Targeting phosphoprotein profiling by combination of hydroxyapatite-based phosphoprotein enrichment and SELDI-TOF MS.
Over the last decade surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) has evolved as a versatile tool in the field of proteomic research. Although the chromatographic matrices on the currently provided SELDI affinity arrays allow efficient on-chip protein enrichment, it can be advantageous to combine SELDI with additional sample pre-fractionation steps. In this study, we demonstrate the potential of combining hydroxyapatite-based phosphoprotein enrichment with SELDI-TOF MS analysis. A straightforward method for the enrichment of phosphoproteins on ceramic hydroxyapatite was developed using fluorescently-labelled model proteins. Hydroxyapatite-based pre-fractionation of proteins derived from cell lysates was performed. SELDI-TOF MS analysis of the pre-fractionation eluate confirmed a considerable reduction of sample complexity and an enhancement of selected protein signals.
Vormbrock
Ingo
I
Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum der Heinrich-Heine-Universität, Düsseldorf, Germany. ingo.vormbrock@uni-duesseldorf.de
Kaber
Gernot
G
Hartwig
Sonja
S
Eckel
Jürgen
J
Schrör
Karsten
K
Lehr
Stefan
S
D000818:Animals; D002478:Cells, Cultured; D005591:Chemical Fractionation / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods; D017886:Durapatite / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D053595:Embryonic Stem Cells / Q000737:chemistry; D005456:Fluorescent Dyes; D005470:Fluorometry; D051379:Mice; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D020033:Protein Isoforms / Q000032:analysis* / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D020543:Proteome / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D005456:Fluorescent Dyes; D010750:Phosphoproteins; D020033:Protein Isoforms; D011506:Proteins; D020543:Proteome; D017886:Durapatite