De Novo Sequencing
De Novo peptide sequencing is a MS/MS data analysis technique. Although both MALDI–TOF/TOF and ESI MS/MS can generate MS/MS spectra, ESI MS/MS spectra are generally of better quality for de novo sequencing.
Recommended for:
- De novo sequencing of proteins that are unlikely to be in the database
- Mapping of post-translational modifications.
Advantages:
- Ability to use the determined sequence for primer design, or database searching against known proteins (cross species matching)
- Provide internal amino acid sequence data
- Can be used for post-translational modification analysis
Disadvantages:
- Leucine and Isoleucine cannot be differentiated. Most peptides will contain at least one Leu/Ile.
- There can be problems calling Phenylalanine and methionine sulphoxide as these amino acids only vary by 0.04Da. Methionine sulphoxide is a common artefact of 2D gel electrophoresis.
- There can also be problems calling Lysine and Glutamine as these amino acids also vary by only 0.04Da.
- Both of these problems can be overcome and are not routinely an issue.
- There is no way of controlling what peptides will be seen in a digest. ie. we cannot specifically target the N-terminus of a protein
For further details or advice on this APAF service please contact us at ms@proteome.org.au.