On-line capillary electrophoresis/microelectrospray ionization-tandem massspectrometry using an ion trap storage/time-of-flight mass spectrometer with SWIFT technology

The development of a system capable of the speed required for on-line capil lary electrophoresis-tandem mass spectrometry (CE-MS/MS) of tryptic digests is described. The ion trap storage/reflectron time-of-flight (IT/reTOF) ma ss spectrometer is used as a nonscanning detector for rapid CE separation, where the peptides are ionized on-line using electrospray ionization (ESI). The ESI produced ions are stored in the ion trap and de pulse injected int o the reTOF-MS at a rate sufficient to maintain the separation achieved by CE. Using methodology generated by software and hardware developed in our l ab, we can produce SWIFT (Stored Waveform Inverse Fourier Transform) ion is olation and TICKLE activation/fragmentation voltage waveforms to generate M S/MS at a rate as high as 10 Hz so that the MS/MS spectra can be optimized on even a 1-2 s eluting peak. In CE separations performed on tryptic digest s of dogfish myelin basic protein (MBP) where eluting peaks 4-8 s wide are observed, it is demonstrated that an acquisition rate of 4 Hz provides >20 spectra/peak and is more than sufficient to provide optimized MS/MS spectra of each of the eluting peaks in the electropherogram. The detailed structu ral analysis of dogfish MBP including several posttranslational modificatio ns using CE-MS and CE-MS/MS is demonstrated using this method with <10 fmol of material consumed.