Micro-high-performance liquid chromatography/Fourier transform mass spectrometry with electron-capture dissociation for the analysis of protein enzymatic digests |
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Authors: | Davidson Walter Frego Lee |
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Institution: | Research and Development Center, Boehringer Ingelheim Pharmaceuticals, 900 Ridgebury Rd., Ridgefield, CT 06877, USA. wdavidso@rdg.boehringer-ingelheim.com |
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Abstract: | Electron-capture dissociation (ECD) Fourier transform mass spectrometry (FTMS) employed to generate comprehensive sequence information for the chromatographic analysis of enzymatic protein digests is described. A pepsin digest of cytochrome c was separated by reversed-phase micro-high-performance liquid chromatography (microHPLC) and ionized 'on-line' by electrospray ionization (ESI). The ions thus formed were transferred to and trapped in the FTMS analyzer cell. Typically, no precursor ion isolation was performed. The trapped ions were subjected to a pulse of electrons to induce fragmentation. Mass spectra were acquired continuously to produce a three-dimensional LC/MS data set. The spectra were dominated by c and, to a lesser degree, z ions, which provided near complete sequence coverage. External calibration provided good mass accuracy and resolution, typical of FTMS. Thus microHPLC/ECD - FTMS is shown to be a highly informative method for the analysis of enzymatic protein digests. |
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