Capillary isoelectric focusing-based multidimensional concentration/separation platform for ultrasensitive proteome analysis

The sensitive characterization of the entire spectrum of proteins, including low abundance proteins, is essential to proteome-wide protein identification and quantitative expression profiling, and still challenges the development of various bioanalytical technologies from sample processing to separation and MS detection. This dissertation is first targeted at the development of a dynamic introduction method for capillary isoelectric focusing, based on electrokinetic injection, to further increase sample loading and therefore the concentrations of focused analytes. Two model systems, a low pI protein calibration kit and tryptic peptides from Saccharomyces cerevisiae , are employed to demonstrate the excellent separation resolution and overall concentration factor achieved using dynamic introduction and focusing.; An integrated proteome concentration/separation approach is further developed, involving on-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC). The self-sharpening effect of CIEF greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of ∼50–240. Besides, CIEF as the first separation dimension resolves proteins/peptides based on their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography. The resolving power of combined CIEF-CRPLC system is demonstrated using the soluble fraction of Drosophila salivary glands taken from two time points following puparium formation.; Furthermore, this multidimensional protein/peptide concentration/separation platform is easily interfaced with ESI-MS/MS for protein/peptide identification. The grouping of two highly resolving techniques of isoelectric focusing and reversed phase chromatography, which consist of completely orthogonal separation mechanisms, together with the electrokinetic concentration effect, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identification of low abundance proteins. The CIEF-based multidimensional separation/concentration platform enables the identification of a greater number of yeast soluble proteins than methods presented in the literature, yet requires a protein loading of only 9.6 μg. This protein loading is two to three orders of magnitude lower than those employed by the reported non-gel based proteome techniques. The distribution of codon adaptation index value for identified yeast proteins approximates to that predicted for the entire yeast proteome and supports the capability of CIEF-based proteome separation technology for achieving comprehensive proteome analysis.