Workflows » De novo sequencing
Not all organisms that are of significance for research are completely sequenced yet i.e. conventional mass spectrometry-based approaches for protein identification may fail. These approaches are based on the correlation of mass spectrometric data with predicted data from sequence databases.
Unknown proteins without sequence information in databases can also be sequenced with the mass spectrometer. In general this requires a manual interpretation of MS/MS-data. However the manual interpretation is error prone and many CID (collision induced dissociation)-spectra do not provide unambiguous sequence information. The available software tools for de novo interpretation of CID MS/MS-spectra are as error prone as manual interpretation.
Edman degradation and amino acid detection is still one of the most reliable methods for de novo sequencing of peptides/proteins. However, it is less sensitive than mass spectrometry, labour-intensive and expensive.
Chemically Assisted Fragmentation is a technique that generates high quality sequence data by MALDI-based tandem mass spectrometry. It is based on a chemical modification of the N-terminal amino group. The modification is negatively charged and suppresses b-ions upon fragmentation by PSD (Post Source Decay) or CID in positive ion mode. The fragment spectra contain only y-ions which simplifies the manual interpretation. We are using a different two step chemistry to firstly block the epsilon amino group of lysines by a guanidination (conversion of lysine to homoarginine) and secondly modify the N-terminus of tryptic peptides by a sulfonation with 4-sulfophenyl-isothiocyanate (SPITC). The method is described in Chen et al. (2004) Rapid Commun. Mass Spectrom, 18:191-198. High quality CID-spectra of SPITC-modified peptides reveal unambiguous sequence information by a clear y-ions series. This approach is faster and more sensitive than Edman sequencing.