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 ionization time-of-flight mass spectrometry (CZE-ESI-TOF-MS) experiments were conducted using uncoated fused-silica capillaries coupled to a reflecting TOF mass spectrometer via a gold-coated sheathless interface. Off-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 level. Sheathless CZE-ESI-TOF-MS was first simulated in an off-line experiment, using a test bench, in order to select a suitable running electrolyte, to find the optimal electrospray potential, and also to test the gold-coated capillary tips. This enabled an ease of transition to on-line measurements. On-line CZE-ESI-TOF-MS measurements of the total ion electropherogram (TIE) and of selected ion electropherograms (SIE) on peptide mixtures demonstrated fmol-level sensitivity, with S/N values 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)(FVHM)) and enabled isotopic resolution of peptide components as well as mass accuracy in the 10 ppm range. These results were comparable with values observed with the usual ESI source on the same mass spectrometer, and thus demonstrated no loss in spectral quality from using the sheathless CE interface. On-line CE separation efficiency was equivalent to that obtained off-line for the separation of a peptide mixture, with N=35000-87000 theoretical plates. Separations of standard proteins yielded equivalent mass spectral resolution and accuracy with separation efficiencies of N=2800-5500 and S/N values of 110-225 on raw data. The gold-coated sheathless CE-ESI interface was found to be relatively easy to prepare with the use of gold vapour deposition. The interface produced a stable electrospray for extended periods of time and was found to be robust and reliable.
McComb
M E
ME
Department of Chemistry, University of Manitoba, Winnipeg, Canada.
Krutchinsky
A N
AN
Ens
W
W
Standing
K G
KG
Perreault
H
H
D019342:Acetic Acid / Q000737:chemistry; D000808:Angiotensinogen / Q000032:analysis / Q000737:chemistry; D000818:Animals; D001920:Bradykinin / Q000032:analysis / Q000737:chemistry; D002417:Cattle; D003574:Cytochrome c Group / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D007987:Gonadotropin-Releasing Hormone / Q000032:analysis; D007328:Insulin / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods*; D009211:Myoglobin / Q000032:analysis; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012015:Reference Standards; D013373:Substance P / Q000032:analysis
D003574:Cytochrome c Group; D007328:Insulin; D009211:Myoglobin; D010455:Peptides; D011506:Proteins; D000808:Angiotensinogen; D013373:Substance P; D007987:Gonadotropin-Releasing Hormone; D019342:Acetic Acid; D001920:Bradykinin