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
peer-reviewed · Journal of Mass Spectrometry · 2007
| Date | 2007-12-01 |
| Type | peer-reviewed |
| Venue | Journal of Mass Spectrometry |
| Publisher | Wiley |
| Contribution | downstream-application |
| DOI | 10.1002/jms.1337 |
| Citations (OpenAlex) | 13 |
| Venue 2-year citedness | 1.85 |
Abstract
We have determined the primary structure of cytochrome c(4) 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.
Methods and tools
- Cytochrome c4 bottom-up sequencing: Determines the primary structure of the 21 kDa cytochrome c4 of Thiocapsa roseopersicina de novo, bottom-up, from three separate digests (trypsin, Lys-C, Glu-C) read by ion-trap and FTICR mass spectrometry.
Cites (4)
- De Novo Peptide Sequencing and Identification with Precision Mass Spectrometry (2007) crossref
- Performance Evaluation of Existing De Novo Sequencing Algorithms (2006) crossref
- “De novo” peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: A preliminary study (2003) crossref
- Peptide and protein de novo sequencing by mass spectrometry (2003) crossref
Cited by (3)
- Lessons in de novo peptide sequencing by tandem mass spectrometry (2015) both
- Extensive <i>De Novo</i> Sequencing of New Parvalbumin Isoforms Using a Novel Combination of Bottom-Up Proteomics, Accurate Molecular Mass Measurement by FTICR−MS, and Selected MS/MS Ion Monitoring (2010) crossref
- De novo sequencing of peptides by MS/MS (2010) crossref