Protein pI shifts due to posttranslational modifications in the separation and characterization of proteins

Proteins from breast cancer cell lines are characterized using a 2-D liquid separation technique in which protein pI is used as the first-dimension separation parameter. To effect this protein pI separation, chromatofocusing(CF) is employed whereby a pH gradient is generated on-column using a weak anion exchange medium with the intact proteins fractionated and collected every 0.2 pH unit. It is demonstrated that the pI for expressed intact proteins as generated by CF is an important parameter for identification and characterization of the actual protein modifications occurring in the cancer cell. For most proteins, the experimentally determined pI is very close to that predicted by the databases. In other cases, however, where the pI is observed to be shifted from the expected value, it is shown that this shift is often correlated to protein modifications. The modifications that cause such shifts include truncations and deletions often observed in cancer cells or phosphorylations that can shift the pI by several pH units. It is also shown that the effects of phosphorylation on the observed shift can vary depending upon the protein and the amount of phosphorylation. Moreover, large changes in the pI are often observed for proteins with a pI above 7.0 upon phosphorylation, whereas little change is observed for proteins with a pI of approximately 5.0. The expressed protein's pI value thus becomes an important parameter together with the intact MW value, peptide map, and MS/MS results for identification of the presence and type of posttranslational modifications occurring in the cancer cell.