Journal
JOURNAL OF PROTEOME RESEARCH
Volume 5, Issue 1, Pages 135-146Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr050278w
Keywords
proteomic analysis; multidimensional chromatography; intact protein; LC-MS; top-down; bottom-up; protein modification; protein processing
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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.
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