Micro-high-performance liquid chromatography/Fourier transform mass spectrometry with electron-capture dissociation for the analysis of protein enzymatic digests.
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.
Davidson
Walter
W
Research and Development Center, Boehringer Ingelheim Pharmaceuticals, 900 Ridgebury Rd., Ridgefield, CT 06877, USA. wdavidso@rdg.boehringer-ingelheim.com
Frego
Lee
L
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000737:chemistry; D017666:Deuterium Oxide; D005583:Fourier Analysis; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010434:Pepsin A; D011492:Protein Hydrolysates / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
D003574:Cytochrome c Group; D011492:Protein Hydrolysates; D010434:Pepsin A; D017666:Deuterium Oxide