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We offer the following Protein sequencing services
N-terminal protein sequencing is offered using ABI’s “Procise-492” model, a fully automated sequencer which employs Edman degradation, PTH derivatization of amino acids and HPLC separation followed by UV detection. The system is qualified as per vendor’s OQ/PV protocol which ensures the optimum performance.
Capabilities:
- Faster turn-around time of 7 working days
- Results are archived for 5yrs
- Samples archived for 1yr
- Samples can be submitted in liquid, lyophilized or blot forms
- Database search to match the identity of proteins performed free of cost (if requested)
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Full protein sequencing is required to provide the entire primary sequence of the given biotherapeutic as part of the CMC document under the quality aspect.
The approach involves chemical or enzymatic digestion of protein/peptide to get the fragments of the product and employing automated sequencing by protein sequencer. Overlapping fragments are generated by multiple enzyme are chemical digests to assign the sequence of the protein.
Capabilities:
- Multiple enzyme approach to elucidate complete a-a sequence.
- Method development to separate the multiple peptides arising out of digestion
- Quick turnaround time with comprehensive documentation which can be used in regulatory dossiers.
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Most microbial and mammalian proteins are blocked at the N-terminus, which prevents it from sequencing by Edman chemistry. Mostly, blocking is by acetylation but formylation and glutamate cyclization is also possible.
There are different de-blocking techniques are developed and established:
- Deacetylation by TFA Vapor
- Deformylation by HCl treatment
- Removal of Pyroglutamate with Pyroglutamte amino peptidase.
If none of these strategies are successful, internal sequencing followed by CNBr digestion can be achieved to get the information related to the identity of the protein.
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