2D LC Protein Fractionation

2D LC Protein Fractionation is performed on a Beckman-Coulter ProteomeLab PF2D system to fractionate intact proteins while comparing expression levels. This system is a multi-dimensional separation approach utilizing chromatofocusing followed by nonporous reverse-phase (RP) HPLC. The first dimension, chromatofocusing, allows intact proteins to be focused into distinct fractions based on each protein’s isoelectric point (pI) (1).

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Typically, first dimension fractions are collected every 0.3pH unit change over the range of 8.5-4.5. The pI-focused fractions are then further separated using nonporous-RP-HPLC which separates proteins based on their surface hydrophobicity. Using this approach 1 complex sample (e.g. plasma) can be separated into ~450 separate fractions.

The comparative analysis is done sequentially with the first dimension of each compared group run on the same day, followed by the second dimension. By running both first dimensions on the same day, we have found excellent reproducibility in the comparative data analysis. Proteins from the collected fractions can also be identified after tryptic digestion and LC-MS/MS analysis. Top down fragmentation is also available.

Figure 1 shows the protein expression level changes between two different cell lines (WT vs Retinoic acid treated). Green bands are up regulated in the WT sample while red are increased in the treated sample. Figure 2 shows the overlay of the 214nm chromatograms from lean or fraction 7.

This protein profiling approach is best run at high levels of sample (0.5 to 1 mg).

Gulcicek, E. E., Colangelo, C. M., McMurray, W., Stone, K., Wu, T., Zhao, H., Spratt, H., Kurosky, A., Wu, B., Williams, K. (2005). Proteomics and the analysis of proteomic data: Overview of protein-profiling technologies In Current Protocols in Bioinformatics (A. D. Baxevanis, D. B. Davison, R. D. M. Page, G. A. Petsko, L. D. Stein and G. D. Stormo, eds) pp 13.1.1 - 13.1.31. John Wiley & Sons Inc, Hoboken, NJ.