Online combination of multidimensional electrokinetic separations with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry for proteome analysis

Novel bioanalytical approaches provide the foundation for new tools and methodologies for the detection and diagnosis of diseases and the identification of targets for drug development. Among these approaches is “proteomics,” the analysis of proteins present in an organism or cell under a given set of physiological or environmental conditions. Automated, high throughput, and reproducible separation techniques are needed to resolve proteins/peptides for large-scale proteome analyses. To this end, a multidimensional electrokinetic separation platform has been developed by online coupling of capillary isoelectric focusing (CIEF) with transient capillary isotachophoresis/zone electrophoresis (CITP/CZE) interfaced with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). A microdialysis junction is employed as the interface for on-line coupling of CIEF with transient CITP/CZE and a sheath liquid interface for CZE-MS. CIEF not only provides high resolution separation of tryptic peptides based on differences in their isoelectric points (pI), but also potentially allows the analysis of low abundant proteins with a typical concentration factor of 50–100 times. Many peptides, which have similar pI, would most likely have different charge to mass ratios, and thus different electrophoretic mobilities in transient CITP/CZE.; The constructed multidimensional separation platform is demonstrated for the microorganism Shewanella oneidensis, which is a potential bioremediation agent. Carrier ampholytes, used to create a pH gradient inside the CIEF capillary, are employed as the leading electrolyte in transient CITP/CZE. The peptides resolved in the second dimension separation are subjected to online ESI-FTICR-MS analysis. Positive ion mass spectra are acquired using a 7-Tesla FTICR mass spectrometer. The components in each CIEF fraction have definite pI values, which together with high mass measurement accuracy offered by FTICR proved to be very useful for confident protein/peptide identification, 26.5% proteome coverage was obtained for Shewanella oneidensis using this technique in a single analysis. Additionally, separation in CLEF for resolving biomolecules is extended to basic regions. The use of N, N, N′, N′-tetramethylethylenediamine (TEMED) and ampholyte 9-11 are investigated for their effects to enhance the separation range to basic regions. The sample mixture, containing bradykinin, the high pI protein calibration kit (pI 5.2–10.30, and cytochrome C digest, is employed to demonstrate the CIEF separation of proteins and peptides over a wide pH range of 3.7–12. The multidimensional system provides a high resolution, high efficiency, high mass measurement accuracy, and ultra high sensitive technique for proteome analysis.