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Proteomics-Based Insights Into the SARS-CoV-2–Mediated COVID-19 Pandemic: A Review of the First Year of Research

Open AccessPublished:June 02, 2021DOI:https://doi.org/10.1016/j.mcpro.2021.100103

      Highlights

      • SARS-CoV-2, which caused the COVID-19 pandemic, depends on features of its proteome and host proteomes for transmissibility and virulence.
      • Qualitative and quantitative proteomic assay development underlies attempts to further understand the virus.
      • Protein–protein interaction and post-translational modification studies inform efforts to develop therapies.
      • In sum, the range of proteomics technologies provides valuable information in the fight against COVID-19.
      In late 2019, a virus subsequently named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China and led to a worldwide pandemic of the disease termed coronavirus disease 2019. The global health threat posed by this pandemic led to an extremely rapid and robust mobilization of the scientific and medical communities as evidenced by the publication of more than 10,000 peer-reviewed articles and thousands of preprints in the first year of the pandemic alone. With the publication of the initial genome sequence of SARS-CoV-2, the proteomics community immediately joined this effort publishing, to date, more than 100 peer-reviewed proteomics studies and submitting many more preprints to preprint servers. In this review, we focus on peer-reviewed articles published on the proteome, glycoproteome, and glycome of SARS-CoV-2. At a basic level, proteomic studies provide valuable information on quantitative aspects of viral infection course; information on the identities, sites, and microheterogeneity of post-translational modifications; and, information on protein–protein interactions. At a biological systems level, these studies elucidate host cell and tissue responses, characterize antibodies and other immune system factors in infection, suggest biomarkers that may be useful for diagnosis and disease-course monitoring, and help in the development or repurposing of potential therapeutics. Here, we summarize results from selected early studies to provide a perspective on the current rapidly evolving literature.

      Graphical Abstract

      Keywords

      Abbreviations:

