Analysis of peptides and proteins by capillary electrophoresis/mass spectrometry using acidic buffers and coated capillaries.
The direct coupling of capillary electrophoresis (CE) and mass spectrometry, combined with ionspray ionization using a coaxial capillary arrangement, is described. The CE/mass spectrometer interface is shown to be effective for the analysis of native and tryptic peptides and of proteins of high molecular weight such as bovine serum apotransferrin (approximately 78 kDa). Adsorption of cationic analytes under acidic buffer conditions is minimized through the use of a non-covalent coated capillary possessing an overall positive charge. Since the direction of the electroosmotic flow is thus reversed, compared to that in conventional CE separation on uncoated capillaries, migration of cations is achieved by applying a negative voltage (typically -30 kV) at the injector end of the capillary. In addition to the inherent advantage of providing pre-formed cationic species for mass spectral detection, this arrangement permits analysis of proteins of high isoelectric points even at low pH. The ability to conduct electrophoresis of globular proteins under acidic conditions also provides a means of monitoring their conformational changes, as reflected both by the variation of migration times and by concurrent changes in the multiply charged ion envelopes.
Thibault
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P
Institute for Marine Biosciences, National Research Council of Canada, Halifax, Nova Scotia.
Paris
C
C
Pleasance
S
S
D000595:Amino Acid Sequence; D002021:Buffers; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin
D002021:Buffers; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D014357:Trypsin