Anchorchip 384 bc plate

Ubiquitination activity was quantified in a time-dependent manner by MALDI-TOF detection of monoubiquitin consumption according to a procedure adapted from De Cesare et al.(De Cesare et al., 2018b) E1 (50 nM), E2 (250 nM), and E3 (500 nM) were combined in reaction buffer composed of 0.25 mg/mL BSA in 10 mM HEPES pH 8.5, 10 mM MgCl₂ and 1 mM ATP. Reactions were incubated at 37 °C and initiated with the addition of ubiquitin (10 µM). At desired time intervals, an aliquot (5 µL) was quenched with 10% TFA (1 µL). Following collection of all time points, samples were doped with DHAP matrix and 4 µM ¹⁵N,¹³C-ubiquitin as an internal standard (3:1:2 matrix:standard:sample) and spotted on an AnchorChip 384 BC plate (Bruker Daltonics). Samples were analyzed by MALDI-TOF (Bruker Autoflex Speed LRF MALDI-TOF System) using the following automated AutoXecute method: Reflector Positive mode with laser intensity at 80%, Laser Fuzzy Control switched off, and accumulation parameters set to 4000 satisfactory shots in 500 shot steps with movement parameters set to “Walk on Spot”. Spectra were accumulated by FlexControl software and processed using FlexAnalysis software. Monoubiquitin signal was normalized to signal from the heavy isotope derivative and plotted as normalized intensity versus time to determine time-dependent ubiquitination activity of the signaling cascade.

Ubiquitination activity was quantified in a time-dependent manner by MALDI-TOF detection of monoubiquitin consumption according to a procedure adapted from De Cesare et al. [28 (link)] E1 (50 nM), E2 (250 nM), and E3 (500 nM) were combined in reaction buffer composed of 0.25 mg/mL BSA in 10 mM HEPES pH 8.5, 10 mM MgCl₂ and 1 mM ATP. Reactions were incubated at 37 °C and initiated with the addition of ubiquitin (10 μM). At desired time intervals, an aliquot (5 μL) was quenched with 10% TFA (1 μL). Following collection of all time points, samples were doped with DHAP matrix and 4 μM ¹⁵N,¹³C-ubiquitin (R&D Systems) as an internal standard (3:1:2 matrix:standard:sample) and spotted on an AnchorChip 384 BC plate (Bruker Daltonics). Samples were analyzed by MALDI-TOF (Bruker Autoflex Speed LRF MALDI-TOF System) using the following automated AutoXecute method: Reflector Positive mode with laser intensity at 80%, Laser Fuzzy Control switched off, and accumulation parameters set to 4000 satisfactory shots in 500 shot steps with movement parameters set to “Walk on Spot”. Spectra were accumulated by FlexControl software and processed using FlexAnalysis software. Monoubiquitin signal was normalized to signal from the heavy isotope derivative and plotted as normalized intensity versus time to determine time-dependent ubiquitination activity of the signaling cascade.