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Ammonia cleaves polypeptides at asparagine proline bonds
1Medical Biomics Centre, St George's Hospital Medical School, London, UK. etarelli@sghms.ac.uk
Summary
Ammonia completely cleaves polypeptides at Asn-Pro sequences, yielding proline and asparagine products. Other Asn-X bonds show varied cleavage or deamidation to aspartic acid.
Area of Science:
- Biochemistry
- Protein Chemistry
- Chemical Biology
Background:
- Asparagine (Asn) and aspartic acid (Asp) residues are common in polypeptides.
- Understanding peptide bond stability and modification is crucial for protein analysis and engineering.
Purpose of the Study:
- To investigate the cleavage and modification of asparagine residues in polypeptides under specific reaction conditions.
- To characterize the products and mechanisms of Asn-Pro bond cleavage and Asn deamidation.
Main Methods:
- Incubation of polypeptides with ammonia.
- Analysis of cleavage products using MALDI-ToF mass spectrometry (MS).
- Assessment of deamidation and isoAsp formation using RP-HPLC and MS.
Main Results:
- Complete cleavage of the Asn-Pro bond by ammonia, forming Pro-N-terminus and Asn/isoAsn-C-terminus products.
- Partial cleavage observed at Asn-Leu, Asn-Thr, and Asn-Ser bonds.
- Deamidation of Asn to Asp and partial conversion of Asp to isoAsp were detected.
Conclusions:
- The Asn-Pro sequence is highly susceptible to ammonia-induced cleavage via a succinimide intermediate.
- Other Asn-X bonds exhibit differential susceptibility to cleavage.
- Deamidation and isoAsp formation are significant side reactions affecting Asn and Asp residues under these conditions.