title: At-line nanofractionation with parallel mass spectrometry and bioactivity assessment for the rapid screening of thrombin and factor Xa inhibitors in snake venoms.
abstract: Snake venoms comprise complex mixtures of peptides and proteins causing modulation of diverse physiological functions upon envenomation of the prey organism. The components of snake venoms are studied as research tools and as potential drug candidates. However, the bioactivity determination with subsequent identification and purification of the bioactive compounds is a demanding and often laborious effort involving different analytical and pharmacological techniques. This study describes the development and optimization of an integrated analytical approach for activity profiling and identification of venom constituents targeting the cardiovascular system, thrombin and factor Xa enzymes in particular. The approach developed encompasses reversed-phase liquid chromatography (RPLC) analysis of a crude snake venom with parallel mass spectrometry (MS) and bioactivity analysis. The analytical and pharmacological part in this approach are linked using at-line nanofractionation. This implies that the bioactivity is assessed after high-resolution nanofractionation (6 s/well) onto high-density 384-well microtiter plates and subsequent freeze drying of the plates. The nanofractionation and bioassay conditions were optimized for maintaining LC resolution and achieving good bioassay sensitivity. The developed integrated analytical approach was successfully applied for the fast screening of snake venoms for compounds affecting thrombin and factor Xa activity. Parallel accurate MS measurements provided correlation of observed bioactivity to peptide/protein masses. This resulted in identification of a few interesting peptides with activity towards the drug target factor Xa from a screening campaign involving venoms of 39 snake species. Besides this, many positive protease activity peaks were observed in most venoms analysed. These protease fingerprint chromatograms were found to be similar for evolutionary closely related species and as such might serve as generic snake protease bioactivity fingerprints in biological studies on venoms.
authors.affiliation: Division of BioAnalytical Chemistry, Amsterdam Institute for Molecules Medicines and Systems, VU University Amsterdam, De Boelelaan 1083, 1081HV, Amsterdam, The Netherlands.
authors.forename: Marija
authors.initials: M
authors.lastname: Mladic
authors.affiliation: Division of BioAnalytical Chemistry, Amsterdam Institute for Molecules Medicines and Systems, VU University Amsterdam, De Boelelaan 1083, 1081HV, Amsterdam, The Netherlands.
authors.forename: Barbara M
authors.initials: BM
authors.lastname: Zietek
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4 117543, Singapore.
authors.forename: Janaki Krishnamoorthy
authors.initials: JK
authors.lastname: Iyer
authors.affiliation: Division of BioAnalytical Chemistry, Amsterdam Institute for Molecules Medicines and Systems, VU University Amsterdam, De Boelelaan 1083, 1081HV, Amsterdam, The Netherlands.
authors.forename: Philip
authors.initials: P
authors.lastname: Hermarij
authors.affiliation: Division of BioAnalytical Chemistry, Amsterdam Institute for Molecules Medicines and Systems, VU University Amsterdam, De Boelelaan 1083, 1081HV, Amsterdam, The Netherlands; Hyphen MassSpec, Herenweg 95 2361, EK, Warmond, The Netherlands.
authors.forename: Wilfried M A
authors.initials: WM
authors.lastname: Niessen
authors.affiliation: Division of BioAnalytical Chemistry, Amsterdam Institute for Molecules Medicines and Systems, VU University Amsterdam, De Boelelaan 1083, 1081HV, Amsterdam, The Netherlands.
authors.forename: Govert W
authors.initials: GW
authors.lastname: Somsen
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4 117543, Singapore.
authors.forename: R Manjunatha
authors.initials: RM
authors.lastname: Kini
authors.affiliation: Division of BioAnalytical Chemistry, Amsterdam Institute for Molecules Medicines and Systems, VU University Amsterdam, De Boelelaan 1083, 1081HV, Amsterdam, The Netherlands. Electronic address: j.kool@vu.nl.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Kool
mesh_terms: D000818:Animals; D000991:Antithrombins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D055808:Drug Discovery / Q000379:methods*; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism; D065427:Factor Xa Inhibitors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D036103:Nanotechnology; D010802:Phylogeny; D030162:Reptilian Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D012235:Rhodamines / Q000737:chemistry; D012910:Snake Venoms / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000991:Antithrombins; D065427:Factor Xa Inhibitors; D005456:Fluorescent Dyes; D030162:Reptilian Proteins; D012235:Rhodamines; D012910:Snake Venoms; C049025:rhodamine 110; D013917:Thrombin; D015951:Factor Xa
keywords: At-line nanofractionation; Liquid chromatographymass spectrometry; Snake venom; Thrombin and factor Xa inhibitors