De novo sequencing of a 21-kDa cytochrome c4 from Thiocapsa roseopersicina by nanoelectrospray ionization ion-trap and Fourier-transform ion-cyclotron resonance mass spectrometry

Authors

  • Rui Miguel Mamede Branca,

    1. Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary
    2. Department Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, USA
    Search for more papers by this author
  • Gabriella Bodó,

    1. Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary
    Search for more papers by this author
  • Csaba Bagyinka,

    1. Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary
    Search for more papers by this author
  • Laszlo Prokai

    Corresponding author
    1. Department Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, USA
    • Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699, USA.
    Search for more papers by this author

  • Paper presented at the 25th Informal Meeting on Mass Spectrometry, Nyiregyháza-Sóstó, Hungary, 6–10 May, 2007

Abstract

We have determined the primary structure of cytochrome c4 from Thiocapsa roseopersicina by de novo protein sequencing using the ‘bottom up’ approach. Three different enzymes (trypsin, endoproteinase Lys-C, and endoproteinase Glu-C) were employed to prepare four different sets of proteolytic digests. The digestion strategy was designed to permit a gradual buildup of smaller peptides into larger ones that were overlapped to yield the complete protein sequence. In this way we countered the main problem: peptides larger than about 1500 Da were difficult to sequence fully by tandem mass spectrometry. Direct infusion and online liquid chromatography were used on a linear ion trap Fourier-transform ion-cyclotron resonance hybrid instrument. The high resolving power, high mass accuracy and the availability of electron capture dissociation and collision-induced dissociation were essential to achieve full sequence coverage. The software DeNovoX complemented by manual interpretation was used to generate sequence information from tandem mass spectra. The predominantly automated nature of data acquisition and handling allowed for a relatively straightforward and fast procedure, which could compete with the mainstream alternative of nucleotide sequence determination. Copyright © 2007 John Wiley & Sons, Ltd.

Ancillary