Abstract
To sensitively analyze complex protein mixtures by mass spectrometry-based shotgun proteomics, researchers have employed platforms that couple orthogonal peptide fractionation methods using nanoscale HPLC. Commonly used platforms have coupled either strong cation exchange (SCX) HPLC or preparative isoelectric focusing (IEF) with nanoscale reversed-phase (nanoRP) HPLC fractionation of peptides. Coupling two dimensions of peptide fractionation, prior to mass spectrometric analysis, increases the sensitivity for identifying low abundance proteins. However, the large dynamic range of protein abundance and high level of complexity of protein mixtures derived from many biological sources, such as bodily fluids, require additional steps of peptide fractionation. To address this shortcoming, we have developed a platform combining three dimensions of peptide fractionation as follows: (1) preparative IEF; (2) SCX HPLC; and (3) nanoRP HPLC. This platform significantly increases the sensitivity of shotgun proteomic analysis in complex protein mixtures. Here, we describe the implementation of this three-dimensional peptide fractionation platform for proteomic studies of complex mixtures.
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Acknowledgments
The authors acknowledge that the work described here was supported in part by National Institutes of Health (USA) Grant 1R01 DE17734, a grant from the Minnesota Medical Foundation, and American Cancer Society Institutional Research Grant IRG-58-001-4.
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Bandhakavi, S., Markowski, T.W., Xie, H., Griffin, T.J. (2011). Three-Dimensional Peptide Fractionation for Highly Sensitive Nanoscale LC-Based Shotgun Proteomic Analysis of Complex Protein Mixtures. In: Toms, S., Weil, R. (eds) Nanoproteomics. Methods in Molecular Biology, vol 790. Humana Press. https://doi.org/10.1007/978-1-61779-319-6_4
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DOI: https://doi.org/10.1007/978-1-61779-319-6_4
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