Improving peptide catabolism interpretation using ion mobility data and server-based data review with HELM integration
A unique challenge in peptide-based drug metabolism for evaluating preclinical candidates is the need for processing and visualization software tools that accommodate both small molecule rules (such as oxidations, synthetic modifications), as well as large molecule modifications (such as amide hydrolysis and oxidative deamination).
As more complex peptide drugs with unnatural modifications are introduced, new software techniques that enable thorough identification, rapid analysis and comparisons of candidate series will be required. Three aspects to be discussed in detail will include: a clearance analysis of multiple peptide candidates involving stepwise substitution of non-natural based amino acid moieties; the utility of collision cross section (CCS) measurements for identifying metabolites; and integrated HELM notation (WebMetabase) to improve visualization of complex peptide analyses.
Eight 14-amino acid analogs of somatostatin were studied. Somatostatin was systematically substituted using both D-amino acids and a non-natural amino acid Msa (mesityl alanine) to determine the analogs’ abilities to slow down human-based metabolism. Analogs were incubated in human serum at eleven time points (0, 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 30 h and 48 h). Data were collected on an ACQUITY UPLC coupled to a benchtop IMS QToF (Waters) acquiring data independent IMS data. Data were transferred and batch processed using MassMetasite (Lead Molecular) via the built-in UNIFI Application Programming Interface (API). All data were uploaded onto WebMetabase version 4.0 ( cloud based/server application, Lead Molecular) and analyzed.
WebMetabase was used to process and visualize all 8 analogs, which enabled thorough interrogations of key metabolic processes. Catabolism primarily occurred at the terminal amino acids outside the disulfide linked ring portion of somatostatin (as reported previously). WebMetabase enabled identification of modifications and their effects on reducing somatostatin degradation, making it clear when specific cleavages were enhanced or blocked via substitution. Reduction (or elimination) of a specific cleavage did not always translate into a clear reduction in overall clearance. It was therefore important to review multiple hot spots in the context of the overall stability plot to ensure that multiple substitutions had the desired effect of stabilizing the original substrate in a holistic manner.
Ion mobility derived collision cross section (CCS) values were recorded for all detected ions. The +3 charge states of intact analogs, along with -Ala and – AlaGly variants, were compared for CCS changes. Preliminary data showed that intact peptides produced a modest difference of >1% in size. However, -Ala variants produced a greater difference of over 3% across analogs, indicating that these modifications impacted gross molecular structure in a measurable way. CCS also provided a routine mechanism to keep track of and differentiate a number of (highly related with numerous isobaric) analogs and their substitutions across multiple timepoints
Ion mobility data processed using the web-based package WebMetabase with HELM notation provided an efficient method to review complex data in a straightforward manner. WebMetabase enabled offloading of key processing time and burden from local hardware installations, allowing data insights to be routinely shared and discussed between several researchers at different sites. Additionally, HELM integration with WebMetabase facilitated a visualization more familiar to biologists (rather than mol or SDF formats).