Routine Metabolite Identification for Complex Peptides Based on IMS Enabled QTof DIA Data Acquisition and Mass-Metasite Data Processing

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Yun W. Alelyunas, Nathan Anderson, Mark D. Wrona
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Purpose: The metabolic fates of peptide drug candidates need to be thoroughly and rapidly investigated in order to assess their potential to advance in discovery and development. Inclusion of unnatural amino acids, backbone modifications, conjugations, and cyclizations are common strategies to both improve efficacy and ADME profiles. To identify catabolites of these peptide types, data rich acquisition modes coupled with tailored software tools capable of effectively dealing with the complicated permutations of peptide LC-MS data are needed. In this study, catabolites of several structurally complex cyclic peptides were investigated using ion-mobility enabled HDMSE acquired data and processed using the Mass-MetaSite and WebMetabase macromolecule software packages (Molecular Discovery Ltd). 

Methods: Cyclic peptides, including daptomycin, dalbavancin, oritavancin, anidulafungin, and lanreotide, were incubated at 10 µM in simulated intestinal fluid (SIF) in the presence of 500 µg/mL chymotrypsin, respectively.1   Each incubate was sampled at 0 min, 5, 15, 45, and 120 min and quenched by adding 2 volumes of acetonitrile containing 1% formic acid.  The LC system used was an ACQUITY H-Class UPLC equipped with an ACQUITY UPLC Peptide BEH C18 Column, 300Å, 1.7 µm, 2.1 mm X 100 mm. A linear gradient from 5 to 40-70 B% in 8 minutes was used (mobile phase A was water + 0.1% formic acid, and mobile phase B was acetonitrile + 0.1% formic acid (v/v)).  Flow rate was 0.4 ml/min and column temperature was 60°C.  Data were acquired in UNIFI using HDMSE (ion mobility enabled MSE) on a Vion IMS QToF with intelligent data capture enabled and processed using Mass-MetaSite and WebMetabase for metabolite identification. 

Results: Ion-mobility enabled, non-targeted MSE acquired data coupled with intelligent data compression and processed using Mass-MetaSite and WebMetabase metabolite ID software was able to identify and elucidate key cyclic peptide metabolites for five FDA approved cyclic peptide drugs of varying non-natural structural complexity. Ion mobility afforded additional resolution that was shown to help further discriminate, characterize and resolve metabolites from matrix ions having similar m/z, but significantly different ion mobility. Intelligent data capture, now directly implemented in raw data acquisition, afforded >70% reduction of file sizes. The macromolecule processing settings for Mass-MetaSite and WebMetabase made possible access to and analysis of IMS-enabled DIA (HDMSE) data directly from the UNIFI software platform.  Key preliminary results for daptomycin incubations in SIF included the observation of a major ring opened metabolite from ester hydrolysis.  The metabolite was confirmed through common fragment identification.  The software also identified minor metabolites from amide hydrolysis at the terminal region of the parent and ring opened metabolites.2 

Conclusion: Ion mobility enabled non-targeted HDMSE acquisition coupled with Mass-MetaSite and WebMetabase provides seamless data acquisition and processing for routine metabolite determination of complex cyclic peptides. 

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