Abstract
Major difficulties in phosphoprotein analysis relate to the presence of a huge number of nonphosphorylated proteins and to the wide concentration dynamic range among them. In order to overcome the analysis complexity, specific clean-up and highly efficient enrichment procedures are mandatory prior to the chromatographic separation and identification by tandem mass spectrometry. In this chapter, a procedure based on immobilized metal affinity chromatography (IMAC)/reversed-phase phosphopeptide purification and analysis by nanoHPLC-ESI-MS/MS with ion trap is described in detail. CID (collision-induced dissociation) and ETD (electron-transfer dissociation) fragmentation techniques are used in combination to specifically determine phosphorylation sites inside the peptide sequences, through the analysis of MS/MS spectra.
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Navajas, R., Paradela, A., Albar, J.P. (2011). Immobilized Metal Affinity Chromatography/Reversed-Phase Enrichment of Phosphopeptides and Analysis by CID/ETD Tandem Mass Spectrometry. In: Walls, D., Loughran, S. (eds) Protein Chromatography. Methods in Molecular Biology, vol 681. Humana Press. https://doi.org/10.1007/978-1-60761-913-0_18
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DOI: https://doi.org/10.1007/978-1-60761-913-0_18
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