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High-resolution capillary isoelectric focusing separations of complex protein mixtures have been obtained for cellular lysates of Saccharomyces cerevisiae, Eschericia coli, and Deinococcus radiodurans. High quality separations are shown to be achievable for total protein concentrations of < 0.1 mg/mL. The separation reproducibility was examined, and the influence of the capillary inner wall coating on resolution investigated using fusedsilica capillaries coated with various hydrophilic polymers including hydroxypropyl cellulose, poly(vinyl alcohol), and linear polyacrylamide. Proteins having an isoelectric point (pI) difference of 0.004 are shown to be separated using a linear carrier ampholyte (linear pH gradient between two electrodes) of 3-10. Approximately 45 discrete peaks in the pI range of 5-7 were obtained for S. cerevisiae, approximately 80 peaks in the pI range of 4.5-8.5 for E. coli, and approximately 210 peaks in the pI range of 3-8.8 for D. radiodurans.

Caco-2 cells offer a means to rapidly screen permeability of drug candidates, allowing pharmaceutical companies to eliminate candidates unable to cross the intestinal barrier early in the discovery process. This screening process is typically performed by conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS), which can require time-consuming method development. An alternative to LC/MS/MS, automated nanoelectrospray tandem mass spectrometry (nanoESI-MS/MS), is introduced. This novel approach requires an off-line ZipTip desalting step followed by automated nanoESI-MS/MS, using the NanoMate 100 and ESI Chip. In addition to reduced method development time, automated nanoESI-MS/MS also offers no carry-over between samples, low sample consumption, and ease-of-use as compared with conventional pulled-capillary nanoelectrospray. Furthermore, the infusion system described has the potential to be high-throughput. A comparison of Caco-2 samples analyzed both by LC/MS/MS and by automated nanoESI-MS/MS is presented. The permeability and recovery data of the two compounds analyzed in this study obtained from conventional LC/MS/MS and by automated nanoESI-MS/MS were in excellent agreement.