title: Coupling Laser Diode Thermal Desorption with Acoustic Sample Deposition to Improve Throughput of Mass Spectrometry-Based Screening.
abstract: The move toward label-free screening in drug discovery has increased the demand for mass spectrometry (MS)-based analysis. Here we investigated the approach of coupling acoustic sample deposition (ASD) with laser diode thermal desorption (LDTD)-tandem mass spectrometry (MS/MS). We assessed its use in a cytochrome P450 (CYP) inhibition assay, where a decrease in metabolite formation signifies CYP inhibition. Metabolite levels for 3 CYP isoforms were measured as CYP3A4-1'-OH-midazolam, CYP2D6-dextrorphan, and CYP2C9-4'-OH-diclofenac. After incubation, samples (100 nL) were acoustically deposited onto a stainless steel 384-LazWell plate, then desorbed by an infrared laser directly from the plate surface into the gas phase, ionized by atmospheric pressure chemical ionization (APCI), and analyzed by MS/MS. Using this method, we achieved a sample analysis speed of 2.14 s/well, with bioanalytical performance comparable to the current online solid-phase extraction (SPE)-based MS method. An even faster readout speed was achieved when postreaction sample multiplexing was applied, where three reaction samples, one for each CYP, were transferred into the same well of the LazWell plate. In summary, LDTD coupled with acoustic sample deposition and multiplexing significantly decreased analysis time to 0.7 s/sample, making this MS-based approach feasible to support high-throughput screening (HTS) assays.
authors.affiliation: Bristol-Myers Squibb, Wallingford, CT, USA zuzana.haarhoff@bms.com.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Haarhoff
authors.affiliation: Bristol-Myers Squibb, Wallingford, CT, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Wagner
authors.affiliation: Bristol-Myers Squibb, Wallingford, CT, USA.
authors.forename: Pierre
authors.initials: P
authors.lastname: Picard
authors.affiliation: Bristol-Myers Squibb, Wallingford, CT, USA.
authors.forename: Dieter M
authors.initials: DM
authors.lastname: Drexler
authors.affiliation: Bristol-Myers Squibb, Wallingford, CT, USA.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Zvyaga
authors.affiliation: Bristol-Myers Squibb, Wallingford, CT, USA.
authors.forename: Wilson
authors.initials: W
authors.lastname: Shou
mesh_terms: D001681:Biological Assay / Q000379:methods; D002138:Calibration; D065607:Cytochrome P-450 Enzyme Inhibitors / Q000737:chemistry*; D055808:Drug Discovery / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D052616:Solid Phase Extraction / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors
keywords: enzyme assays; label-free technologies; liquid handling; mass spectrometry; multiplex assays and technology