Accurate quantification of modified cyclic peptides without the need for authentic standards

Abstract

There is a growing interest in the use of cyclic peptides as therapeutics, but their efficient production is often the bottleneck in taking them forward in the development pipeline. We have recently developed a method to synthesise azole-containing cyclic peptides using enzymes derived from different cyanobactin biosynthetic pathways. Accurate quantification is crucial for calculation of the reaction yield and for the downstream biological testing of the products. In this study, we demonstrate the development and validation of two methods to accurately quantify these compounds in the reaction mixture and after purification. The first method involves the use of a HPLC coupled in parallel to an ESMS and an ICPMS, hence correlating the calculated sulfur content to the amount of cyclic peptide. The second method is an NMR ERETIC method for quantifying the solution concentration of cyclic peptides. These methods make the quantification of new compounds much easier as there is no need for the use of authentic standards when they are not available.

Establishment of measurement traceability for peptide and protein quantification through rigorous purity assessment—a review

“…It is therefore important to differentiate both small-molecule and related peptide impurities containing sulfur atoms from the target peptide to eliminate any bias. This approach has been applied to several proteins [149,150,154] and has successfully been benchmarked in the key comparison CCQM-K151 and pilot study CCQM-P191 for the determination of the insulin analog, underpinning the accuracy of two orthogonal SI traceable methods in absolute quantification of proteins.…”

Ascidian Toxins with Potential for Drug Development

“…With broad substrate tolerance, these diversity-generating pathways may allow the organism to adapt to changing predators by altering toxic metabolites [ 42 ]. The discovery of these pathways opens the way for large scale production of cyanobactins by protein engineering and in vitro [ 43 ]. New methods for accurate quantitation of synthesized cyanobactins have been developed without the need for authentic standards [ 43 ].…”

“…The discovery of these pathways opens the way for large scale production of cyanobactins by protein engineering and in vitro [ 43 ]. New methods for accurate quantitation of synthesized cyanobactins have been developed without the need for authentic standards [ 43 ].…”