Sequencing of proteins and peptides is often used as a generic term to summarise different techniques. Depending on your project's requirements we can apply the following strategies:
N-terminal sequencing by Edman degradation
Chemical sequencing by automated phenylisothiocyanate cleavage chemistry was developed by Pehr Edman. It affords the N-terminal protein sequence in repetitive cycles. Its strengths are the discrimination of leucine and isoleucine and the straightforward elucidation of N-terminal processing. Samples need to be relatively pure and the first amino acids must not be blocked which is the case in roughly 25% of mammalian proteins.
When you need to know the identity of a protein, take a look at protein identification by LCMS.
After cleavage with a suitable protease (trypsin is the first choice in most cases) the generated peptides are separated and detected by LCMS. Peptide ions are automatically fragmented and resulting spectra are submitted to searches against large databases like NCBInr and Swissprot. As genome information is available for many commonly studied organisms and species or homologues of them, this methodology can be applied to most sample origins.
De novo sequencing of proteins where no DNA sequence is known
When the genome of an organism or other databases are not available, LCMS data can be used for de novo sequencing. Sequence tags are generated from which DNA primers can be deducted.
To confirm that a recombinant protein matches a given sequence, a number of different proteolytic digests is analysed by LCMS for matching against the supplied sequence. Sequence coverage of higher than 95% is reached in most cases by analysis of several proteolytic digests.
Full de novo sequencing e. g. of a monoclonal antibody
When complete sequence information of a protein is required without prior knowledge, different proteolytic digests are analysed by LCMS followed by de novo sequencing. If necessary Edman sequencing of HPLC separated peptides can be done to fill sequence gaps or discriminate leucine from isoleucine.
Please contact us to discuss your project with our specialists.