Applications of peptide immobilised pH gradient isoelectric focusing (IPG-IEF) for proteomics experiments

Johan Lengqvist, Hanna Eriksson, Maria Pernemalm and Sara Ståhl.

Peptide IPG-IEF has recently emerged as an alternative to strong cation-exchange (SCX) chromatography for pre-fractionation of complex peptide samples. It offers high resolution, reproducibility and loadability, which may be more problematic in chromatography based methods.

Most importantly, it also provides additional information since peptides are fractionated according to their pI (isoelectric point) values. This offers an independent and readily automated way of verifying the output from automated database searches in the peptide (protein) identification step. From the assigned peptide sequence, the pI can be calculated using pI-prediction algorithms. Outliers (i.e. misidentifications) can then be easily removed through filtering based on pI. We are implementing the peptide IPG-IEF method into proteomics projects in KBC. The group has demonstrated the compatibility of the iTRAQ stable isotope labeling method with not only IPG-IEF separation but also with pI-filtering of identification results. The project is now focused on the effect of post-translational modifications (PTMs) on the observed peptide pI in order to extend the use of the method to PTMs such as protein phosphorylation.

In conclusion, peptide IPG-IEF as a pre-fractionation step enhances the information content of the sample and simplifies the data analysis process. This is of great importance for high-throughput proteomics projects.

Last updated: 2008-01-10