Almost all regulatory processes in biology ultimately lead to or originate from protein modifications. However, it is unclear to which extent each mechanism of regulation actually affects proteins and thus phenotypes. Currently we know that the N-terminal truncation is one of resources of impurities in manufacturing, purification and storage, and it may affect the designed medical efficiency. So far the scientists have found that most proteins have N-terminally truncated proteoforms, originated from protein cleavage, alternative translation and alternative splicing. Meanwhile, alternative cleavage of signal peptides are also one reason for isoform generation.
Based on the N-terminal sequencing service, Creative Proteomics can help to analyze the N-terminal sequence of the protein therapeutics. Generally our tech support can provide 3 analytical approaches to characterize the N-termini of the protein drugs:
1 Edman Degradation
Phenyl isothiocyanate is applied to react with an uncharged N-terminal amino group, leading to the release of amino acid one by one in the repeated circles. The released amino acids would be used to predict, or verify the N-terminal sequence of the protein.
2 Top-Down Analysis
The protein therapeutics, such as monoclonal antibodies, would be ionized for subsequent analysis with MALDI-TOF or even tandem mass spectrometry. The ionized proteins and corresponding fragmented ions would be detected with high accuracy mass analyzer, to obtain accurate and precise m/z values, to calculate the molecular weight of relevant peptides. Since the primary sequence is known to us, it’s easy to nominate the peptides with specific sequences.
3 Bottom-Up Analysis
The target protein would be cleaved with proteases, to release the peptides. With the assistance of high resolution/precision mass spectrometry and proteomics databases available, the peptides, especially the fragments from N-terminus, would be mapped.
Creative Proteomics are glad to discuss your projects, to figure out the optimal analytical workflow, to characterize the N-terminal sequences.