High-Performance Ion-Exchange Chromatography for Analysis of Protein Phosphorylation

Pranathi R. Perati, Valoran P. Hanko, and Jeffrey S. Rohrer, Dionex Corporation

Protein phosphorylation is one of the most prevalent intracellular protein modifications, regulating numerous cellular processes including cell differentiation, proliferation, and migration. Approximately 30% of cell proteins are phosphorylated at any given time and changes in protein phosphorylation often signal developmental or pathological disorders (1). To better understand the role of protein phosphorylation, it is important to separate the phosphorylated forms of a given protein.

In this study, we used the ProPac® SAX-10 column (Dionex), a uniquely constructed strong anion exchange column, to separate protein variants due to phosphorylation. Protein phosphorylation confers variability in the protein's charge and therefore ion-exchange chromatography is ideal for separation of these variants. The high efficiency of the ProPac SAX-10 anion-exchange resin enables resolution of different protein isoforms using a simple salt gradient. This study shows the separation of phosphorylated variants of the phosphoproteins, casein, and ovalbumin.

Experimental

A bio-inert liquid chromatography system (Dionex ICS-3000 or the UltiMate® 3000 Titanium) was used to eliminate the possibility of metal complexes or redox reactions with proteins. Ovalbumin was dephosphorylated and the phosphate content determined as described in Dionex AN214 (1).

Results

Figure 1 shows high-resolution strong anion-exchange separations of ovalbumin and casein and a dephosphorylated preparation of each.

Figure 1

In Figure 1a, the phosphate content analysis of ovalbumin showed that peaks 2, 4, and 7 in the phosphorylated trace were ovalbumin isoforms with zero, one, and two phosphates respectively. Figure 1b shows casein is a complex sample with considerable heterogeneity, and the majority of the peaks in the purchased dephosphorylated casein show less retention than the peaks in the untreated casein preparation. The net loss in negative charge in the dephosphorylated preparation causes this shift in retention.

Conclusion

This application uses high-resolution strong anion-exchange chromatography to separate protein isoforms with differences as small as one phosphate on a single amino acid residue.

Reference

(1) "Separation of Protein Phosphoisoforms Using Anion-Exchange Chromatography," Application Note 214, LPN 2134: Dionex Corporation, Sunnyvale, CA.

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