Protein calibration standard 2 mixture

Recombinant USP14 protein was desalted to remove interfering reducing agent on 30 kDa cut-off spin columns (Merck) and resuspended in water. For MALDI-TOF analysis of compound binding, 10 µg USP14 protein was incubated with 10 µM of selected compounds for 45 minutes at room temperature. Samples were then desalted to remove unbound compound on 30 kDa cut-off spin columns and resuspended in water. Approximately 0.25 µg sample was loaded onto polished stainless steel MALDI-TOF plates with sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid, Sigma) as matrix substance and air-dried until completely dry. Whole protein mass spectra were acquired on an UltrafleXtreme MALDI-TOF mass spectrometer (Bruker Daltonics) instrument operated in the linear positive ion mode with flexControl software (Version 3.4, Bruker Daltonics). Each spectrum was the accumulation of approx. 5000 laser shots for the sample spots. The MS spectra were externally calibrated using the Protein Calibration Standard II mixture (Bruker Daltonics). The MS spectra obtained were analyzed using flexAnalysis software (Version 3.4, Bruker Daltonik).

10 µg (7 µM) of recombinant USP14 was reacted with DMSO or 10 µM of b-AP15 at 30°C for 1 hour in 10 mM Tris-HCl pH 8.0. The samples were then desalted using 30 kDa cut-off Microcon-30 Centrifugal filters (Millipore) and reconstituted with deionized water. Approximately 0.25 µg of USP14 was loaded onto polished stainless steel MALDI-TOF plates with 20 µg sinapic acid (Sigma) in 70% acetonitrile/0.03% Formic Acid solution and air-dried. Whole protein mass spectra were acquired on an UltrafleXtreme MALDI-TOF mass spectrometer (Bruker Daltonics) operated in the linear positive ion mode with flexControl software v 3.4. Each spectrum was the cumulative signal from approximately 5000 laser shots. The MS spectra were externally calibrated using the Protein Calibration Standard II mixture (Bruker Daltonics). The MS spectra were analyzed using flexAnalysis v 3.4 (Bruker Daltonics).