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

Rui Miguel Mamede Branca, Gabriella Bodó, Csaba Bagyinka, Laszlo Prokai

Research output: Contribution to journalArticle

8 Scopus citations

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.

Original languageEnglish
Pages (from-to)1569-1582
Number of pages14
JournalJournal of Mass Spectrometry
Volume42
Issue number12
DOIs
StatePublished - 1 Dec 2007

Keywords

  • Bottom up
  • Collision-induced dissociation
  • Cytochrome c
  • De novo protein sequencing
  • Electron-capture dissociation
  • Electrospray ionization
  • Fourier-transform ion-cyclotron resonance
  • Ion trap
  • Liquid chromatography-mass spectrometry

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