Zic-Hilic Zwitterionic stationary phase is a powerful tool for the separation of complex proteomic samples via 2D liquid chromatography with MudPit (multidimensional protein identification technology)
When Zic-Hilic, a versatile stationary phase that is based on sulfobetaine groups, is used as the first dimension and a reverse phase column is used as the second dimension, superb separation of a protein mixture can be obtained.
The zwitterionic stationary phase provides better chromatographic resolution and avoids the clustering of +2 and +3 proteins that occurs when a strong cation exchange stationary phase is employed.
A major benefit of the zwitterionic stationary phase is that the charge of the chromatographic medium is constant between pH=3 and pH=8, thus any change in the pH only has an effect on the charge of the peptide residue.
At pH=3, a Zic-Hilic separation resembles that obtained from a strong cation exchanger and a significant enrichment of N-Acetylated peptides is observed.
At higher pH, better resolution is obtained, thereby allowing for identification of more proteins.
In a recent study, the combination of the Zic-Hilic stationary phase with a reverse phase column allowed scientists at Utrecht University and the Netherlands Cancer Institute to analyze the nuclear fraction of a cellular lysate.
The Zic-Hilic separation was performed at pH=3 (which provided the best orthogonality with the reverse phase), and also at pH=6.8 which showed the best separation.
The pH=3/RP separation allowed to the identification of 1040 proteins with 4973 peptides, while the pH=6.8/RP separation led to 1284 proteins and 6625 peptides.
The use of a Zic-Hilic column as the first dimension provides a powerful and flexible tool for the separation of complex protein mixtures and is a very good alternative to the use of the commonly employed strong cation exchangers used for MudPit.
