Abstract
Understanding how phosphorylation regulates the behavior of individual proteins is critical to understanding signaling pathways. These studies usually involve knowledge of which amino acid residues are phosphorylated on a given protein and the extent of such a modification. This is often a rather difficult task in that most phosphoproteins contain multiple substoichiometric sites of phosphorylation.
Here we describe the multidimensional electrospray (ES) mass spectrometry (MS)-based phosphopeptide-mapping strategy developed in our laboratory. In the first dimension of the process, phosphopeptides present in a protein digest are selectively detected and collected into fractions during on-line liquid chromatography (LC)/ES/MS, which monitors for phosphopeptide-specific marker ions. This analysis generates a phosphorylation profile that can be used to assess changes in the phosphorylation state of a protein pointing to those phosphopeptides that require further investigation. The phosphopeptide-containing fractions are then analyzed in the second dimension by nano-ES with precursor-ion scan for the marker ion m/z 79. As the final step, direct sequencing of the phosphopeptides is performed by LC/ES/MS/MS. Merits and limitations of the strategy, as well as experimental details and suggestions, are described here.
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© 2004 Humana Press Inc., Totowa, NJ
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Zappacosta, F., Huddleston, M.J., Annan, R.S. (2004). Comparative Phosphorylation Site Mapping From Gel-Derived Proteins Using a Multidimensional ES/MS-Based Approach. In: Dickson, R.C., Mendenhall, M.D. (eds) Signal Transduction Protocols. Methods in Molecular Biology, vol 284. Humana Press. https://doi.org/10.1385/1-59259-816-1:091
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DOI: https://doi.org/10.1385/1-59259-816-1:091
Publisher Name: Humana Press
Print ISBN: 978-1-58829-245-2
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