workflow combines protein fractionation (multidimensional chromatographic separation) with parallel online ESI-TOF-MS intact protein analysis, and fraction collection. Collected fractions were digested and protein identifications were produced using MALDI Q-TOF-MS analysis. These identifications were then linked with corresponding ESI-TOF-MS intact protein mass data to permit full protein characterization. This methodology was applied to an E. coli cytosolic protein fraction, and enabled the identification and characterization of proteins exhibiting co-translational processing, post-translational modification, and proteolytic processing events. The approach also provided the ability to distinguish between closely related protein isoforms. The summary of results from this study indicated that roughly one-third of all detected components generated corresponding data from both top-down and bottom-up analyses, and that significant and novel information can be derived from this application of the hybrid analytical methodology.
Millea
Kevin M
KM
Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA.
Krull
Ira S
IS
Cohen
Steven A
SA
Gebler
John C
JC
Berger
Scott J
SJ
D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D003600:Cytosol / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D020033:Protein Isoforms / Q000032:analysis / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D029968:Escherichia coli Proteins; D020033:Protein Isoforms