Discovermp 2

To determine the oligomeric state of UBR5, mass photometry measurements were performed on the Refeyn TwoMP using Refeyn AcquireMP 2.3.0 software. Mass calibration was achieved by measurement of a protein mixture, providing a range of molecular masses as follows: conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa) and thyroglobulin (669 kDa) in a final concentration of ~50 nM of each component. Measurements of either UBR5 or UBR5^Dimer were carried out by diluting UBR5 to a final concentration of ~140 nM in the same buffer used for focus-finding (25 mM HEPES (pH 7.5), 150 mM NaCl and 1 mM DTT). Videos were collected for 1 min. Data were then analyzed using DiscoverMP 2.3.0 (Refeyn) software and the collected mass calibration as a reference.

Data were processed and analyzed using Refeyn Discover^MP 2.3.0 software by performing three main steps: (1) background removal, (2) identification of particle landing events on the coverslip acquisition field, and (3) particle fitting to extract maximum contrast. The following default fitting parameters were used; threshold 1 (related to the particle contrast relative to the background noise) was set to 1.5, threshold 2 (related to the radial symmetry of the particle signature) was set to 0.25, and 5 ratiometric frames were binned. Individual particle contrasts from each individual movie were converted to mass using a contrast-to-mass (C2M) calibration. From these data, kernel density estimates (KDEs) were generated for each sample using a Gaussian kernel with a fixed bandwidth of 25 kDa.
Calibration procedure: Contrast-to-mass calibration was performed in the acquisition buffer. NativeMark™ unstained protein standard (# LC0725, Thermo Scientific), which contains proteins in the range of 20 to 1200 kDa, was diluted 1:100 in acquisition buffer, and 2.5 μL were added to an acquisition well for measurement. The following masses were used to generate a standard calibration curve in the Discover^MP software: 66, 146, 480 and 1048 kDa.