Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 1 - Large increases in isoform resolution of human transferrin by use of dual simultaneous independent gradients of pH & acetonitril

•Creation of simultaneous independent pH & acetonitrile gradients in quad HPLC.•Simultaneous pH & acetonitrile gradients work on mixed bed RP-IEX columns.•pH & acetonitrile gradients on mixed bed RP-IEX columns are a new proteomics tool.•More than 60 Human Transferrin isoforms are demonstrated.

We have previously described a liquid chromatographic (LC) method for uncoupling controlled, wide range pH gradients and simultaneous controlled gradients of a non-buffering solute on ion exchange resins (Hirsh and Tsonev, 2012) [1]. Here we report the application of this two dimensional LC technique to the problem of resolving Human Transferrin (HT) isoforms. This important iron transporting protein should theoretically occur in several thousand glycoforms, but only about a dozen have been reported. Using dual simultaneous independent gradients (DSIGs) of acetonitrile (ACN) and pH on a mixed bed stationary phase (SP) consisting of a mixture of an anion exchange resin and a reversed phase (RP) resin we partially resolve about 60 isoforms. These are likely to be partially refolded glycoforms generated by interaction of HT with the highly hydrophobic RP SP, as well as distinct folded glycoforms. Thus this study should have interesting implications for both glycoform separation and the study of protein folding.