Ltq orbitrap velos spectrometer

A FTIR Equinox 55 instrument from Bruker (Billerica, MA, USA) equipped with an attenuated total reflection accessory with a zinc selenide crystal (Harrick Scientific Products, Pleasantville, NY, United States) and a mercury cadmium telluride detector was used to perform Fourier transform infrared spectroscopy. The following condition were used for FTIR measurements: wavelength range between 4000 cm⁻¹ and 550 cm⁻¹, 10 KHz and 32 scans per sample. The evaluation was carried out with the software OPUS from Bruker (version 4.0.24).
¹H-NMR measurements were performed with AV III HD 300N and AV 600 instruments (Bruker, Billerica, MA, USA), with magnetic field strength of 300 and 600 MHz. The different deuterated solvents were purchased from Deutero. The evaluation was carried out with the analytical software MestReNova from MESTRELAB.
Electrospray ionization mass spectrometry (ESI-MS) was conducted on a LTQ-Orbitrap Velos spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). A voltage of 2.3 to 2.8 kV (positive) or 1.7 to 2.5 kV (negative) was applied. The sample was dissolved in methanol (c = 50 µg/mL) and 0.1 mg/mL trimethyltetradecylammonium bromide was added. The flow rate was adjusted to 0.1 µL/min.

¹H-NMR spectra were acquired on an AVANCE III 400 MHz NMR spectrometer (Bruker, Rheinstetten, Germany) in CDCl₃. Optical rotations were acquired on a Polaar 3005 Polarimeter (Optical Activity, Huntingdon, Great Britain) using a 2.5 cm cell with a Na 589 nm filter and the concentration of samples was denoted as c. Mass spectra data were acquired on a TSQ Quantum Access Max Mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). High-resolution mass spectra (HRMS) were acquired on a LTQ Orbitrap Velos spectrometer (Thermo Scientific) and on a Bruker MicrOTOF. FTIR spectra were acquired on an IR Affinity-1 spectrometer (Shimadzu, Thermo Scientific). Organic solvents used were dried by standard methods when necessary. Commercially available reagents were used without further purification. All reactions were monitored by TLC with silica gel coated plates (EMD/Merck KGaA, Darmstadt, Germany), with visualization by UV light and by charring with 0.1% ninhydrin in EtOH. Column chromatography was performed using Merck 60 Å 70–230 mesh silica gel. The optical density was determined using a Multiskan FC spectrophotometer (Thermo Scientific) at a wavelength of 540 nm when using the MTT assay.

Anti-MDM2 immunoprecipitates were separated and visualized on NuPAGE 4–12% Bis-Tris gels (Invitrogen) followed by Coomassie staining with 0.12% Coomassie Brilliant Blue G-250 (Tokyo Chemical Industry Tokyo, Japan), 10% phosphoric acid, 10% ammonium sulfate, and 20% methanol. Protein bands were retrieved and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Digested peptides were cleaned using a Pierce C18Spin Column (Thermo Fisher Scientific) and analyzed on a LTQ-Orbitrap Velos spectrometer (Thermo Fisher Scientific). For protein identification, spectra were processed with Proteome Discoverer version 1.3 software (Thermo Fisher Scientific) using the Mascot algorithm and the Swiss-Prot human protein database. Peptide data were filtered using a Mascot significance threshold of less than 0.05 and Peptide Probability (FDR < 0.01).