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.