title: Screening for proteolytic activities in snake venom by means of a multiplexing electrospray ionization mass spectrometry assay scheme.
abstract: A multiplexed mass spectrometry based assay scheme for the simultaneous determination of five different substrate/product pairs was developed as a tool for screening of proteolytic activities in snake venom fractions from Bothrops moojeni. The assay scheme was employed in the functional characterization of eight model proteases. Time-resolved reaction profiles were generated and the relative reaction progress at each time point was determined. These were used to semi-quantitatively sort the catalytic activities of each enzyme towards the respective substrates into six classes. The resulting activity pattern served as an activity fingerprint for each enzyme. The multiplex assay scheme was then applied to a screening for proteolytic activities in fractions of the pre-separated venom from B. moojeni. Activity patterns of each fraction were generated and used to sort the fractions into three different categories of activity. By comparison of the fingerprint activity patterns of the venom fractions and the model enzymes, a compound with proteolytic properties similar to activated protein C was detected.
authors.affiliation: University of Twente, Chemical Analysis Group and MESA+ Institute for Nanotechnology, P.O. Box 217, 7500 AE Enschede, The Netherlands.
authors.forename: André
authors.initials: A
authors.lastname: Liesener
authors.forename: Anna-Maria
authors.initials: AM
authors.lastname: Perchuc
authors.forename: Reto
authors.initials: R
authors.lastname: Schöni
authors.forename: Marianne
authors.initials: M
authors.lastname: Wilmer
authors.forename: Uwe
authors.initials: U
authors.lastname: Karst
mesh_terms: D000818:Animals; D017837:Bothrops; D002417:Cattle; D003435:Crotalid Venoms / Q000032:analysis* / Q000737:chemistry*; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D010447:Peptide Hydrolases / Q000032:analysis* / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D003435:Crotalid Venoms; D010447:Peptide Hydrolases