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Title: Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2
Abstract

Coronaviruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a nucleotidyl transferase in the N-terminal (NiRAN) domain of thenonstructuralprotein (nsp) 12 protein within the RNA dependent RNA polymerase. Here we show the detection of guanosine monophosphate (GMP) and uridine monophosphate-modified amino acids in nidovirus proteins using heavy isotope-assisted mass spectrometry (MS) and MS/MS peptide sequencing. We identified lysine-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of in vitro GMP attachment via a phosphoramide bond. In SARS-CoV-2 replicase proteins, we demonstrate nsp12-mediated nucleotidylation of nsp7 lysine-2. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.

 
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Award ID(s):
2010789
NSF-PAR ID:
10218022
Author(s) / Creator(s):
; ; ;
Publisher / Repository:
Nature Publishing Group
Date Published:
Journal Name:
Communications Chemistry
Volume:
4
Issue:
1
ISSN:
2399-3669
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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    N6‐Formyl lysine is a well‐known modification of histones and other proteins. It can also be formed as a damaged product from direct formylation of free lysine and accompanied by other lysine derivatives such as acetylated or methylated forms. In relation to the activity of cellular repair enzymes in protein turnover and to lysine metabolism, it is important to accurately quantify the overall ratio of modified lysine to free lysine.

    Methods

    N6‐Formyl lysine was quantified using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with data collected in a non‐targeted manner using positive mode electrospray ionization on a Q‐Exactive HF+Orbitrap mass spectrometer. Studies were performed with lysine and deuterated lysine spiked into protein digests and solvents to investigate the extent of spontaneous formation and matrix effects of formation ofN6‐formyl lysine.

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    We show thatN6‐formyl lysine,N2‐formyl lysine,N6‐acetyl lysine, andN2‐acetyl lysine are all formed spontaneously during sample preparation and LC/MS/MS analysis, which complicates quantification of these metabolites in biological samples.N6‐Formyl lysine was spontaneously formed and correlated to the concentration of lysine. In the sample matrix of protein digests, 0.03% of lysine was spontaneously converted intoN6‐formyl lysine, and 0.005% of lysine was converted intoN6‐formyl lysine in pure run solvent.

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    Spontaneous formation ofN6‐formyl lysine,N6‐acetyl lysine,N2‐formyl lysine, andN2‐acetyl lysine needs to be subtracted from biologically formed lysine modifications when quantifying these epimetabolites in biological samples.

     
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