On-Line Capillary Electrophoresis/ Microelectrospray Ionization-Tandem Mass Spectrometry Using an Ion Trap Storage/ Time-of-Flight Mass Spectrometer with SWIFT Technology Xiaoying Jin, Jeongkwon Kim, Stephen Parus, and David M. Lubman* Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055 Robert Zand Department of Biological Chemistry and Biophysics Research Division, The University of Michigan, Ann Arbor, Michigan 48109-1055 The development of a system capable of the speed required for on-line capillary electrophoresis-tandem mass spectrometry (CE-MS/ MS) of tryptic digests is described. The ion trap storage/ reflectron time-of-flight (IT/ reTOF) mass 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 dc pulse injected into the reTOF-MS at a rate sufficient to maintain the separation achieved by CE. Using methodology gener- ated by software and hardware developed in our lab, we can produce SWIFT (Stored Waveform Inverse Fourier Transform) ion isolation and TICKLE activation/ fragmen- tation voltage waveforms to generate MS/ 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 per- formed on tryptic digests 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 structural analysis of dogfish MBP including several posttranslational modifica- tions using CE-MS and CE-MS/ MS is demonstrated using this method with <10 fmol of material consumed. Capillary electrophoresis-mass spectrometry (CE-MS) provides distinct advantages as a method for the separation and analysis of complex peptide mixtures resulting from protein digestion. 1-22 Capillary electrophoresis is able to provide rapid separations of complex mixtures with high resolution in which more than 30 peptides can be separated in less than 15 min. 16 The separation is based on the electrophoretic mobility which depends on the mass and charge of the peptide, so that even modifications such as phosphorylation and deamidations can be readily detected and observed in the separation. 22 Also, a very small amount of often- limited sample from biological sources is consumed in analysis. Moreover, the use of CE is particularly well-suited to a mass spectrometer detector because of the sharp, highly concentrated bands that result since the mass spectrometer is a concentration- dependent detector. 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