      ACE2 (angiotensin-converting enzyme 2), CK2 (casein kinase II), COVID-19 (coronavirus disease 2019), DDA (data-dependent acquisition), nsp (nonstructural protein), PIKFyve (a FYVE finger–containing phosphoinositide kinase), PPI (protein–protein interaction), PRM (parallel reaction monitoring), PTM (post-translational modification), qPCR (quantitative PCR), SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), TMPRSS2 (transmembrane serine protease 2), TMT (tandem mass tag)
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a betacoronavirus that began infecting people in 2019 with the index case identified as a hospitalized patient who initially became ill on December 1, 2019 (
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      ). The SARS-CoV-2 genome reported (29,903 bases, single-stranded RNA) was annotated as encoding 26 or more proteins and has high sequence similarity (~80% identity at the nucleotide level) to the extensively studied SARS-CoV-1 responsible for SARS outbreaks in 2002 and 2003 as well as to multiple animal coronaviruses (
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      ).
      The SARS-CoV-2 National Center for Biotechnology Information reference genome was released shortly after publication of the initial genome sequences and contains annotations for 28 encoded proteins. Starting from the 5’-end, the annotated proteins consist of 16 nonstructural proteins (denoted as nsp1–nsp16), translated as components of large polyproteins and then separated by viral proteases, followed by structural proteins and additional ORFs at the 3’-end (Fig. 1). Putative functions of the encoded proteins were initially inferred by sequence homology to previously studied coronaviruses (
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      • Yuen K.Y.
      Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan.
      ,
      • Wu A.
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      Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China.
      ). The nsp proteins include two with protease functions essential for polyprotein processing—Mpro, also called 3CLpro (3C-like protease, nsp5) and PLpro (papain-like protease, nsp3)—as well as the viral replication–transcription complex subunits. Structural proteins encoded include spike (S), envelope (E), membrane (M), and nucleocapsid (N). A number of accessory proteins that have (partially) determined roles in host defense interference, intracellular trafficking, transcription, and replication in related coronaviruses are also encoded (
      • Chan J.F.
      • Kok K.H.
      • Zhu Z.
      • Chu H.
      • To K.K.
      • Yuan S.
      • Yuen K.Y.
      Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan.
      ,
      • Wu C.
      • Liu Y.
      • Yang Y.
      • Zhang P.
      • Zhong W.
      • Wang Y.
      • Wang Q.
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      • Zheng M.
      • Chen L.
      • Li H.
      Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods.
      ,
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      The proteins of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2 or n-COV19), the cause of COVID-19.
      ). In the context of proteomics, it is important to note that such homology-based annotations provide useful initial models but are necessarily subject to more definitive empirical characterization. This is particularly essential for most RNA viruses because of their comparatively compact genomes and the resulting multiform and multifunction nature of their encoded proteins driven by evolutionary constraints (
      • Holmes E.C.
      Error thresholds and the constraints to RNA virus evolution.
      ). Consequently, different groups have used a variety of marginally different annotations in their studies. Researchers have, therefore, continued to work on refining annotations of regulatory elements and encoded polypeptides that may not have been completely characterized by homology-based methods (
      • Finkel Y.
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      The coding capacity of SARS-CoV-2.
      ,
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      • Yang J.S.
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      • Chang H.
      The architecture of SARS-CoV-2 transcriptome.
      ,
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      • Meyerson M.
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      Pervasive generation of non-canonical subgenomic RNAs by SARS-CoV-2.
      ,
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      The SARS-CoV-2 ORF10 is not essential in vitro or in vivo in humans.
      ,
      • Davidson A.D.
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      • Carroll M.W.
      • Heesom K.J.
      • Zambon M.
      • Ellis J.
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      • Hiscox J.A.
      • Matthews D.A.
      Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein.
      ). For example, Finkel et al. (
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      • Mizrahi O.
      • Nachshon A.
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      • Morgenstern D.
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      • Stein D.
      • Israeli O.
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      • Paran N.
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      The coding capacity of SARS-CoV-2.
      ) reported 23 unannotated ORFs in their study using ribosome profiling, and Davidson et al. (
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      • Lewis S.
      • Shoemark D.
      • Carroll M.W.
      • Heesom K.J.
      • Zambon M.
      • Ellis J.
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      • Matthews D.A.
      Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein.
      ) reported that 14% of the transcripts detected in their study do not code for a known ORF and subsequently identified peptides from these transcripts. To our knowledge, there is no current definitive database compiling this information, and interested readers are directed to the original research articles.
      Figure thumbnail gr1
      Fig. 1The SARS-CoV-2 proteome and its post-translational modifications (PTMs). The SARS-CoV-2 NCBI reference sequence proteome delineated along its genome (A). The 28 proteins annotated in the NCBI reference sequence are represented as boxes with the starting base corresponding to each protein in the genome listed later along with most protein names (pp1ab and pp1a are labeled inside boxes). Note that the nsp proteins are expressed as parts of large polyproteins (pp1ab and pp1a), which are subsequently cleaved by proteases contained in the polyproteins themselves. A summary of PTMs detected in proteomics studies is listed above each protein except for N and S, which are shown in detail in panels B and C. Numbers in parentheses indicate the residue number in pp1ab as given in the study by Klann et al. (
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      Growth factor receptor signaling inhibition prevents SARS-CoV-2 replication.
      ). The PTMs of S. A partial domain structure is shown for orientation with coloring for contrast and start residue numbers. The most abundant N-glycans from the most abundant Oxford class at each site are shown as reported by Zhao et al. (
      • Zhao P.
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      • Grant O.C.
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      Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.
      ). The class abundances at each site reported by Watanabe et al. (
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      Site-specific glycan analysis of the SARS-CoV-2 spike.
      ) are similar although the protein they aonalyzed showed a small but clear tendency toward slightly less processed glycoforms. Articles have reported varying amounts of O-glycosylation on S almost exclusively at T323, occupancy generally ~10% or less. Note also that Davidson et al. (
      • Davidson A.D.
      • Williamson M.K.
      • Lewis S.
      • Shoemark D.
      • Carroll M.W.
      • Heesom K.J.
      • Zambon M.
      • Ellis J.
      • Lewis P.A.
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      • Matthews D.A.
      Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein.
      ) identified 13 sites of phosphorylation on S; however, most were not cytoplasmic. Secretory pathway kinases have been confirmed (e.g., FAM20C), but it is not clear that these sites fit with known specificity determinants. The PTMs of N and ORF9b. Domain structure shown with coloring for contrast and start residue numbers. ORF9b is an alternative ORF in the N coding sequence that is not annotated in the NCBI reference sequence. FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; NCBI, National Center for Biotechnology Information; nsp, nonstructural protein; RBD, receptor binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
      While the genome and preliminary information on the proteome of the SARS-CoV-2 itself were being defined, researchers also began determining the host cell proteins required for or facilitative of infection. Angiotensin-converting enzyme 2 (ACE2) was known to be the host cell surface receptor for several other coronaviruses, including SARS-CoV-1, and its identity as the host cell surface receptor for SARS-CoV-2 was quickly confirmed (
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      A pneumonia outbreak associated with a new coronavirus of probable bat origin.
      ,
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      ,
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      Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein.
      ,
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      • Drosten C.
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      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      ). Mature membrane-bound ACE2 is a 788-amino acid single-pass type I membrane protein (~91 kDa without post-translational modifications [PTMs]) consisting of an N-terminal peptidase domain and a C-terminal collectrin-like domain that causes homodimerization at the cell surface and contains the transmembrane helix (
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      • Jahn O.
      • Pöhlmann S.
      TMPRSS2 and ADAM17 cleave ACE2 differentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein.
      ,
      • Yan R.
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      • Xia L.
      • Guo Y.
      • Zhou Q.
      Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2.
      ,
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      • Acton S.
      A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9.
      ). Many viruses in addition utilize host cell proteases and other host cell machinery to enable and facilitate initial cell infection. The protease transmembrane serine protease 2 (TMPRSS2) was confirmed as a key factor in SARS-CoV-2 infection in one of the early articles confirming the identity of the cell surface receptor as ACE2 (
      • Hoffmann M.
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      • Schroeder S.
      • Krüger N.
      • Herrler T.
      • Erichsen S.
      • Schiergens T.S.
      • Herrler G.
      • Wu N.H.
      • Nitsche A.
      • Müller M.A.
      • Drosten C.
      • Pöhlmann S.
      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      ). Researchers have since continued to pursue potential alternative host cell surface receptors, related or alternative proteases (TMPRSS4, cathepsin B and L), and proteins involved in processes such as endosome maturation (a FYVE finger–containing phosphoinositide kinase [PIKFyve], two-pore channel 2) that are critical for infection (
      • Hoffmann M.
      • Kleine-Weber H.
      • Schroeder S.
      • Krüger N.
      • Herrler T.
      • Erichsen S.
      • Schiergens T.S.
      • Herrler G.
      • Wu N.H.
      • Nitsche A.
      • Müller M.A.
      • Drosten C.
      • Pöhlmann S.
      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      ,
      • Ou X.
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      • Lei X.
      • Li P.
      • Mi D.
      • Ren L.
      • Guo L.
      • Guo R.
      • Chen T.
      • Hu J.
      • Xiang Z.
      • Mu Z.
      • Chen X.
      • Chen J.
      • Hu K.
      • et al.
      Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV.
      ,
      • Zang R.
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      • Zeng Q.
      • Rothlauf P.W.
      • Sonnek N.M.
      • Liu Z.
      • Brulois K.F.
      • Wang X.
      • Greenberg H.B.
      • Diamond M.S.
      • Ciorba M.A.
      • Whelan S.P.J.
      • Ding S.
      TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes.
      ,
      • Matsuyama S.
      • Nao N.
      • Shirato K.
      • Kawase M.
      • Saito S.
      • Takayama I.
      • Nagata N.
      • Sekizuka T.
      • Katoh H.
      • Kato F.
      • Sakata M.
      • Tahara M.
      • Kutsuna S.
      • Ohmagari N.
      • Kuroda M.
      • et al.
      Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells.
      ,
      • Daniloski Z.
      • Jordan T.X.
      • Wessels H.H.
      • Hoagland D.A.
      • Kasela S.
      • Legut M.
      • Maniatis S.
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      • Lu L.
      • Geller E.
      • Danziger O.
      • Rosenberg B.R.
      • Phatnani H.
      • Smibert P.
      • Lappalainen T.
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      Identification of required host factors for SARS-CoV-2 infection in human cells.
      ,
      • Wei J.
      • Alfajaro M.M.
      • DeWeirdt P.C.
      • Hanna R.E.
      • Lu-Culligan W.J.
      • Cai W.L.
      • Strine M.S.
      • Zhang S.M.
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      • Mankowski M.C.
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      • Gasque V.
      • et al.
      Genome-wide CRISPR screens reveal host factors critical for SARS-CoV-2 infection.
      ). However, many questions still remain regarding the role and behavior of host proteases and proteins during infection.
      While experimental studies have continued, furthering understanding of the biology of SARS-CoV-2, important parallel efforts have focused on cataloging and increasing the accessibility of this information. Of particular note to the proteomics community are resources compiling genome sequences, annotations, protein–protein interactions (PPIs), PTMs, and proteomics datasets. Genome sequences for SARS-CoV-2 from which protein sequences may be derived are available from the Global Initiative on Sharing Avian Influenza Data (
      • Shu Y.
      • McCauley J.
      GISAID: Global initiative on sharing all influenza data - from vision to reality.
      ), National Center for Biotechnology Information (
      • Coordinators N.R.
      Database resources of the national center for biotechnology information.
      ), European Molecular Biology Laboratory's European Bioinformatics Institute (
      • Cantelli G.
      • Cochrane G.
      • Brooksbank C.
      • McDonagh E.
      • Flicek P.
      • McEntyre J.
      • Birney E.
      • Apweiler R.
      The European bioinformatics institute: Empowering cooperation in response to a global health crisis.
      ), and other organizations. The coronavirus disease 2019 (COVID-19) data portal (https://www.covid19dataportal.org/) from European Molecular Biology Laboratory's European Bioinformatics Institute maintains an updated curated collection of SARS-CoV-2 and host proteins and their relationships and information on pathways and from gene expression studies (
      • Cantelli G.
      • Cochrane G.
      • Brooksbank C.
      • McDonagh E.
      • Flicek P.
      • McEntyre J.
      • Birney E.
      • Apweiler R.
      The European bioinformatics institute: Empowering cooperation in response to a global health crisis.
      ). A database specific for PPIs is available from the Biological General Repository for Interaction Datasets curation project (https://thebiogrid.org/project/3) and may be consulted for continuously updated information (
      • Oughtred R.
      • Rust J.
      • Chang C.
      • Breitkreutz B.J.
      • Stark C.
      • Willems A.
      • Boucher L.
      • Leung G.
      • Kolas N.
      • Zhang F.
      • Dolma S.
      • Coulombe-Huntington J.
      • Chatr-Aryamontri A.
      • Dolinski K.
      • Tyers M.
      The BioGRID database: A comprehensive biomedical resource of curated protein, genetic, and chemical interactions.
      ). Many proteomics datasets from relevant studies are available through ProteomeXchange and its subsidiary databases (http://www.proteomexchange.org/) (
      • Deutsch E.W.
      • Csordas A.
      • Sun Z.
      • Jarnuczak A.
      • Perez-Riverol Y.
      • Ternent T.
      • Campbell D.S.
      • Bernal-Llinares M.
      • Okuda S.
      • Kawano S.
      • Moritz R.L.
      • Carver J.J.
      • Wang M.
      • Ishihama Y.
      • Bandeira N.
      • et al.
      The ProteomeXchange consortium in 2017: Supporting the cultural change in proteomics public data deposition.
      ). A number of additional databases contain proteomics data particularly useful for analyzing (or developing assays to analyze) host cell factors, for example, proteomicsDB (https://www.proteomicsdb.org/) (
      • Samaras P.
      • Schmidt T.
      • Frejno M.
      • Gessulat S.
      • Reinecke M.
      • Jarzab A.
      • Zecha J.
      • Mergner J.
      • Giansanti P.
      • Ehrlich H.C.
      • Aiche S.
      • Rank J.
      • Kienegger H.
      • Krcmar H.
      • Kuster B.
      • et al.
      ProteomicsDB: A multi-omics and multi-organism resource for life science research.
      ), PAXdb (https://pax-db.org/) (
      • Wang M.
      • Herrmann C.J.
      • Simonovic M.
      • Szklarczyk D.
      • von Mering C.
      Version 4.0 of PaxDb: Protein abundance data, integrated across model organisms, tissues, and cell-lines.
      ), the Clinical Proteomic Tumor Analysis Consortium Data Portal (https://cptac-data-portal.georgetown.edu/cptacPublic/) (
      • Edwards N.J.
      • Oberti M.
      • Thangudu R.R.
      • Cai S.
      • McGarvey P.B.
      • Jacob S.
      • Madhavan S.
      • Ketchum K.A.
      The CPTAC data portal: A resource for cancer proteomics research.
      ), Human Proteome Map (http://www.humanproteomemap.org/) (
      • Kim M.S.
      • Pinto S.M.
      • Getnet D.
      • Nirujogi R.S.
      • Manda S.S.
      • Chaerkady R.
      • Madugundu A.K.
      • Kelkar D.S.
      • Isserlin R.
      • Jain S.
      • Thomas J.K.
      • Muthusamy B.
      • Leal-Rojas P.
      • Kumar P.
      • Sahasrabuddhe N.A.
      • et al.
      A draft map of the human proteome.
      ), and The Human Protein Atlas (https://www.proteinatlas.org/) (
      • Uhlen M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • Sivertsson Å.
      • Kampf C.
      • Sjöstedt E.
      • Asplund A.
      • Olsson I.
      • Edlund K.
      • Lundberg E.
      • Navani S.
      • Szigyarto C.A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      ). Glycan information from various studies is compiled in GlyGen (https://www.glygen.org/) (
      • York W.S.
      • Mazumder R.
      • Ranzinger R.
      • Edwards N.
      • Kahsay R.
      • Aoki-Kinoshita K.F.
      • Campbell M.P.
      • Cummings R.D.
      • Feizi T.
      • Martin M.
      • Natale D.A.
      • Packer N.H.
      • Woods R.J.
      • Agarwal G.
      • Arpinar S.
      • et al.
      GlyGen: Computational and informatics resources for glycoscience.
      ). In addition, glycan and other PTM information is available through PhosphoSitePlus (https://www.phosphosite.org/) (
      • Hornbeck P.V.
      • Zhang B.
      • Murray B.
      • Kornhauser J.M.
      • Latham V.
      • Skrzypek E.
      PhosphoSitePlus, 2014: Mutations, PTMs and recalibrations.
      ).
      In the first half of this article, we focus on the virus and its cell entry factors including the host cell receptor ACE2. The literature in this area may be further divided among (1) studies examining the basic qualitative behavior of viral and host cell entry factor peptides in mass spectrometric experiments; (2) quantitative proteomics studies that either detail viral protein expression over time or examine the distribution of host cell entry factors in human tissues and cells; and (3) studies of viral and ACE2 structure and PTMs. In the second half of this review, we provide an overview of studies focused on the proteomes of host (primarily human) cells and tissues and their responses and interactions with the SARS-CoV-2 virus. These studies encompass PPI mapping experiments, the quantitative proteomics of host cell protein expression during infection, determination of putative biomarkers, and characterization of immune system responses and SARS-CoV-2–directed antibodies during infection. Another review of SARS-CoV-2 proteomics was published during preparation of this article and may be of interest for further reading (
      • Mahmud I.
      • Garrett T.J.
      Mass spectrometry techniques in emerging pathogens studies: COVID-19 perspectives.
      ).

      Virus and Host Cell Entry Factor Studies

      Basic Qualitative Proteomics and Potential Clinical Diagnostics

      A number of studies have been published containing information on the basic qualitative proteomics of the virus and the potential of proteomics technology–based assays for clinical diagnostics development. Important results include the determination of peptides suitable for targeted method development in LC–MS experiments in terms of level of detection and quantification, specificity and stability of amino acid sequences in reported genomes, and the presence or absence of PTMs. Testing of developed methods with relevant clinical samples has also been reported by several groups.
      In an early study, Gouveia et al. (
      • Gouveia D.
      • Grenga L.
      • Gaillard J.C.
      • Gallais F.
      • Bellanger L.
      • Pible O.
      • Armengaud J.
      Shortlisting SARS-CoV-2 peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data.
      ) detected 101 (tryptic) peptides across six viral proteins (N, S, M, ORF1ab, ORF3a, and ORF8) from virus-infected Vero cells and further recommended 14 peptides for targeted assays (Table 1 in the article of Gouveia et al.). Zecha et al. (
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ) characterized both tryptic viral peptides (from cells or cell culture supernatant independently) and tryptic host cell peptides from four relevant cell line models (discussed further later in this review). Parallel reaction monitoring (PRM) assays for viral proteins were developed for 23 peptides with favorable properties in nanoflow PRM and 21 peptides in microflow PRM. For each peptide, the top six transitions are also detailed (supplemental Table S3 in the article by Zecha et al. (
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ), spectral libraries are available at Panorama Public (
      • Sharma V.
      • Eckels J.
      • Schilling B.
      • Ludwig C.
      • Jaffe J.D.
      • MacCoss M.J.
      • MacLean B.
      Panorama public: A public repository for quantitative data sets processed in skyline.
      )). Gouveia et al. and Zecha et al. recommended similar lists of peptides for targeted method development, sharing two peptides for M, five peptides for N, and one peptide for S. Additional articles have been published more recently and may be consulted for further reference (
      • Singh P.
      • Chakraborty R.
      • Marwal R.
      • Radhakrishan V.S.
      • Bhaskar A.K.
      • Vashisht H.
      • Dhar M.S.
      • Pradhan S.
      • Ranjan G.
      • Imran M.
      • Raj A.
      • Sharma U.
      • Singh P.
      • Lall H.
      • Dutta M.
      • et al.
      A rapid and sensitive method to detect SARS-CoV-2 virus using targeted-mass spectrometry.
      ,
      • Cazares L.H.
      • Chaerkady R.
      • Samuel Weng S.H.
      • Boo C.C.
      • Cimbro R.
      • Hsu H.E.
      • Rajan S.
      • Dall'Acqua W.
      • Clarke L.
      • Ren K.
      • McTamney P.
      • Kallewaard-LeLay N.
      • Ghaedi M.
      • Ikeda Y.
      • Hess S.
      Development of a parallel reaction monitoring mass spectrometry assay for the detection of SARS-CoV-2 spike glycoprotein and nucleoprotein.
      ,
      • Iles R.K.
      • Zmuidinaite R.
      • Iles J.K.
      • Carnell G.
      • Sampson A.
      • Heeney J.L.
      Development of a clinical MALDI-ToF mass spectrometry assay for SARS-CoV-2: Rational design and multi-disciplinary team work.
      ) (Table 1).
      Table 1Selected peptides for SARS-CoV-2 detection and quantification reported in at least two publications
      AccessionProteinPeptide sequenceTheoretical unmodified precursor [M + H]+Observed precursor zCommon modsReferences
      VME1_SARS2MEITVATSR876.482None(
      • Gouveia D.
      • Grenga L.
      • Gaillard J.C.
      • Gallais F.
      • Bellanger L.
      • Pible O.
      • Armengaud J.
      Shortlisting SARS-CoV-2 peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data.
      ,
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      )
      VAGDSGFAAYSR1200.572None(
      • Gouveia D.
      • Grenga L.
      • Gaillard J.C.
      • Gallais F.
      • Bellanger L.
      • Pible O.
      • Armengaud J.
      Shortlisting SARS-CoV-2 peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data.
      ,
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      )
      NCAP_SARS2NADETQALPQR1128.572Deamidation (NQ)(
      • Gouveia D.
      • Grenga L.
      • Gaillard J.C.
      • Gallais F.
      • Bellanger L.
      • Pible O.
      • Armengaud J.
      Shortlisting SARS-CoV-2 peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data.
      ,
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ,
      • Singh P.
      • Chakraborty R.
      • Marwal R.
      • Radhakrishan V.S.
      • Bhaskar A.K.
      • Vashisht H.
      • Dhar M.S.
      • Pradhan S.
      • Ranjan G.
      • Imran M.
      • Raj A.
      • Sharma U.
      • Singh P.
      • Lall H.
      • Dutta M.
      • et al.
      A rapid and sensitive method to detect SARS-CoV-2 virus using targeted-mass spectrometry.
      ,
      • Cazares L.H.
      • Chaerkady R.
      • Samuel Weng S.H.
      • Boo C.C.
      • Cimbro R.
      • Hsu H.E.
      • Rajan S.
      • Dall'Acqua W.
      • Clarke L.
      • Ren K.
      • McTamney P.
      • Kallewaard-LeLay N.
      • Ghaedi M.
      • Ikeda Y.
      • Hess S.
      Development of a parallel reaction monitoring mass spectrometry assay for the detection of SARS-CoV-2 spike glycoprotein and nucleoprotein.
      ,
      • Gouveia D.
      • Miotello G.
      • Gallais F.
      • Gaillard J.-C.
      • Debroas S.
      • Bellanger L.
      • Lavigne J.-P.
      • Sotto A.
      • Grenga L.
      • Pible O.
      • Armengaud J.
      Proteotyping SARS-CoV-2 virus from nasopharyngeal swabs: A proof-of-concept focused on a 3 min mass spectrometry window.
      )
      AYNVTQAFGR1126.572Deamidation (NQ)(
      • Gouveia D.
      • Grenga L.
      • Gaillard J.C.
      • Gallais F.
      • Bellanger L.
      • Pible O.
      • Armengaud J.
      Shortlisting SARS-CoV-2 peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data.
      ,
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      )
      GFYAEGSR886.412None(
      • Gouveia D.
      • Grenga L.
      • Gaillard J.C.
      • Gallais F.
      • Bellanger L.
      • Pible O.
      • Armengaud J.
      Shortlisting SARS-CoV-2 peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data.
      ,
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ,
      • Gouveia D.
      • Miotello G.
      • Gallais F.
      • Gaillard J.-C.
      • Debroas S.
      • Bellanger L.
      • Lavigne J.-P.
      • Sotto A.
      • Grenga L.
      • Pible O.
      • Armengaud J.
      Proteotyping SARS-CoV-2 virus from nasopharyngeal swabs: A proof-of-concept focused on a 3 min mass spectrometry window.
      )
      IGMEVTPSGTWLTYTGAIK2025.042, 3Oxidation (M)(
      • Gouveia D.
      • Grenga L.
      • Gaillard J.C.
      • Gallais F.
      • Bellanger L.
      • Pible O.
      • Armengaud J.
      Shortlisting SARS-CoV-2 peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data.
      ,
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      )
      NPANNAAIVLQLPQGTTLPK2060.152, 3Deamidation (NQ)(
      • Gouveia D.
      • Grenga L.
      • Gaillard J.C.
      • Gallais F.
      • Bellanger L.
      • Pible O.
      • Armengaud J.
      Shortlisting SARS-CoV-2 peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data.
      ,
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      )
      SPIKE_SARS2SFQTLLALHR1098.643None(
      • Gouveia D.
      • Grenga L.
      • Gaillard J.C.
      • Gallais F.
      • Bellanger L.
      • Pible O.
      • Armengaud J.
      Shortlisting SARS-CoV-2 peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data.
      ,
      • Cazares L.H.
      • Chaerkady R.
      • Samuel Weng S.H.
      • Boo C.C.
      • Cimbro R.
      • Hsu H.E.
      • Rajan S.
      • Dall'Acqua W.
      • Clarke L.
      • Ren K.
      • McTamney P.
      • Kallewaard-LeLay N.
      • Ghaedi M.
      • Ikeda Y.
      • Hess S.
      Development of a parallel reaction monitoring mass spectrometry assay for the detection of SARS-CoV-2 spike glycoprotein and nucleoprotein.
      )
      LQSLQTYVTQQLIR1690.952, 3None(
      • Gouveia D.
      • Grenga L.
      • Gaillard J.C.
      • Gallais F.
      • Bellanger L.
      • Pible O.
      • Armengaud J.
      Shortlisting SARS-CoV-2 peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data.
      ,
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      )
      Host cell entry factor peptides have also been qualitatively characterized. Zecha et al. (
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ) developed PRM assays for ACE2 and TMPRSS2 (human and, partly nonshared, monkey) targeting 16 tryptic human ACE2 sequences and six tryptic human TMPRSS2 sequences (supplemental Table S1 in the article by Zecha et al., spectral libraries are available at Panorama Public (
      • Sharma V.
      • Eckels J.
      • Schilling B.
      • Ludwig C.
      • Jaffe J.D.
      • MacCoss M.J.
      • MacLean B.
      Panorama public: A public repository for quantitative data sets processed in skyline.
      )). They found that ACE2 could be detected in all four of their model cell lines (ACE2-A549, an ACE2 overexpressor, Vero E6, Calu-3, and Caco-2) using PRM methods but only in two using a data-dependent acquisition (DDA)–based method. TMPRSS2 was only detectable in two of the cell lines tested (Calu-3 and Caco-2). Other known and potential viral entry factors including TMPRSS4, CTSB, cathepsin L, BSG (CD147), and FURIN variously appeared across cell lines in DDA data (
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ). In addition, numerous articles have reported analyses of de novo or publicly available proteomics datasets aimed at characterizing the cell, tissue, and bodily fluid distribution of relevant proteins and may be useful as further references (
      • Hikmet F.
      • Méar L.
      • Edvinsson Å.
      • Micke P.
      • Uhlén M.
      • Lindskog C.
      The protein expression profile of ACE2 in human tissues.
      ,
      • Wang Y.
      • Wang Y.
      • Luo W.
      • Huang L.
      • Xiao J.
      • Li F.
      • Qin S.
      • Song X.
      • Wu Y.
      • Zeng Q.
      • Jin F.
      • Wang Y.
      A comprehensive investigation of the mRNA and protein level of ACE2, the putative receptor of SARS-CoV-2, in human tissues and blood cells.
      ,
      • Feng L.
      • Yin Y.Y.
      • Liu C.H.
      • Xu K.R.
      • Li Q.R.
      • Wu J.R.
      • Zeng R.
      Proteome-wide data analysis reveals tissue-specific network associated with SARS-CoV-2 infection.
      ,
      • Stanley K.E.
      • Thomas E.
      • Leaver M.
      • Wells D.
      Coronavirus disease-19 and fertility: Viral host entry protein expression in male and female reproductive tissues.
      ).
      One longstanding goal in the proteomics field is the development of clinical diagnostics utilizing proteomics, and particularly MS-based, methods. This goal is of particular interest during a time of supply-chain disruptions and shortages of necessary reagents for PCR-based assays and other frequently used clinical laboratory methods. However, MS-based proteomics often suffers from sensitivity, specificity, and throughput issues. Zecha et al. (
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ) concluded that the PRM methods developed by their group were inadequate to serve as a reasonable clinical diagnostic platform. Furthermore, considering the literature more broadly (including preprints), they found wide variability among studies suggesting caution in attempting to apply these methods in a clinical setting. Additional studies may be consulted for further information on current progress in developing SARS-CoV-2 diagnostics from nasopharyngeal swabs, gargle solutions, other human samples, and simulated (mock) samples (from in vitro–derived mucus and inactivated virus) (
      • Singh P.
      • Chakraborty R.
      • Marwal R.
      • Radhakrishan V.S.
      • Bhaskar A.K.
      • Vashisht H.
      • Dhar M.S.
      • Pradhan S.
      • Ranjan G.
      • Imran M.
      • Raj A.
      • Sharma U.
      • Singh P.
      • Lall H.
      • Dutta M.
      • et al.
      A rapid and sensitive method to detect SARS-CoV-2 virus using targeted-mass spectrometry.
      ,
      • Cazares L.H.
      • Chaerkady R.
      • Samuel Weng S.H.
      • Boo C.C.
      • Cimbro R.
      • Hsu H.E.
      • Rajan S.
      • Dall'Acqua W.
      • Clarke L.
      • Ren K.
      • McTamney P.
      • Kallewaard-LeLay N.
      • Ghaedi M.
      • Ikeda Y.
      • Hess S.
      Development of a parallel reaction monitoring mass spectrometry assay for the detection of SARS-CoV-2 spike glycoprotein and nucleoprotein.
      ,
      • Iles R.K.
      • Zmuidinaite R.
      • Iles J.K.
      • Carnell G.
      • Sampson A.
      • Heeney J.L.
      Development of a clinical MALDI-ToF mass spectrometry assay for SARS-CoV-2: Rational design and multi-disciplinary team work.
      ,
      • Gouveia D.
      • Miotello G.
      • Gallais F.
      • Gaillard J.-C.
      • Debroas S.
      • Bellanger L.
      • Lavigne J.-P.
      • Sotto A.
      • Grenga L.
      • Pible O.
      • Armengaud J.
      Proteotyping SARS-CoV-2 virus from nasopharyngeal swabs: A proof-of-concept focused on a 3 min mass spectrometry window.
      ,
      • Ihling C.
      • Tänzler D.
      • Hagemann S.
      • Kehlen A.
      • Hüttelmaier S.
      • Arlt C.
      • Sinz A.
      Mass spectrometric identification of SARS-CoV-2 proteins from gargle solution samples of COVID-19 patients.
      ,
      • Rivera B.
      • Leyva A.
      • Portela M.M.
      • Moratorio G.
      • Moreno P.
      • Durán R.
      • Lima A.
      Quantitative proteomic dataset from oro- and naso-pharyngeal swabs used for COVID-19 diagnosis: Detection of viral proteins and host's biological processes altered by the infection.
      ,
      • Nachtigall F.M.
      • Pereira A.
      • Trofymchuk O.S.
      • Santos L.S.
      Detection of SARS-CoV-2 in nasal swabs using MALDI-MS.
      ).
      Additional studies not substantially focused on characterization of viral or host cell entry factor peptides but containing lists of detected peptides and further relevant information (often with deposited datasets available in various proteomics databases) have also been published (
      • Davidson A.D.
      • Williamson M.K.
      • Lewis S.
      • Shoemark D.
      • Carroll M.W.
      • Heesom K.J.
      • Zambon M.
      • Ellis J.
      • Lewis P.A.
      • Hiscox J.A.
      • Matthews D.A.
      Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein.
      ,
      • Ihling C.
      • Tänzler D.
      • Hagemann S.
      • Kehlen A.
      • Hüttelmaier S.
      • Arlt C.
      • Sinz A.
      Mass spectrometric identification of SARS-CoV-2 proteins from gargle solution samples of COVID-19 patients.
      ,
      • Nikolaev E.N.
      • Indeykina M.I.
      • Brzhozovskiy A.G.
      • Bugrova A.E.
      • Kononikhin A.S.
      • Starodubtseva N.L.
      • Petrotchenko E.V.
      • Kovalev G.I.
      • Borchers C.H.
      • Sukhikh G.T.
      Mass-spectrometric detection of SARS-CoV-2 virus in scrapings of the epithelium of the nasopharynx of infected patients via nucleocapsid N protein.
      ,
      • Villar M.
      • Fernández de Mera I.G.
      • Artigas-Jerónimo S.
      • Contreras M.
      • Gortázar C.
      • de la Fuente J.
      Coronavirus in cat flea: Findings and questions regarding COVID-19.
      ,
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ,
      • Bojkova D.
      • Klann K.
      • Koch B.
      • Widera M.
      • Krause D.
      • Ciesek S.
      • Cinatl J.
      • Münch C.
      Proteomics of SARS-CoV-2-infected host cells reveals therapy targets.
      ,
      • Grenga L.
      • Gallais F.
      • Pible O.
      • Gaillard J.C.
      • Gouveia D.
      • Batina H.
      • Bazaline N.
      • Ruat S.
      • Culotta K.
      • Miotello G.
      • Debroas S.
      • Roncato M.A.
      • Steinmetz G.
      • Foissard C.
      • Desplan A.
      • et al.
      Shotgun proteomics analysis of SARS-CoV-2-infected cells and how it can optimize whole viral particle antigen production for vaccines.
      ).

