Peptides incubations with trypsin, chymotrpsin, elastase, pepsinA, DPP4 and neprilysin

ABSTRACT: The first set (dataset-1) included ten commercially available peptides (secretin, calcitonin, oxytocin, octreotide, deslorelin, histrelin, goserelin, buserelin, leuprolide and gonadorelin) and four marker substrate peptides with known cleavage sites as positive controls for each of the selected proteases - trypsin, chymotrypsin, pancreatic elastase and pepsin (Table 1). Five out of the ten compounds had unnatural amino acids and three of them were cyclic peptides. Moreover, to investigate the effect of small chemical/monomer changes in the peptide structure with respect to the proteases catalyzed reactions, the set was selected to also contain five synthetic analogues for the same peptide series, luteinizing-hormone releasing hormone (LHRH). All test compounds were prepared as a stock in concentration of 10 mM in dimethyl sulfoxide (DMSO). For the second set of peptides (dataset-2) metabolite identification was performed for 4 commercially available peptides (glucagon-like peptide-1 (GLP-1) and 3 synthetic analogues for the GLP-1: taspoglutide, exenatide and liraglutide) for the selected proteases (Table 1). Taspoglutide peptide had non-natural amino acids and liraglutide had C-16 fatty acid side chain (palmitic acid). GLP-1, taspoglutide and exenatide were prepared as a stock in concentration of 10 mM and liraglutide was prepared in concentration of 5 mM in DMSO. For the first dataset, chromatographic separation of metabolites was performed using the ACQUITY ultra-performance liquid chromatography (UPLC) system (Waters, Milford, MA, USA). The ACQUITY UPLC BEH C18 column. MS/MS analyses were run on a Thermo Scientific Q-Exactive Plus mass spectrometer operated in positive electrospray ionization (ESI) mode. MS/MS was a data dependent acquisition (DDA) using peptide specific inclusion lists containing amide hydrolysis ions of multiple charge states (z =1 to 3). Thermo Scientific Dionex UltiMate 3000 RS HPLC system in combination with a Pal autosampler (CTC Analytics AG, Zwingen, Switzerland was used. Chromatographic separation was performed using an Acquity CSHT Phenyl-Hexyl Column. MS/MS analyses were run on a Q Exactive? Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) operated in positive electrospray ionization (ESI) mode. MS/MS was a data dependent acquisition (DDA) using peptide specific inclusion lists containing amide hydrolysis ions of multiple charge states (z =1 to 5).

SUBMITTER: Tatiana Radchenko 

PROVIDER: S-BSST33 | biostudies-other |

REPOSITORIES: biostudies-other