This study demonstrates the use of on-line isoelectric focusing/asymmetrical flow field-flow fractionation (IEF-AF4), a non-gel based high speed two dimensional (isoelectric point and hydrodynamic diameter) protein separation device used for the isolation/separation of phosphoproteins. IEF-AF4 performance was evaluated by first fractionating α-casein molecules at different pIs and sizes. Collected proteins were analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS n) to determine various isoforms of the phosphopeptides as well as the relative ratio of phosphorylated and unmodified peptides. A narrow pH cut (δpH=0.5) of carrier ampholyte was used in IEF-AF4 to finely resolve phosphoproteins by pI. When the channel lane of multilane AF4 became acidic, the relative ratio of phosphorylated to unmodified or less phosphorylated peptides increased. The current method was applied to prostate cancer cell lysates to demonstrate that IEF-AF4 can examine the relative abundances of specific phosphoproteins, known as biomarkers, in prostate cancer. While affinity-based enrichment methods remove unmodified peptides, IEF-AF4 offers intact phosphoprotein separation at the protein level without removing unmodified proteins. IEF-AF4 enables quantitative analysis without isotope labeling.
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