Multidimensional liquid phase protein separations in conjunction with stable isotope labelling for quantitative proteomics

A method for quantitative protein profiling has been developed utilising multidimensional liquid phase protein separations in conjunction with stable isotope labelling. This approach combines the advantages of high throughput, automated, reproducible protein separations with accurate protein quantitation performed in the mass spectrometer. Escherichia coli cells were grown in the presence and absence of the DNA methylation inhibitor 5-Azacytidine on N-14 and N-15 enriched media. Protein separations were performed using ion exchange chromatography in the first dimension and RP capillary chromatography in the second dimension. UV absorbance measurements were used for the initial semiquantitative identification of differentially expressed proteins. Selected peaks from the mixed N-15/N-14 lysates were used for the accurate quantitation performed in the mass spectrometer using the ratios of the stable isotopes. Using this approach, a number of differentially expressed proteins have been identified. Moreover, this approach overcomes a number of caveats associated with multidimensional liquid phase protein separations, including the presence of multiple proteins present in a single chromatographic peak.