Reversed-phase HPLC separation of human serum employing a novel saw-tooth gradient: Toward multidimensional proteome analysis

A novel reversed-phase (RP-) HPLC gradient profile applicable to multidimensional separations of complex protein mixtures is reported. This gradient profile elutes small numbers of proteins from the RP-HPLC column in discrete intervals while minimizing the amount of band broadening between elution intervals and maintaining constant flow through the HPLC column. Eluting the proteins in discrete intervals eases the instrumental requirements necessary for performing multidimensional separations and can be used to aid in the collection of well-defined fractions. The saw-tooth gradient was applied to the successful isolation of albumin from less abundant proteins in whole human serum and provides adequate separation of proteins in a low-molecular weight (LMW) fraction of human serum with resolution comparable to that achieved using a typical linear gradient profile.