SENSITIVE HIGH-RESOLUTION ANALYSIS OF BIOLOGICAL MOLECULES BY CAPILLARY-ZONE-ELECTROPHORESIS COUPLED WITH REFLECTING TIME-OF-FLIGHT MASS-SPECTROMETRY

Off-line and on line capillary zone electrophoresis-electrospray ioniz ation time-of-flight mass spectrometry (CZE-ESI-TOF-MS) experiments we re conducted using uncoated fused-silica capillaries coupled to a refl ecting TOF mass spectrometer via a gold-coated sheathless interface. O ff-line and on-line experiments were performed on standard mixtures of proteins and peptides. Samples collected off-line electrokinetically in plastic vials were analyzed by standard ESI-TOF-MS at the pmol leve l. Sheathless CZE-ESI-TOF-MS was first simulated in an off-line experi ment, using a test bench, in order to select a suitable running electr olyte, to find the optimal electrospray potential, and also to test th e gold-coated capillary tips. This enabled an ease of transition to on -line measurements. On-line CZE-ESI-TOF-MS measurements of the total i on electropherogram (TIE) and of selected ion electropherograms (SIE) on peptide mixtures demonstrated fmol-level sensitivity, with S/N valu es of 250-400 on raw data (TIE mode) and of 30-760 (SIE mode). The use of reflecting TOF-MS afforded mass resolution values R>6000 (m/Delta m(FWHM)) and enabled isotopic resolution of peptide components as well as mass accuracy in the 10 ppm range. These results were comparable w ith values observed with the usual ESI source on the same mass spectro meter, and thus demonstrated no loss in spectral quality from using th e sheathless CE interface. On-line CE separation efficiency was equiva lent to that obtained off-line for the separation of a peptide mixture , with N=35000-87000 theoretical plates. Separations of standard prote ins yielded equivalent mass spectral resolution and accuracy with sepa ration efficiencies of N=2800-5500 and S/N values of 110-225 on raw da ta. The gold-coated sheathless CE-ESI interface was found to be relati vely easy to prepare with the use of gold vapour deposition. The inter face produced a stable electrospray for extended periods of time and w as found to be robust and reliable. (C) Elsevier Science B.V.