Abstract
Protein profiling enables the qualitative characterization of a proteome of interest. Phosphorylation is a post-translational modification with regulatory functions in a plethora of cell processes. We present an experimental workflow for simultaneous analysis of the proteome and phosphoproteome with no additional enrichment for phosphoproteins/phosphopeptides. Our approach is based on isoelectric focusing (IEF) which allows the separation of peptide mixtures on an immobilized pH gradient (IPG) according to their isoelectric point. Due to the negative charge of the phosphogroup, most of the phosphopeptides migrate toward acidic pH values. Peptides and phosphopeptides are then identified by mass spectrometry (MS) and phosphopeptide spectra are manually checked for the assignment of phosphorylation sites. Here, we apply this methodology to investigate synaptosome extracts from whole mouse brain. IEF-based peptide separation is an efficient method for peptide and phosphopeptide identification.
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Abbreviations
- CID:
-
Collision-induced dissociation
- IEF:
-
Isoelectric focusing
- IPG:
-
Immobilized pH gradient
- LC-ESI-MS/MS:
-
Liquid chromatography-electrospray ionization-tandem mass spectrometry
- MS:
-
Mass spectrometry
- pI:
-
Isoelectric point
- S:
-
Serine
- T:
-
Threonine
- Y:
-
Tyrosine
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Acknowledgments
This work is funded by the Max Planck Society. M.D.F. is supported by a grant from the Deutsche Forschungsgemeinschaft (FI 1895/1-1). We thank Chris Turck for useful comments.
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Maccarrone, G., Filiou, M.D. (2015). Protein Profiling and Phosphoprotein Analysis by Isoelectric Focusing. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 1295. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2550-6_22
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DOI: https://doi.org/10.1007/978-1-4939-2550-6_22
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2549-0
Online ISBN: 978-1-4939-2550-6
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