The use of on-line capillary electrophoresis/electrospray ionization with detection via an ion trap storage/reflectron time-of-flight mass spectrometer for rapid mutation-site analysis of hemoglobin variants.

Capillary electrophoresis/electrospray ionization using an ion trap storage/reflectron time-of-flight mass spectrometer detector (CE/ESI-IT/reTOF) is used to provide a rapid and sensitive method for analyzing structural variants in the hemoglobin (Hb) beta-chain. The Hb alpha- and beta-chains are separated and the beta-chain is digested by trypsin. The digest is analyzed by CE/ESI-IT/reTOF where a comparison of the total ion electrophorograms and mass spectra of the mutant and normal hemoglobins (Hbs) can detect the presence of a mutation site. In addition, collision-induced dissociation in the vacuum interface-skimmer region can be used to pinpoint the identity of such a site. The unique capability of the CE/ESI-IT/reTOF system for accurately detecting fast separations with narrow peaks that may be under 1 s full width at half maximum is demonstrated. The speed of this system is essential for resolution of the large number of peaks that are separated in a short time duration using CE separations.