Capillary zone electrophoresis–mass spectrometry with electrospray ionization of peptides and proteins

Abstract

Abstract. Capillary zone electrophoresis (CZE) coupled with an on-line mass spectrometric (MS) detection system by an atmospheric pressure electrospray ionization (ESI) interface has been used successfully for separations of peptide and protein mixtures. For CZE-MS, ESI produces multiply charged molecular ions and thus allows m a s spectrometers with a limited mass-to-charge range to analyze proteins having molecular weights greater than 100,000. Various pH buffer systems have been investigated, including buffer systems in which pH > p1 (isoelectric point of analytes) and pH << pl are designed to reduce protein adsorption onto silica capillary surfaces and thus improve separation efficiencies. More than 125,000 theoretical plates have been obtained from a CZE-MS separation of less than 1 pmol per component of leucine enkephalin and horse heart myoglobin. CZE-MS of other large peptides and proteins are demonstrated.

Nanoscale separations combined with electrospray ionization mass spectrometry: Sulfonamide determination

“…They demonstrated the on-line separation of quaternary ammonium salts, nucleos ides, and peptides where the total column effluent was introduced to the mass spectrometer with no additional makeup flow [53,54]. Subsequent interfaces made use of additional makeup flow, by using either a coaxial flow delivery system [37,[55][56][57][58][59][60][61][62][63] or a liquid junction coupling (64)(65)(66)(67)(68)(69), to provide extra flow because electrospray requires a minimum flow rate greater than the usual operating flow rates of CZE [55). The on-line separations of peptides and proteins [38,[56][57][58][59][60][61][62][63][64][65][66][67]69), tryptic digests [66,67,69], quaternary phosphonium salts (55), amines [55), sulfonated azo dyes [66,68,69], acid pesticides [66], and benzodiazepines (69) have been shown.…”

Application of electrospray mass spectrometry in biology

“…The high resolving power of capillary electrophoresis (CE) and the low flow rates involved make it an ideal technique to interface with ESI-MS; this was first demonstrated for proteins in 1989. 70 There are a number of problems associated with the very low flow rates and high buffer concentrations required for CE in some instances, and also in raising the potential on the capillary to a suitable voltage to generate the electrospray. These have been overcome by a number of methods 68 including the addition of a second solvent at the probe tip via a larger coaxial capillary, or coating the capillary in gold to make the contact.…”

Liquid chromatography: theory and methodology

“…Smith and co-workers developed an electrospray interface for CE/MS (94, [158][159][160][161][162][163]. The electrospray interface makes electrical contact with the electrophoretic buffer via either a small needle (158) or a thin film of metal deposited on the surface of the capillary (94).…”

“…Selected ion monitorin was used to follow CE separations with 125 OOO plates. Capikry isotachophoresis was employed to increase concentration detection limits for peptides and proteins (163). MS/MS spectra of melittin were shown, demonstrating complex fragmentation for each charge state.…”

“…hctrospray MS/MS produced high-resolution spectra of enkephalins (MW 600), myoglobin (MW 18 000), and glycoproteins (e.g. transferrins, MW 80000, 163). Mosely et al showed that use of a continuous flow FAB interface for CE MS allowed the generation separation efficiencies of over 400000 plates (167).…”