Immobilized metal affinity chromatography/reversed-phase enrichment of phosphopeptides and analysis by CID/ETD tandem mass spectrometry.
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.
Laboratorio de Proteómica, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Rosana
R
Navajas
Alberto
A
Paradela
Juan Pablo
JP
Albar
D000595:Amino Acid Sequence; D053000:Analytic Sample Preparation Methods; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D030562:Databases, Protein; D004579:Electron Transport; D006801:Humans; D055767:Immobilized Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008670:Metals / Q000737:chemistry*; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D055767:Immobilized Proteins; D008670:Metals; D010748:Phosphopeptides