Sciex 4800 maldi tof tof ms

All reagents and solvents were purchased from commercial sources (Fisher, Aldrich, and Chem-Impex) and used directly. Final compounds were purified on preparative high-pressure liquid chromatography (RP-HPLC) was performed on either Waters 1525 or Agilent 1260 Series system. Systems were run with 0–20% methanol/water gradient with 0.1% TFA. Matrix-assisted laser desorption ionization mass spectra (MALDI-MS) data were acquired in positive-ion mode using a Sciex 4800 MALDI TOF/TOF MS. The peptides (PKR, PKRIA and PRRRS) were synthesized on a CEM Liberty Blue Automated Microwave Peptide Synthesizer with the manufacturers standard coupling cycles at 0.1 mmol scale. All compounds were cleaved from the resin in a cocktail of trifluoroacetic acid/2,2’-(ethylenedioxy)diethanethiol/H₂O/triisopropylsilane (94:2.5:2.5:1) and confirmed by mass spectrometry. The purity of final compounds was confirmed through Agilent 1260 Series HPLC system by running with 5% to 30% methanol/water gradient with 0.1% TFA. All the purity of target compounds showed >95%.

MALDI-MS methylation inhibition assay was performed and analyzed via a Sciex 4800 MALDI TOF/TOF MS. Compounds were tested at 30 μM in a final well volume of 100 μL: 0. 2 μM NTMT1, 20 mM Tris (pH = 7.5), 50 mM KCl, 1 μM AdoMet and incubated at 37°C for 10 mins before the addition of 2 μM APKRQSPLPP peptide to initiate the reaction. After 20 mins, the reactions were quenched in a 1:1 ratio by addition of 100 μL a quenching solution (20 mM NH₄H₂PO₄, 0.4% (v/v) TFA in 1:1 acetonitrile/water). Samples were analyzed by the MALDI-MS with α-Cyano-4-hydroxycinnamic acid (CHCA) matrix solution. Data were processed in Data Explorer. Those compounds that showed over 50% inhibition at 30 μM will be further validated at both 10 and 100 μM.

MALDI-MS methylation and inhibition assay were performed and analyzed via a Sciex 4800 MALDI TOF/TOF MS. MALDI-MS methylation assay was performed in a final well volume of 40 μL: 0.1 μM NTMT1, 20 mM Tris (pH = 7.5), 50 mM KCl, 3 μM SAM and 10 μM compounds. After incubation overnight at 37 °C, the samples were quenched in a 1:1 ratio with a quenching solution (20 mM NH₄H₂PO₄, 0.4% (v/v) TFA in 1:1 acetonitrile/water). Samples were analyzed by MALDI-MS with 2,5-dihydroxybenzoic acid matrix solution.
The inhibition assay was performed in a final well volume of 40 μL: 0.1 μM NTMT1, 20 mM Tris (pH = 7.5), 50 mM KCl, 3 μM SAM and various concentrations of compounds at 37 °C for 10 min before the addition of 0.5 μM GPKRIA peptide to initiate the reaction. After incubation for 20 min, the samples were quenched in a 1:1 ratio with a quenching solution (20 mM NH₄H₂PO₄, 0.4% (v/v) TFA in 1:1 acetonitrile/water). Samples were analyzed by MALDI-MS with 2,5-dihydroxybenzoic acid matrix solution. Duplicate was performed for all experiments. Data were processed in Data Explorer.