Fig. 5: SARS-CoV-2 and EAV radiolabeling with UMP.
From: Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2
a Radiolabeling of SARS-CoV-2 (nsp7 and nsp8) or EAV (nsp7 and nsp9) proteins using either α-32P-GTP or α -32P-UTP at a total nucleotide concentration of ~0.2 μM or 200 μM. The total percentage of nsp proteins labeled in each experiment is shown below autoradiographs, given 0.6–2.7 μM of each labeled protein per reaction. Identical exposures are shown for left and right panels. Results represent a single experiment. b Competition of α-32P-GTP labeling (at 10 μM) with 316 μM of the indicated cold nucleotide. Autoradiographs are shown of an SDS-PAGE protein analysis. c The percentage 32P-GMP labeling remaining after direct competition with 316 μM GTP, UTP, ATP or CTP. Three separate experiments (n = 3) were performed for both SARS-CoV-2 (white diamonds) and EAV (black diamonds), and individual data points are shown. d LC–MS spectra at 39.7 min for SARS-CoV-2 samples that were labeled with UTP or not. Spectra show peaks at m/z of ~908.897 and 606.267, which correspond the SARS-CoV-2 nsp7 1–14 peptide (GSKMSDVKCTSVVL) in charge states of +2 and +3, respectively, that has been modified with UMP (306.0253 amu mass addition). e Top peptide spectrum match for UMP-labeled GSKMSDVKCTSVVL peptide that was derived from chymotrypsin-digested SARS-CoV-2 nsp7 protein. EThcD fragmentation of the 606.266 peptide ion was used with 25% normalized supplemental collisional energy (NCE). Detection of the UMP-modified peptide was observed in a single experiment with two technical replicates.
