Comprehensive two-dimensional separation system by coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection.
A comprehensive two-dimensional (2-D) separation system, coupling capillary reverse-phase liquid chromatography (cRPLC) to capillary isoelectric focusing (CIEF), is described for protein and peptide mapping. cRPLC, the first dimension, provided high-resolution separations for salt-free proteins. CIEF, the second dimension with an orthogonal mechanism to cRPLC afforded excellent resolution capability for proteins with efficient protein enrichment. Since all sample fractions in cRPLC effluents could be transferred to the CIEF dimensions, the combination of the two high-efficiency separations resulted in maximal separation capabilities of each dimension. Separation effectiveness of this approach was demonstrated using complex protein/peptide samples, such as yeast cytosol and a BSA tryptic digest. A peak capacity of more than 10 000 had been achieved. A laser-induced fluorescence (LIF) detector, developed for this system, allowed for high-sensitive detection, with a fmol level of peptide detection for the BSA digest. FITC and BODIPY maleimide were used to tag the proteins, and the latter was found better both for separation and detection in our 2-D system.
D001896:Boron Compounds; D002853:Chromatography, Liquid / Q000379:methods*; D003600:Cytosol / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D007834:Lasers; D008301:Maleimides / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000032:analysis; D014357:Trypsin; D015003:Yeasts / Q000737:chemistry
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D008301:Maleimides; C043592:maleimide; D012710:Serum Albumin, Bovine; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate