DOI: 10.25345/c5zp3w37q
title: MassIVE Reanalysis RMSV000000359.1 - Open modification searching of SARS-CoV-2-human protein interaction data reveals novel viral modification sites
title: We performed a reanalysis of a SARS-CoV-2-human protein-protein interaction map from MSV000085144/PXD018117. Open modification searching was used to investigate the presence of PTMs in the context of the SARS-CoV-2 virus-host PPI network. Based on an over two-fold increase in identified spectra, our detected protein interactions show a high overlap with independent mass spectrometry-based SARS-CoV-2 studies and virus-host interactions for alternative viruses, as well as previously unknown protein interactions. Additionally, we identified several novel modification sites on SARS-CoV-2 proteins that we investigated in relation to their interactions with host proteins. A detailed analysis of relevant modifications, including phosphorylation, ubiquitination, and S-nitrosylation, provides important hypotheses about the functional role of these modifications during viral infection by SARS-CoV-2. In the original study affinity purification was performed using 27 SARS-CoV-2 proteins that were individually tagged and expressed in triplicate in HEK-293T cells. Bead-bound proteins were denatured, reduced, carbamidomethylated, and enzymatically digested using trypsin, and each sample was injected via an Easy-nLC 1200 (Thermo Fisher Scientific) into a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific). The SARS-CoV-2 proteins that were included are: all mature nonstructural proteins (Nsps), except for Nsp3 and Nsp16; a mutated version of Nsp5 to disable its proteolytic activity (Nsp5_C145A); and all predicted SARS-CoV-2 open reading frames (Orfs), including the spike (S), membrane (M), nucleocapsid (N), and envelope (E) protein. First, the downloaded raw files were converted to MGF files using ThermoRawFileParser (version 1.2.3). Next, OMS was performed using the ANN-SoLo spectral library search engine (version 0.2.4). A combined human-SARS-CoV-2 spectral library was used for searching. The MassIVE-KB library (version 2018/06/15) was used as human spectral library. SARS-CoV-2 spectra were simulated by generating all possible tryptic peptide sequences from the SARS-CoV-2 protein sequences downloaded from UniProt (version 2020/03/05) using Pyteomics (version 4.3.2) and predicting the corresponding spectra using Prosit (version prosit_intensity_2020_hcd; collision energy 33 as determined by Prosit collision energy calibration). A simulated spectral library for the green fluorescent protein was generated in a similar fashion. A final spectral library was compiled by merging all spectra using SpectraST (version 5.0) and adding decoy spectra in a 1:1 ratio using the shuffle-and-reposition method. ANN-SoLo was configured to use a 20 ppm precursor mass tolerance during the first step of its cascade search and a 500 Da precursor mass tolerance during its open search. Other search settings were to filter peaks below 101 m/z, above 1500 m/z, and in a 0.5 m/z window around the precursor mass; a 0.02 m/z fragment mass tolerance; and a bin size of 0.05 m/z. The remaining settings were kept at their default values. Peptide-spectrum matches (PSMs) were filtered at 1% FDR.