      Quantitative Proteomics

      Quantitative proteomics experiments involving SARS-CoV-2 and SARS-CoV-2 host cell entry factors generally fall into three categories: experiments quantifying virus (proteins), experiments examining the cell type and tissue distribution of host cell entry factors, and experiments quantifying changes in host cell (and other) factors during infection. Many of these experiments are natural extensions of the qualitative experiments outlined in the previous section.
      Quantification of SARS-CoV-2 proteins has been carried out to understand the kinetics of viral infection, examine the effect of administration of potential therapeutics, and assess viral abundance in infected patients. Grenga et al. (
      • Grenga L.
      • Gallais F.
      • Pible O.
      • Gaillard J.C.
      • Gouveia D.
      • Batina H.
      • Bazaline N.
      • Ruat S.
      • Culotta K.
      • Miotello G.
      • Debroas S.
      • Roncato M.A.
      • Steinmetz G.
      • Foissard C.
      • Desplan A.
      • et al.
      Shotgun proteomics analysis of SARS-CoV-2-infected cells and how it can optimize whole viral particle antigen production for vaccines.
      ) characterized viral kinetics by assaying viral protein expression at several time points (day 1, 2, 3, 4, or 7) for cultures infected either at multiplicity of infection 0.01 or 0.001 on day 0. These results were compared with quantitative PCR (qPCR) measurements of viral RNA and found to be consistent validating their methods in this context (Grenga et al.; Fig. 2). Appelberg et al. (
      • Appelberg S.
      • Gupta S.
      • Svensson Akusjärvi S.
      • Ambikan A.T.
      • Mikaeloff F.
      • Saccon E.
      • Végvári Á.
      • Benfeitas R.
      • Sperk M.
      • Ståhlberg M.
      • Krishnan S.
      • Singh K.
      • Penninger J.M.
      • Mirazimi A.
      • Neogi U.
      Dysregulation in Akt/mTOR/HIF-1 signaling identified by proteo-transcriptomics of SARS-CoV-2 infected cells.
      ) also looked at viral RNA levels by qPCR as compared with viral protein abundance over time (24, 48, or 72 h after infection) from SARS-CoV-2–infected Huh7 cells using a tandem mass tag (TMT)–based method (TMT-MS) and found similar trend agreement between qPCR results and their developed TMT-MS method (Appelberg et al.; Fig. 1). Gordon et al. (
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ) quantified virus protein expression at 8 h after infection in cell culture in relation to treatment (singly) with three potential therapeutic compounds previously identified in their study through other methods (two ligands of sigma-1 and sigma-1 receptors and one protein biogenesis inhibitor), confirming the effectiveness of these compounds in putatively disrupting viral replication. Zecha et al. applied their PRM-based methods to quantify viral proteins in patient samples, although their assays were designed for repeatability rather than for accurate and precise quantification. For samples in which SARS-CoV-2 peptides were detected, peptide intensities were generally in good correspondence with PCR results. However, the PRM assay had a prohibitive rate of false negatives in patient samples (43 of 54 or approximately 80% false negative), a difficulty encountered with MS-based assays in general (
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ). Gouveia et al. further developed and tested a method based on their first article (on SARS-CoV-2 peptide analytical characteristics) establishing a lower limit of detection and concluding that two tryptic peptides from the nucleocapsid protein provide the best basis for a DDA (with inclusion list) reversed-phase LC–MS/MS-based diagnostic platform of the type described. However, these experiments similarly achieved a low rate of detection with diagnosed patient samples (two of nine patients or ~22% from a PCR validated cohort) (
      • Gouveia D.
      • Miotello G.
      • Gallais F.
      • Gaillard J.-C.
      • Debroas S.
      • Bellanger L.
      • Lavigne J.-P.
      • Sotto A.
      • Grenga L.
      • Pible O.
      • Armengaud J.
      Proteotyping SARS-CoV-2 virus from nasopharyngeal swabs: A proof-of-concept focused on a 3 min mass spectrometry window.
      ). Relevant peptides from these studies are summarized in Table 1.
      Figure thumbnail gr2
      Fig. 2Views of the SARS-CoV-2 spike protein and its glycosylation. Images courtesy of Oliver C. Grant (unused graphics from Zhao et al. (
      • Zhao P.
      • Praissman J.L.
      • Grant O.C.
      • Cai Y.
      • Xiao T.
      • Rosenbalm K.E.
      • Aoki K.
      • Kellman B.P.
      • Bridger R.
      • Barouch D.H.
      • Brindley M.A.
      • Lewis N.E.
      • Tiemeyer M.
      • Chen B.
      • Woods R.J.
      • et al.
      Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.
      )). Protein models courtesy of Professor Bing Chen. A, the interface of SARS-CoV-2 S (white) bound to ACE2 (red) showing glycans involved in glycan–peptide and glycan–glycan interactions. B, the postfusion structure of SARS-CoV-2 S showing its distinctive columnar structure and regular spacing of N-glycans. ACE2, angiotensin-converting enzyme 2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
      Several studies have been published to date examining the tissue and cell-type distribution of host cell entry factors. One of the classic studies is by Hamming et al. (
      • Hamming I.
      • Timens W.
      • Bulthuis M.L.
      • Lely A.T.
      • Navis G.
      • van Goor H.
      Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis.
      ), published in 2004 shortly after the original SARS outbreaks and still a very relevant resource to consider newer research against, although not MS based. In more recent research, Zecha et al. (
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ) were able to relatively quantify ACE2 in their four cell line models, ACE2-A549, Vero E6, Calu-3, and Caco-2, using a PRM-based method they developed (discussed in more detail previously), finding expression to be more than 1000 times lower in Calu-3 and Caco-2 cells compared with ACE2-A549 cells. TMPRSS2 and other factors involved or putatively involved in host cell entry also varied widely in expression (Fig. 1, supplemental Fig. S1C, and supplemental Table S1 in the article by Zecha et al.). Hikmet et al. (
      • Hikmet F.
      • Méar L.
      • Edvinsson Å.
      • Micke P.
      • Uhlén M.
      • Lindskog C.
      The protein expression profile of ACE2 in human tissues.
      ) and Aguiar et al. (
      • Aguiar J.A.
      • Tremblay B.J.
      • Mansfield M.J.
      • Woody O.
      • Lobb B.
      • Banerjee A.
      • Chandiramohan A.
      • Tiessen N.
      • Cao Q.
      • Dvorkin-Gheva A.
      • Revill S.
      • Miller M.S.
      • Carlsten C.
      • Organ L.
      • Joseph C.
      • et al.
      Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.
      ) carried out immunohistochemical analysis of tissue and protein profiling by Western blot and from two publicly available MS datasets (one in common to both articles, Kim et al., 2014 (
      • Kim M.S.
      • Pinto S.M.
      • Getnet D.
      • Nirujogi R.S.
      • Manda S.S.
      • Chaerkady R.
      • Madugundu A.K.
      • Kelkar D.S.
      • Isserlin R.
      • Jain S.
      • Thomas J.K.
      • Muthusamy B.
      • Leal-Rojas P.
      • Kumar P.
      • Sahasrabuddhe N.A.
      • et al.
      A draft map of the human proteome.
      )). Both articles extensively analyzed tissue and cell distribution finding a similar pattern of ACE2 expression (high abundance in kidney and testis, lower abundance in gallbladder, and so on; Figs. 4–6 in the article by Aguiar et al. (
      • Aguiar J.A.
      • Tremblay B.J.
      • Mansfield M.J.
      • Woody O.
      • Lobb B.
      • Banerjee A.
      • Chandiramohan A.
      • Tiessen N.
      • Cao Q.
      • Dvorkin-Gheva A.
      • Revill S.
      • Miller M.S.
      • Carlsten C.
      • Organ L.
      • Joseph C.
      • et al.
      Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.
      ), Fig. 6 in the article by Hikmet et al. (
      • Hikmet F.
      • Méar L.
      • Edvinsson Å.
      • Micke P.
      • Uhlén M.
      • Lindskog C.
      The protein expression profile of ACE2 in human tissues.
      )). Aguiar et al. (
      • Aguiar J.A.
      • Tremblay B.J.
      • Mansfield M.J.
      • Woody O.
      • Lobb B.
      • Banerjee A.
      • Chandiramohan A.
      • Tiessen N.
      • Cao Q.
      • Dvorkin-Gheva A.
      • Revill S.
      • Miller M.S.
      • Carlsten C.
      • Organ L.
      • Joseph C.
      • et al.
      Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.
      ) also profiled TMPRSS2, BSG (CD147), and HSPA5 in detail (Figs. 4–6 in the article by Aguiar et al.). Wang et al. (
      • Wang Y.
      • Wang Y.
      • Luo W.
      • Huang L.
      • Xiao J.
      • Li F.
      • Qin S.
      • Song X.
      • Wu Y.
      • Zeng Q.
      • Jin F.
      • Wang Y.
      A comprehensive investigation of the mRNA and protein level of ACE2, the putative receptor of SARS-CoV-2, in human tissues and blood cells.
      ) carried out a similar analysis entirely using previously published data and generating similar results (Fig. 2 in the article by Wang et al.). Finally, Stanley et al. looked specifically at reproductive cells using data from the Human Protein Atlas (
      • Uhlen M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • Sivertsson Å.
      • Kampf C.
      • Sjöstedt E.
      • Asplund A.
      • Olsson I.
      • Edlund K.
      • Lundberg E.
      • Navani S.
      • Szigyarto C.A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      ) and the Human Proteome Map (
      • Kim M.S.
      • Pinto S.M.
      • Getnet D.
      • Nirujogi R.S.
      • Manda S.S.
      • Chaerkady R.
      • Madugundu A.K.
      • Kelkar D.S.
      • Isserlin R.
      • Jain S.
      • Thomas J.K.
      • Muthusamy B.
      • Leal-Rojas P.
      • Kumar P.
      • Sahasrabuddhe N.A.
      • et al.
      A draft map of the human proteome.
      ) and concluded that reproductive consequences of SARS-CoV-2 infection are low given a lack of detectable coexpression of ACE2 and TMPRSS2 at the protein level (
      • Stanley K.E.
      • Thomas E.
      • Leaver M.
      • Wells D.
      Coronavirus disease-19 and fertility: Viral host entry protein expression in male and female reproductive tissues.
      ). One of the most interesting results from these and prior studies, considering the primary respiratory route of viral transmission, has been the difficulty of detecting ACE2 and TMPRSS2 in upper airway samples (
      • Aguiar J.A.
      • Tremblay B.J.
      • Mansfield M.J.
      • Woody O.
      • Lobb B.
      • Banerjee A.
      • Chandiramohan A.
      • Tiessen N.
      • Cao Q.
      • Dvorkin-Gheva A.
      • Revill S.
      • Miller M.S.
      • Carlsten C.
      • Organ L.
      • Joseph C.
      • et al.
      Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.
      ) and other lung tissue samples (
      • Hikmet F.
      • Méar L.
      • Edvinsson Å.
      • Micke P.
      • Uhlén M.
      • Lindskog C.
      The protein expression profile of ACE2 in human tissues.
      ,
      • Wang Y.
      • Wang Y.
      • Luo W.
      • Huang L.
      • Xiao J.
      • Li F.
      • Qin S.
      • Song X.
      • Wu Y.
      • Zeng Q.
      • Jin F.
      • Wang Y.
      A comprehensive investigation of the mRNA and protein level of ACE2, the putative receptor of SARS-CoV-2, in human tissues and blood cells.
      ,
      • Aguiar J.A.
      • Tremblay B.J.
      • Mansfield M.J.
      • Woody O.
      • Lobb B.
      • Banerjee A.
      • Chandiramohan A.
      • Tiessen N.
      • Cao Q.
      • Dvorkin-Gheva A.
      • Revill S.
      • Miller M.S.
      • Carlsten C.
      • Organ L.
      • Joseph C.
      • et al.
      Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.
      ). Researchers have typically explained this by suggesting that alternative entry factors may exist, noting that ACE2 expression appears to be restricted to a subset of (generally) epithelial cells, and by noting that interferon can upregulate ACE2 expression once infection is established (
      • Hamming I.
      • Timens W.
      • Bulthuis M.L.
      • Lely A.T.
      • Navis G.
      • van Goor H.
      Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis.
      ,
      • Aguiar J.A.
      • Tremblay B.J.
      • Mansfield M.J.
      • Woody O.
      • Lobb B.
      • Banerjee A.
      • Chandiramohan A.
      • Tiessen N.
      • Cao Q.
      • Dvorkin-Gheva A.
      • Revill S.
      • Miller M.S.
      • Carlsten C.
      • Organ L.
      • Joseph C.
      • et al.
      Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.
      ,
      • Ziegler C.G.K.
      • Allon S.J.
      • Nyquist S.K.
      • Mbano I.M.
      • Miao V.N.
      • Tzouanas C.N.
      • Cao Y.
      • Yousif A.S.
      • Bals J.
      • Hauser B.M.
      • Feldman J.
      • Muus C.
      • Wadsworth M.H.
      • Kazer S.W.
      • Hughes T.K.
      • et al.
      SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues.
      ). Among other tissues notable in pathogenesis and symptom presentation, ACE2 and TMPRSS2 were codetected in multiple intestinal samples (
      • Hikmet F.
      • Méar L.
      • Edvinsson Å.
      • Micke P.
      • Uhlén M.
      • Lindskog C.
      The protein expression profile of ACE2 in human tissues.
      ,
      • Wang Y.
      • Wang Y.
      • Luo W.
      • Huang L.
      • Xiao J.
      • Li F.
      • Qin S.
      • Song X.
      • Wu Y.
      • Zeng Q.
      • Jin F.
      • Wang Y.
      A comprehensive investigation of the mRNA and protein level of ACE2, the putative receptor of SARS-CoV-2, in human tissues and blood cells.
      ,
      • Aguiar J.A.
      • Tremblay B.J.
      • Mansfield M.J.
      • Woody O.
      • Lobb B.
      • Banerjee A.
      • Chandiramohan A.
      • Tiessen N.
      • Cao Q.
      • Dvorkin-Gheva A.
      • Revill S.
      • Miller M.S.
      • Carlsten C.
      • Organ L.
      • Joseph C.
      • et al.
      Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.
      ), and intestine has long been known to be particularly enriched in ACE2 expression (
      • Hamming I.
      • Timens W.
      • Bulthuis M.L.
      • Lely A.T.
      • Navis G.
      • van Goor H.
      Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis.
      ).
      Studies have also examined changes in host cell entry factor abundance during infection. Understanding the role of ACE2 modulation during infection is clinically significant because ACE2 is both the viral receptor as well as a lung protective factor (notably in SARS-CoV-1 infection) (
      • Hoffmann M.
      • Kleine-Weber H.
      • Schroeder S.
      • Krüger N.
      • Herrler T.
      • Erichsen S.
      • Schiergens T.S.
      • Herrler G.
      • Wu N.H.
      • Nitsche A.
      • Müller M.A.
      • Drosten C.
      • Pöhlmann S.
      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      ,
      • Ziegler C.G.K.
      • Allon S.J.
      • Nyquist S.K.
      • Mbano I.M.
      • Miao V.N.
      • Tzouanas C.N.
      • Cao Y.
      • Yousif A.S.
      • Bals J.
      • Hauser B.M.
      • Feldman J.
      • Muus C.
      • Wadsworth M.H.
      • Kazer S.W.
      • Hughes T.K.
      • et al.
      SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues.
      ,
      • Ingraham N.E.
      • Barakat A.G.
      • Reilkoff R.
      • Bezdicek T.
      • Schacker T.
      • Chipman J.G.
      • Tignanelli C.J.
      • Puskarich M.A.
      Understanding the renin-angiotensin-aldosterone-SARS-CoV axis: A comprehensive review.
      ,
      • Liu M.Y.
      • Zheng B.
      • Zhang Y.
      • Li J.P.
      Role and mechanism of angiotensin-converting enzyme 2 in acute lung injury in coronavirus disease 2019.
      ,
      • Kai H.
      • Kai M.
      Interactions of coronaviruses with ACE2, angiotensin II, and RAS inhibitors-lessons from available evidence and insights into COVID-19.
      ,
      • Samavati L.
      • Uhal B.D.
      ACE2, much more than just a receptor for SARS-COV-2.
      ,
      • Lumbers E.R.
      • Delforce S.J.
      • Pringle K.G.
      • Smith G.R.
      The lung, the heart, the novel coronavirus, and the renin-angiotensin system; the need for clinical trials.
      ,
      • Li Y.
      • Zeng Z.
      • Cao Y.
      • Liu Y.
      • Ping F.
      • Liang M.
      • Xue Y.
      • Xi C.
      • Zhou M.
      • Jiang W.
      Angiotensin-converting enzyme 2 prevents lipopolysaccharide-induced rat acute lung injury via suppressing the ERK1/2 and NF-kappaB signaling pathways.
      ). However, characterizing this modulation has proven challenging as ACE2 (cell surface) levels are variously upregulated or downregulated by different factors in infection including interferon signaling and proteases (e.g., ADAM17) (
      • Verdecchia P.
      • Cavallini C.
      • Spanevello A.
      • Angeli F.
      The pivotal link between ACE2 deficiency and SARS-CoV-2 infection.
      ,
      • Glowacka I.
      • Bertram S.
      • Herzog P.
      • Pfefferle S.
      • Steffen I.
      • Muench M.O.
      • Simmons G.
      • Hofmann H.
      • Kuri T.
      • Weber F.
      • Eichler J.
      • Drosten C.
      • Pöhlmann S.
      Differential downregulation of ACE2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus NL63.
      ). In SARS-CoV-2 infection, Bojkova et al. (and Bock et al. (
      • Bock J.O.
      • Ortea I.
      Re-analysis of SARS-CoV-2-infected host cell proteomics time-course data by impact pathway analysis and network analysis: A potential link with inflammatory response.
      ) based on the same dataset) have now reported reduction of ACE2 abundance in the Caco-2 cell line (a human colon carcinoma line) (
      • Bojkova D.
      • Klann K.
      • Koch B.
      • Widera M.
      • Krause D.
      • Ciesek S.
      • Cinatl J.
      • Münch C.
      Proteomics of SARS-CoV-2-infected host cells reveals therapy targets.
      ). In contrast to these data, however, Zecha et al. (
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ) did not find ACE2 decrease in their cell line models (ACE2-A549, Vero E6, Calu-3, and notably also Caco-2), although they found a decrease in abundance of cathepsin L over time. Continued work will further refine our understanding of host cell entry factor changes during the course of infection.

      PTMs

      Coronavirus proteins, receptors, and other relevant host cell proteins are often post-translationally modified (
      • Fung T.S.
      • Liu D.X.
      Post-translational modifications of coronavirus proteins: Roles and function.
      ,
      • Gupta R.
      • Charron J.
      • Stenger C.L.
      • Painter J.
      • Steward H.
      • Cook T.W.
      • Faber W.
      • Frisch A.
      • Lind E.
      • Bauss J.
      • Li X.
      • Sirpilla O.
      • Soehnlen X.
      • Underwood A.
      • Hinds D.
      • et al.
      SARS-CoV-2 (COVID-19) structural and evolutionary dynamicome: Insights into functional evolution and human genomics.
      ). Review of the SARS-CoV-2 proteome literature revealed studies of glycosylation, phosphorylation, and at least one report detailing (lysine, arginine, and glutamic acid) methylation and proline oxidation on SARS-CoV-2 S and human ACE2 produced in insect cells (
      • Sun Z.
      • Ren K.
      • Zhang X.
      • Chen J.
      • Jiang Z.
      • Jiang J.
      • Ji F.
      • Ouyang X.
      • Li L.
      Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications.
      ). The methylation results are somewhat surprising since most methyltransferases are localized to the nucleus although there are reports of aspartic and glutamic acid methylation in the secretory pathway (
      • Sprung R.
      • Chen Y.
      • Zhang K.
      • Cheng D.
      • Zhang T.
      • Peng J.
      • Zhao Y.
      Identification and validation of eukaryotic aspartate and glutamate methylation in proteins.
      ). No studies have yet confirmed other modifications typically observed with coronaviruses such as ADP ribosylation, sumoylation, palmitoylation, or ubiquitination although sites have been predicted by bioinformatics (
      • Gupta R.
      • Charron J.
      • Stenger C.L.
      • Painter J.
      • Steward H.
      • Cook T.W.
      • Faber W.
      • Frisch A.
      • Lind E.
      • Bauss J.
      • Li X.
      • Sirpilla O.
      • Soehnlen X.
      • Underwood A.
      • Hinds D.
      • et al.
      SARS-CoV-2 (COVID-19) structural and evolutionary dynamicome: Insights into functional evolution and human genomics.
      ,
      • Requena D.
      • Médico A.
      • Chacón R.D.
      • Ramírez M.
      • Marín-Sánchez O.
      Identification of novel candidate epitopes on SARS-CoV-2 proteins for South America: A review of HLA frequencies by country.
      ,
      • Sardar R.
      • Satish D.
      • Birla S.
      • Gupta D.
      Integrative analyses of SARS-CoV-2 genomes from different geographical locations reveal unique features potentially consequential to host-virus interaction, pathogenesis and clues for novel therapies.
      ), and relevant protein interactions for such modifications have been demonstrated (
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ,
      • Bock J.O.
      • Ortea I.
      Re-analysis of SARS-CoV-2-infected host cell proteomics time-course data by impact pathway analysis and network analysis: A potential link with inflammatory response.
      ,
      • Li J.
      • Guo M.
      • Tian X.
      • Wang X.
      • Yang X.
      • Wu P.
      • Liu C.
      • Xiao Z.
      • Qu Y.
      • Yin Y.
      • Wang C.
      • Zhang Y.
      • Zhu Z.
      • Liu Z.
      • Peng C.
      • et al.
      Virus-host interactome and proteomic survey reveal potential virulence factors influencing SARS-CoV-2 pathogenesis.
      ).

      Glycoproteomics and Glycomics

      Several SARS-CoV-2 proteins (S, M, E, and certain “orf” proteins such as Orf8 (
      • Wang X.
      • Lam J.Y.
      • Wong W.M.
      • Yuen C.K.
      • Cai J.P.
      • Au S.W.
      • Chan J.F.
      • To K.K.W.
      • Kok K.H.
      • Yuen K.Y.
      Accurate diagnosis of COVID-19 by a novel immunogenic secreted SARS-CoV-2 orf8 protein.
      ,
      • Sicari D.
      • Chatziioannou A.
      • Koutsandreas T.
      • Sitia R.
      • Chevet E.
      Role of the early secretory pathway in SARS-CoV-2 infection.
      )—a viroporin—and likely Orf7 (
      • Zhang J.
      • Cruz-Cosme R.
      • Zhuang M.W.
      • Liu D.
      • Liu Y.
      • Teng S.
      • Wang P.H.
      • Tang Q.
      A systemic and molecular study of subcellular localization of SARS-CoV-2 proteins.
      ), as with related coronaviruses), as well as host cell factors important in infection, transit the secretory pathway during expression and thus may be glycosylated by secretory pathway glycosyltransferases. The “S” protein (also called “spike” or “surface glycoprotein”) assembles as homotrimers and coats SARS-CoV-2 virions (
      • Wrapp D.
      • Wang N.
      • Corbett K.S.
      • Goldsmith J.A.
      • Hsieh C.L.
      • Abiona O.
      • Graham B.S.
      • McLellan J.S.
      Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.
      ,
      • Walls A.C.
      • Park Y.J.
      • Tortorici M.A.
      • Wall A.
      • McGuire A.T.
      • Veesler D.
      Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein.
      ). The primary binding partner of spike for host cell entry, ACE2, is also a glycoprotein (
      • Zhou P.
      • Yang X.L.
      • Wang X.G.
      • Hu B.
      • Zhang L.
      • Zhang W.
      • Si H.R.
      • Zhu Y.
      • Li B.
      • Huang C.L.
      • Chen H.D.
      • Chen J.
      • Luo Y.
      • Guo H.
      • Jiang R.D.
      • et al.
      A pneumonia outbreak associated with a new coronavirus of probable bat origin.
      ,
      • Hoffmann M.
      • Kleine-Weber H.
      • Schroeder S.
      • Krüger N.
      • Herrler T.
      • Erichsen S.
      • Schiergens T.S.
      • Herrler G.
      • Wu N.H.
      • Nitsche A.
      • Müller M.A.
      • Drosten C.
      • Pöhlmann S.
      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      ,
      • Letko M.
      • Marzi A.
      • Munster V.
      Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses.
      ). Representing natural targets for both antibodies and inhibitors, there has been substantial interest in both these proteins and their protein-linked carbohydrate moieties that may shield or otherwise alter PPIs and protein accessibility. In reviewing glycoproteomic and glycomic studies (particularly preprints), it is important to note that recombinant protein design may lead to non-native modifications (e.g., reports of secretory pathway glycosylation of N).
      To date, there have been six glycoproteomics and glycomics studies published on the carbohydrates covalently attached to SARS-CoV-2 spike, two of which also characterized the glycosylation of ACE2 (
      • Sun Z.
      • Ren K.
      • Zhang X.
      • Chen J.
      • Jiang Z.
      • Jiang J.
      • Ji F.
      • Ouyang X.
      • Li L.
      Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications.
      ,
      • Watanabe Y.
      • Allen J.D.
      • Wrapp D.
      • McLellan J.S.
      • Crispin M.
      Site-specific glycan analysis of the SARS-CoV-2 spike.
      ,
      • Shajahan A.
      • Supekar N.T.
      • Gleinich A.S.
      • Azadi P.
      Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2.
      ,
      • Zhou D.
      • Tian X.
      • Qi R.
      • Peng C.
      • Zhang W.
      Identification of 22 N-glycosites on spike glycoprotein of SARS-CoV-2 and accessible surface glycopeptide motifs: Implications for vaccination and antibody therapeutics.
      ,
      • Zhao P.
      • Praissman J.L.
      • Grant O.C.
      • Cai Y.
      • Xiao T.
      • Rosenbalm K.E.
      • Aoki K.
      • Kellman B.P.
      • Bridger R.
      • Barouch D.H.
      • Brindley M.A.
      • Lewis N.E.
      • Tiemeyer M.
      • Chen B.
      • Woods R.J.
      • et al.
      Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.
      ,
      • Antonopoulos A.
      • Broome S.
      • Sharov V.
      • Ziegenfuss C.
      • Easton R.L.
      • Panico M.
      • Dell A.
      • Morris H.R.
      • Haslam S.M.
      Site-specific characterisation of SARS-CoV-2 spike glycoprotein receptor binding domain.
      ). All results published to date confirm that the spike protein is predominantly modified by N-glycans (at 22 sites) and that there may be varying amounts of O-glycans present at one site (T323). Distinguishing parameters of primary importance among these results are the cell model, recombinant protein design, and purification strategy used. Two of the studies utilized experimental designs (human cell line expression and trimer purification) that have previously been widely shown with viruses in general to produce proteins very close in character to those derived from actual viral infections (
      • Watanabe Y.
      • Allen J.D.
      • Wrapp D.
      • McLellan J.S.
      • Crispin M.
      Site-specific glycan analysis of the SARS-CoV-2 spike.
      ,
      • Zhao P.
      • Praissman J.L.
      • Grant O.C.
      • Cai Y.
      • Xiao T.
      • Rosenbalm K.E.
      • Aoki K.
      • Kellman B.P.
      • Bridger R.
      • Barouch D.H.
      • Brindley M.A.
      • Lewis N.E.
      • Tiemeyer M.
      • Chen B.
      • Woods R.J.
      • et al.
      Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.
      ). These studies, by Watanabe et al. (
      • Watanabe Y.
      • Allen J.D.
      • Wrapp D.
      • McLellan J.S.
      • Crispin M.
      Site-specific glycan analysis of the SARS-CoV-2 spike.
      ) and Zhao et al. (
      • Zhao P.
      • Praissman J.L.
      • Grant O.C.
      • Cai Y.
      • Xiao T.
      • Rosenbalm K.E.
      • Aoki K.
      • Kellman B.P.
      • Bridger R.
      • Barouch D.H.
      • Brindley M.A.
      • Lewis N.E.
      • Tiemeyer M.
      • Chen B.
      • Woods R.J.
      • et al.
      Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.
      ), were in substantial agreement regarding the identities of glycans present and the occupancy of each glycosite and in addition demonstrate the importance of multiple protease digestion and the use of different types of fragmentation activation for comprehensive glycan and glycosite characterization. Zhao et al. (
      • Zhao P.
      • Praissman J.L.
      • Grant O.C.
      • Cai Y.
      • Xiao T.
      • Rosenbalm K.E.
      • Aoki K.
      • Kellman B.P.
      • Bridger R.
      • Barouch D.H.
      • Brindley M.A.
      • Lewis N.E.
      • Tiemeyer M.
      • Chen B.
      • Woods R.J.
      • et al.
      Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.
      ) were also one of only two studies to date in which glycomics was carried out to refine the topologies of the glycans present (
      • Antonopoulos A.
      • Broome S.
      • Sharov V.
      • Ziegenfuss C.
      • Easton R.L.
      • Panico M.
      • Dell A.
      • Morris H.R.
      • Haslam S.M.
      Site-specific characterisation of SARS-CoV-2 spike glycoprotein receptor binding domain.
      ). Other studies cited previously either utilized proteins not produced in human cell lines (
      • Sun Z.
      • Ren K.
      • Zhang X.
      • Chen J.
      • Jiang Z.
      • Jiang J.
      • Ji F.
      • Ouyang X.
      • Li L.
      Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications.
      ,
      • Zhou D.
      • Tian X.
      • Qi R.
      • Peng C.
      • Zhang W.
      Identification of 22 N-glycosites on spike glycoprotein of SARS-CoV-2 and accessible surface glycopeptide motifs: Implications for vaccination and antibody therapeutics.
      ) or protein other than full-length trimer purified spike (
      • Shajahan A.
      • Supekar N.T.
      • Gleinich A.S.
      • Azadi P.
      Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2.
      ,
      • Antonopoulos A.
      • Broome S.
      • Sharov V.
      • Ziegenfuss C.
      • Easton R.L.
      • Panico M.
      • Dell A.
      • Morris H.R.
      • Haslam S.M.
      Site-specific characterisation of SARS-CoV-2 spike glycoprotein receptor binding domain.
      ), raising additional questions as to the biological relevance of the glycosylation results obtained with respect to actual SARS-CoV-2 virions in human hosts. The apparent resulting differences provide valuable information to researchers considering antibody or vaccine candidate production in nonhuman cell lines or using nontrimer purified protein. Finally, it is worth noting that SARS-CoV-2 glycosylation is significantly more host like than the glycosylation found on many other viruses such as HIV when considering N-glycan processing and density, although high mannose glycans still occur with greater prevalence than on most host proteins (
      • Watanabe Y.
      • Allen J.D.
      • Wrapp D.
      • McLellan J.S.
      • Crispin M.
      Site-specific glycan analysis of the SARS-CoV-2 spike.
      ,
      • Zhao P.
      • Praissman J.L.
      • Grant O.C.
      • Cai Y.
      • Xiao T.
      • Rosenbalm K.E.
      • Aoki K.
      • Kellman B.P.
      • Bridger R.
      • Barouch D.H.
      • Brindley M.A.
      • Lewis N.E.
      • Tiemeyer M.
      • Chen B.
      • Woods R.J.
      • et al.
      Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.
      ,
      • Watanabe Y.
      • Berndsen Z.T.
      • Raghwani J.
      • Seabright G.E.
      • Allen J.D.
      • Pybus O.G.
      • McLellan J.S.
      • Wilson I.A.
      • Bowden T.A.
      • Ward A.B.
      • Crispin M.
      Vulnerabilities in coronavirus glycan shields despite extensive glycosylation.
      ).
      The two recent studies characterizing the glycosylation of ACE2 form a subset of the SARS-CoV-2 spike articles (
      • Sun Z.
      • Ren K.
      • Zhang X.
      • Chen J.
      • Jiang Z.
      • Jiang J.
      • Ji F.
      • Ouyang X.
      • Li L.
      Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications.
      ,
      • Zhao P.
      • Praissman J.L.
      • Grant O.C.
      • Cai Y.
      • Xiao T.
      • Rosenbalm K.E.
      • Aoki K.
      • Kellman B.P.
      • Bridger R.
      • Barouch D.H.
      • Brindley M.A.
      • Lewis N.E.
      • Tiemeyer M.
      • Chen B.
      • Woods R.J.
      • et al.
      Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.
      ) and provide a much more complete picture than earlier articles (
      • Towler P.
      • Staker B.
      • Prasad S.G.
      • Menon S.
      • Tang J.
      • Parsons T.
      • Ryan D.
      • Fisher M.
      • Williams D.
      • Dales N.A.
      • Patane M.A.
      • Pantoliano M.W.
      ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis.
      ,
      • Kristiansen T.Z.
      • Bunkenborg J.
      • Gronborg M.
      • Molina H.
      • Thuluvath P.J.
      • Argani P.
      • Goggins M.G.
      • Maitra A.
      • Pandey A.
      A proteomic analysis of human bile.
      ,
      • Chen R.
      • Jiang X.
      • Sun D.
      • Han G.
      • Wang F.
      • Ye M.
      • Wang L.
      • Zou H.
      Glycoproteomics analysis of human liver tissue by combination of multiple enzyme digestion and hydrazide chemistry.
      ,
      • Wu K.
      • Li W.
      • Peng G.
      • Li F.
      Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor.
      ). In particular, Zhao et al. (
      • Zhao P.
      • Praissman J.L.
      • Grant O.C.
      • Cai Y.
      • Xiao T.
      • Rosenbalm K.E.
      • Aoki K.
      • Kellman B.P.
      • Bridger R.
      • Barouch D.H.
      • Brindley M.A.
      • Lewis N.E.
      • Tiemeyer M.
      • Chen B.
      • Woods R.J.
      • et al.
      Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.
      ) carried out comprehensive glycomics-informed glycoproteomic analysis on a purified soluble version of the protein. ACE2 has seven “canonical” N-glycosylation sequons, six of which were included in the expression construct used in the article and analyzed in depth. The N-glycosylation of ACE2 was found to be broadly similar to that seen with other human proteins that traffic through the secretory pathway. Only a small amount of O-glycosylation was detected. This detailed analysis of glycosylation also allowed the authors to carry out molecular dynamics simulations of SARS-CoV-2 S glycoprotein bound to ACE2 glycoprotein suggesting that several N-linked glycans on each protein are intimately involved in glycoprotein–glycoprotein interaction (Fig. 2). In addition, a model of the postfusion SARS-CoV-2 S glycoprotein was developed showing its distinctive columnar structure and even N-glycan spacing (Fig. 2). A number of other articles containing molecular dynamics simulation results and molecular modeling work have been published based on different glycoproteomics studies and are valuable additional references (
      • Woo H.
      • Park S.J.
      • Choi Y.K.
      • Park T.
      • Tanveer M.
      • Cao Y.
      • Kern N.R.
      • Lee J.
      • Yeom M.S.
      • Croll T.I.
      • Seok C.
      • Im W.
      Developing a fully glycosylated full-length SARS-CoV-2 spike protein model in a viral membrane.
      ,
      • Ke Z.
      • Oton J.
      • Qu K.
      • Cortese M.
      • Zila V.
      • McKeane L.
      • Nakane T.
      • Zivanov J.
      • Neufeldt C.J.
      • Cerikan B.
      • Lu J.M.
      • Peukes J.
      • Xiong X.
      • Kräusslich H.G.
      • Scheres S.H.W.
      • et al.
      Structures and distributions of SARS-CoV-2 spike proteins on intact virions.
      ,
      • Turonova B.
      • Sikora M.
      • Schurmann C.
      • Hagen W.
      • Welsch S.
      • Blanc F.
      • Bulow S.
      • Gecht M.
      • Bagola K.
      • Horner C.
      • Zandbergen G.
      • Landry J.
      • Azevedo N.
      • Mosalaganti S.
      • Schwarz A.
      • et al.
      In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges.
      ,
      • Grant O.C.
      • Montgomery D.
      • Ito K.
      • Woods R.J.
      Analysis of the SARS-CoV-2 spike protein glycan shield reveals implications for immune recognition.
      ,
      • Prates E.T.
      • Garvin M.R.
      • Pavicic M.
      • Jones P.
      • Shah M.
      • Demerdash O.
      • Amos B.K.
      • Geiger A.
      • Jacobson D.
      Potential pathogenicity determinants identified from structural proteomics of SARS-CoV and SARS-CoV-2.
      ,
      • Wintjens R.
      • Bifani A.M.
      • Bifani P.
      Impact of glycan cloud on the B-cell epitope prediction of SARS-CoV-2 Spike protein.
      ,
      • Yao H.
      • Song Y.
      • Chen Y.
      • Wu N.
      • Xu J.
      • Sun C.
      • Zhang J.
      • Weng T.
      • Zhang Z.
      • Wu Z.
      • Cheng L.
      • Shi D.
      • Lu X.
      • Lei J.
      • Crispin M.
      • et al.
      Molecular architecture of the SARS-CoV-2 virus.
      ,
      • Casalino L.
      • Gaieb Z.
      • Goldsmith J.A.
      • Hjorth C.K.
      • Dommer A.C.
      • Harbison A.M.
      • Fogarty C.A.
      • Barros E.P.
      • Taylor B.C.
      • McLellan J.S.
      • Fadda E.
      • Amaro R.E.
      Beyond shielding: The roles of glycans in the SARS-CoV-2 spike protein.
      ). Sun et al. (
      • Sun Z.
      • Ren K.
      • Zhang X.
      • Chen J.
      • Jiang Z.
      • Jiang J.
      • Ji F.
      • Ouyang X.
      • Li L.
      Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications.
      ) reported very similar results on the N-glycosylation of ACE2. Although they were unable to detect N-glycans at N053 and N322, by using a construct including the N-glycosylation site N690, Sun et al. (
      • Sun Z.
      • Ren K.
      • Zhang X.
      • Chen J.
      • Jiang Z.
      • Jiang J.
      • Ji F.
      • Ouyang X.
      • Li L.
      Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications.
      ) were able to characterize glycosylation at this seventh site in contrast with Zhao et al. (
      • Zhao P.
      • Praissman J.L.
      • Grant O.C.
      • Cai Y.
      • Xiao T.
      • Rosenbalm K.E.
      • Aoki K.
      • Kellman B.P.
      • Bridger R.
      • Barouch D.H.
      • Brindley M.A.
      • Lewis N.E.
      • Tiemeyer M.
      • Chen B.
      • Woods R.J.
      • et al.
      Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.
      ). In total, the glycosylation patterns of the SARS-CoV-2 spike protein and its host cell surface receptor have been characterized in detail by multiple groups using different biological models providing important information for future research and particularly informing modeling that may be crucial in understanding and addressing the emergence of potential vaccine and antibody escape variants.

