Phosphopeptides enrichment using on-line two-dimensional strong cation exchange followed by reversed-phase liquid chromatography/mass spectrometry

We have developed a method to isolate and enhance the detection of phosphopeptides using liquid chromatography (LC)/mass spectrometry on a tryptic-digested protein sample. The method uses an on-line two-dimensional chromatography approach that consists of strong cation exchange (SCX) followed by reversed-phase (RP) chromatography with mass spectrometric detection. At pH 2.6 or lower, tryptic phosphopeptides are not retained during the first-dimension SCX chromatography step. Thus the capture of these peptides in the flow-through by the second-dimension RP trap can dramatically reduce the complexity of the phosphopeptide chromatography, resulting in little or no suppression of the signal often caused by the coeluting nonphosphorylated peptides. The method provides higher phosphopeptide recovery and less nonspecific biding of acidic peptides than the commonly used enrichment methods, such as immobilized metal affinity chromatography. Since the widely adopted multidimensional LC strategy in shotgun proteomics uses a similar SCX-RP approach, the method can be adapted to detect and characterize phosphopeptides from a complex mixture in a single experiment. Limitations of the method are also discussed.