04 Jun WebMetabase: High Resolution Mass Spectrometry tool to investigate peptide metabolism and investigate peptides cleavage site based on frequency

Tatiana Radchenko(1,2), Christopher Kochansky(3), Alison Bateman(3), Fabien Fontaine(1), Luca Morettoni,(4) Ismael Zamora(1,2)
1. Pompeu Fabra University, Dr, Aiguader 80, Barcelona Spain. 2. Lead Molecular Design, S.L, Vallés 96-102 (L27), Sant Cugat del Vallés, Spain. 3. Merck & Co, 770 Sumneytown Pike, West Point, PA, 19486-0004, USA. 4.Molecular Discovery Ltd, London, UK

Introduction The interest in peptide derived drugs is increasing because of their high selectivity and low toxicity. In general peptides are not stable in the lumen of the gastrointestinal tract due to the high protease activity within. Therefore, during drug design it is required to identify the scissile bonds in the investigated peptide for different proteases and apply structural modifications to improve the stability. The aim of this article is to present a new approach that uses LC-MS to obtain the analytical data from incubations of peptides with different matrices to predict the specific metabolic cleavage site and store results in a chemical aware database. Finally frequency analysis of the sites of cleavage based on the stored data will be shown. Methods Incubations were performed on 13 commercial peptide compounds and 4 positive substrates for the four selected proteases (trypsin, chymotrypsin, pancreatic elastase and pepsin). Samples were analyzed using a Thermo Scientific Q-Exactive Plus Mass Spectrometer (MS) in data dependent scan mode. All data acquired from the LC/MS system were processed using Mass-Metasite and uploaded into WebMetabase to detect and display the peptide related chromatographic peaks together with the structural elucidation data for parent and predicted metabolites. A new algorithm was introduced into WebMetabase to store information about peptides in a chemically aware searchable format, including a system to perform frequency analysis of the site of cleavage for the analysed peptides to reveal the most metabolically labile amide bonds. Preliminary Data Metabolite identification was performed with 13 commercially available peptides (secretin, glucagon, calcitonin, terlipressin, vasopressin, oxytocin, octreotide, deslorelin, histrelin, goserelin, buserelin, leuprolide, gonadorelin) and 4 substrate peptides for the selected proteases. Six compounds had non-natural amino acids and five are cyclic peptides. The smallest peptide (oxytocin) was digested slower than the biggest (calcitonin) by chymotrypsin and trypsin. For the elastase, the smaller octreotide was digested significantly faster than the bigger secretin and calcitonin and slower than oxytocin, these results agreed with the literature. In the investigated peptides all the metabolites identified are coming from amide hydrolysis. The analysis resulted in 45 metabolites that were annotated in the database. These metabolite structural assignments were checked manually and were considered as reliable because the m/z difference between the observed and the computed values was lower than 3 ppm. For all positive substrates one metabolite coming from losing leaving group (7-amino-4-methylcoumarin or 4-nitroanilide, depending on substrate). These results were in agreement with the literature. The frequency analysis revealed 26 sites of cleavage, with the Trp-Ser being the most frequently cleaved bond for all cases. Selectivity was identified for pancreatic elastase and trypsin/chymotrypsin because the Ser-Tyr and Leu-Ser were revealed as a most frequently cleaved bond, respectively. These results were in agreement with previous studies. The effect of small chemical changes in peptide structures for a set of 5 synthetic analogues for the luteinizing-hormone releasing hormone along with natural gonadorelin itself were analyzed with respect to the proteases catalyzed reactions. All LHRH analogues were digested at similar rates by both chymotrypsin and trypsin except for leuprolide, which was hydrolyzed slower. Our approach revealed that elastase cleaved the Ser-Tyr bond and trypsin cleaved the Trp-Ser and Arg-Pro-Nhet except of cases when D-Ser(tBu) was positioned on P2-prime and the C-terminal was modified to Pro-NHet. Novel Aspect This approach is an effective tool to investigate peptide metabolism and to elucidate cleavage sites based on the identified metabolites.