Enrichment of phosphoproteins for proteomic analysis using immobilized Fe(III)-affinity adsorption chromatography.
We described an efficient protocol to strongly enrich phosphoproteins from mixtures of total cellular proteins using homemade, recyclable Fe(III)-affinity columns. An integral feature of the method is the use of a detergent cocktail that allows use of different pHs for total protein extraction (pH 6.8) and for subsequent affinity capture of phosphoproteins (pH 3.4). Affinity captured proteins from rat fibroblasts were fractionated on 2D gels and random selection was identified by mass spectrometry. More than 85% of identified proteins were previously known to be phosphorylated. The specificity of the method was further validated by isolating proteins from (32)P labeled cells. Our comparison of the clusters of acidic residues in the captured proteins with acidic clusters in proteins of the rat genome indicates that affinity for phosphate groups dominates over adsorption of proteins with acidic clusters.
Guerrera
Ida Chiara
IC
Centre for Molecular Medicine, Department of Medicine, University College London, 5 University Street, WC1E 6JJ London, United Kingdom.
Predic-Atkinson
Jelena
J
Kleiner
Oliver
O
Soskic
Vukic
V
Godovac-Zimmermann
Jasminka
J
D000327:Adsorption; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts / Q000378:metabolism; D016678:Genome; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry*; D007668:Kidney / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D016622:Silver Staining
D010750:Phosphoproteins; D011506:Proteins; D007501:Iron