Microfluidic Chip-Based Online Screening Coupled to Mass Spectrometry: Identification of Inhibitors of Thrombin and Factor Xa.
Thrombin and factor Xa (FXa) are critical enzymes of the blood coagulation cascade and are excellent targets of anticoagulant agents. Natural sources present an array of anticoagulants that can be developed as antithrombotic drugs. High-resolution, online screening techniques have been developed for the identification of drug leads from complex mixtures. In this study, we have developed and optimized a microfluidic online screening technique coupled to nano-liquid chromatography (LC) and in parallel with a mass spectrometer for the identification of thrombin and FXa inhibitors in mixtures. Inhibitors eluting from the nano-LC were split postcolumn in a 1:1 ratio; half was fed into a mass spectrometer (where its mass is detected), and the other half was fed into a microfluidic chip (which acts as a microreactor for the online assays). With our platform, thrombin and FXa inhibitors were detected in the assay in parallel with their mass identification. These methods are suitable for the identification of inhibitors from sample amounts as low as sub-microliter volumes.
Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore.
Janaki Krishnamoorthy
JK
Iyer
AIMMS Division of BioAnalytical Chemistry, Faculty of Sciences, VU University Amsterdam, Amsterdam, Netherlands.
Reka A
RA
Otvos
AIMMS Division of BioAnalytical Chemistry, Faculty of Sciences, VU University Amsterdam, Amsterdam, Netherlands.
Jeroen
J
Kool
Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore dbskinim@nus.edu.sg.
R Manjunatha
RM
Kini
D000925:Anticoagulants / Q000737:chemistry / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects; D002853:Chromatography, Liquid / Q000379:methods; D015951:Factor Xa / Q000737:chemistry*; D065427:Factor Xa Inhibitors / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D044085:Microfluidics / Q000379:methods; D009862:Online Systems; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry*
D000925:Anticoagulants; D065427:Factor Xa Inhibitors; D013917:Thrombin; D015951:Factor Xa
FXa inhibitor; argatroban; benzamidine; thrombin inhibitor; thrombosis