A combination of affinity chromatography, 2D DIGE, and mass spectrometry to analyze the phosphoproteome of liver progenitor cells.
Reversible protein phosphorylation is a ubiquitous posttranslational modification that regulates cellular signaling pathways in multiple biological processes. A comprehensive analysis of protein phosphorylation patterns can only be achieved by employing different complementary experimental strategies all aiming at selective enrichment of phosphorylated proteins/peptides. In this chapter, we describe a method that utilizes a phosphoprotein affinity chromatography (Qiagen) to isolate intact phosphoproteins. These are subsequently detected by difference in two-dimensional gel electrophoresis and identified by mass spectrometry techniques. Additional experiments using a specific stain for phosphoproteins demonstrated that phosphoprotein affinity column was an effective method for enriching phosphate-containing proteins. Further validating the method, this workflow was applied to probe changes in the activation patterns of intermediates involved in different signaling pathways, such as NDRG1 and stathmin, in liver progenitor cells (MLP-29) upon proteasome inhibition.
Proteomics Unit, Biomedical Research Center, Navarra Health Service, Pamplona, Spain. esantamma@cfnavarra.es
Enrique
E
Santamaría
Virginia
V
Sánchez-Quiles
Joaquín
J
Fernández-Irigoyen
Fernando J
FJ
Corrales
D000818:Animals; D002457:Cell Extracts / Q000737:chemistry / Q000302:isolation & purification; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000166:cytology*; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013234:Stem Cells / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry; D057825:Two-Dimensional Difference Gel Electrophoresis / Q000379:methods
D002457:Cell Extracts; D010446:Peptide Fragments; D010750:Phosphoproteins; D020543:Proteome; D014357:Trypsin