Two dimensional (pI & ds) separation of phosphorylated proteins by isoelectric focusing/asymmetrical flow field-flow fractionation: Application to prostatic cancer cell line

► Two dimensional (pI & size) separation of phosphoproteins by IEF-AF4. ► Isoelectric focusing/asymmetrical flow field-flow fractionation (IEF-AF4). ► Characterization of phosphopeptide isoforms by nLC-MSn. ► Relative ratio of phosphorylated to unmodified peptide is determined. ► Application to phosphorylated biomarker from prostate cancer cell line.