Summary
Mass spectrometry is the tool of choice for sequencing peptides and determining the sites of posttrans-lational modifications; however, this bottom-up approach lacks in providing global information about the modification states of proteins including the number and types of isoforms and their stoichiometry. Recently, various techniques and mass spectrometers, such as high-field Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometers, have been developed to study intact proteins (top-down proteomics). While the protein molecular mass and the qualitative and quantitative information about protein isoforms can be revealed by FTICR-MS analysis, their primary structure (including the identification of modifications and their exact locations in the amino acid sequence) can directly be determined using the MS/MS capability offered by the FTICR mass spectrometer. The distinct advantage of top-down methods are that modifications can be determined for a specific protein isoform rather than for peptides belonging to one or several isoforms. In this chapter, we describe different top-down proteomic approaches enabled by high-field (7, 9.4, and 12 T) FTICR mass spectrometers, and their applicability to answer biological and biomedical questions. We also describe the use of the free flow electrophoresis (FFE) to separate proteins prior to top-down mass spectrometric characterization.
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Acknowledgments
This work was supported in part by a Ford Foundation Diversity Fellowship(M.T.), by the Cystic Fibrosis Foundation (CFFT-IBORCHE05U0) and CDC H75/CCH424675 (S.O.-P.) and a grant sponsored by Genome BC and Genome Canada. A portion of this work was done at the UNC-Duke Proteomics Centre, which was supported by a gift from an anonymous donor to support research in proteomics at UNC. The Bruker 12 T FTICR mass spectrometer at the UNC-Duke Michael Hooker Proteomics Centre, UNC-CH, Chapel Hill, NC, was purchased with funding from the North Carolina Biotechnology Center (NCBC 2005-IDG-1015) and from the National Institutes of Health (1-S10-RR019889–01).
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Ouvry-Patat, S.A. et al. (2009). Top-Down Proteomics on a High-field Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. In: Lipton, M.S., Paša-Tolic, L. (eds) Mass Spectrometry of Proteins and Peptides. Methods In Molecular Biology, vol 492. Humana Press. https://doi.org/10.1007/978-1-59745-493-3_12
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