In-depth analysis of protein phosphorylation by multidimensional ion exchange chromatography and mass spectrometry.
Protein phosphorylation controls fundamental biological functions that are often deregulated in disease. Therefore, system-level understanding of complex pathophysiological processes requires methods that can be used to profile and quantify protein phosphorylation as comprehensively as possible. Here we present a detailed protocol to enrich phosphopeptides from total cell lysates in a form amenable to downstream analysis by mass spectrometry. Using these techniques, we have detected several thousands of phosphorylation sites in the NIH-3T3 cell line.
Alcolea
Maria P
MP
Analytical Signalling Group, Centre for Cell Signalling, Institute of Cancer, Bart's and the London School of Medicine, Queen Mary University of London, London, UK.
Cutillas
Pedro R
PR
D053000:Analytic Sample Preparation Methods; D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008670:Metals / Q000737:chemistry; D051379:Mice; D041681:NIH 3T3 Cells; D010446:Peptide Fragments / Q000032:analysis; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics; D052616:Solid Phase Extraction; D014025:Titanium / Q000737:chemistry
D008670:Metals; D010446:Peptide Fragments; D010750:Phosphoproteins; D011506:Proteins; C009495:titanium dioxide; D014025:Titanium