Introduction: De novo sequencing of peptides is an effective tool for the identification of post-translational modifications (PTM) and sequence similarity. The data acquisition has become highly automated with LC/MS/MS instrumentation. The high throughput of data analyses requires de novo sequencing to be performed automatically with minimum user input.
Methods: A sample of three bovine proteins was treated with iodoacetic acid, and digested with trypsin. The mixture of peptides was separated on a reverse phase column, and the fragmentation data were acquired with Finnigan LCQ Deca ion trap in automated data dependent acquisition mode. DeNovoX, a new de novo sequencing program based on PARSEK II algorithm, was used to sequence the data in a fully automated fashion.
Preliminary Data: The combination of de novo sequencing with the database searching results in a high level of confidence in identification of proteins and their modifications. In a fully automated search DeNovoX identified 63 peptides belonging to three major protein components of the mixture, and an unexpected minor impurity. Eight modified peptides were characterised.
Further evolution of this combined approach will concentrate on more sophisticated level of communication between a database search engine and de novo sequencing program.
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