      Phosphorylation

      Currently, three published studies have specifically examined the phosphorylation of SARS-CoV-2 proteins (Fig. 1) and host cell entry factors (
      • Davidson A.D.
      • Williamson M.K.
      • Lewis S.
      • Shoemark D.
      • Carroll M.W.
      • Heesom K.J.
      • Zambon M.
      • Ellis J.
      • Lewis P.A.
      • Hiscox J.A.
      • Matthews D.A.
      Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein.
      ,
      • Bouhaddou M.
      • Memon D.
      • Meyer B.
      • White K.M.
      • Rezelj V.V.
      • Correa Marrero M.
      • Polacco B.J.
      • Melnyk J.E.
      • Ulferts S.
      • Kaake R.M.
      • Batra J.
      • Richards A.L.
      • Stevenson E.
      • Gordon D.E.
      • Rojc A.
      • et al.
      The global phosphorylation landscape of SARS-CoV-2 infection.
      ,
      • Klann K.
      • Bojkova D.
      • Tascher G.
      • Ciesek S.
      • Münch C.
      • Cinatl J.
      Growth factor receptor signaling inhibition prevents SARS-CoV-2 replication.
      ). Davidson et al. (
      • Davidson A.D.
      • Williamson M.K.
      • Lewis S.
      • Shoemark D.
      • Carroll M.W.
      • Heesom K.J.
      • Zambon M.
      • Ellis J.
      • Lewis P.A.
      • Hiscox J.A.
      • Matthews D.A.
      Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein.
      ) used TiO2 followed by ferric nitrilotriacetate phosphopeptide enrichment on in-gel trypsin-digested peptides from whole cell lysate enabling the mapping of 44 phosphorylation sites among five viral proteins (Figs. 3–5 and Table 4 in the article by Davidson et al.: nsp3, nsp9, M, N, and S—nsp12 and ORF3a also produced phosphorylated peptides, but sites could not be confidently assigned) in a typical DDA LC–MS/MS higher-energy collisional dissociation experiment (
      • Davidson A.D.
      • Williamson M.K.
      • Lewis S.
      • Shoemark D.
      • Carroll M.W.
      • Heesom K.J.
      • Zambon M.
      • Ellis J.
      • Lewis P.A.
      • Hiscox J.A.
      • Matthews D.A.
      Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein.
      ). The report of phosphorylation sites distributed across spike is surprising, as noted by Davidson et al. in their article, given its secretory pathway expression, and it will be interesting to see if future studies confirm this result. Bouhaddou et al. (
      • Bouhaddou M.
      • Memon D.
      • Meyer B.
      • White K.M.
      • Rezelj V.V.
      • Correa Marrero M.
      • Polacco B.J.
      • Melnyk J.E.
      • Ulferts S.
      • Kaake R.M.
      • Batra J.
      • Richards A.L.
      • Stevenson E.
      • Gordon D.E.
      • Rojc A.
      • et al.
      The global phosphorylation landscape of SARS-CoV-2 infection.
      ) comprehensively examined phosphorylation of both viral proteins and host proteins, enriching from cell lysate tryptic peptides via iron affinity and analyzing via LC–MS/MS (DDA and data-independent acquisition) with higher-energy collisional dissociation. Using this approach, they identified 25 phosphorylation sites (omitting results from Davidson et al. (
      • Davidson A.D.
      • Williamson M.K.
      • Lewis S.
      • Shoemark D.
      • Carroll M.W.
      • Heesom K.J.
      • Zambon M.
      • Ellis J.
      • Lewis P.A.
      • Hiscox J.A.
      • Matthews D.A.
      Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein.
      ) that they also presented) on SARS-CoV-2 viral proteins (Fig. 2 and supplemental Table S2 in the article by Bouhaddou et al. (
      • Bouhaddou M.
      • Memon D.
      • Meyer B.
      • White K.M.
      • Rezelj V.V.
      • Correa Marrero M.
      • Polacco B.J.
      • Melnyk J.E.
      • Ulferts S.
      • Kaake R.M.
      • Batra J.
      • Richards A.L.
      • Stevenson E.
      • Gordon D.E.
      • Rojc A.
      • et al.
      The global phosphorylation landscape of SARS-CoV-2 infection.
      ): on nsp3, nsp14, orf9b, M, and N). The phosphorylation of N has since been shown to be functionally important in nucleocapsid assembly and viral replication and transcription (
      • Carlson C.R.
      • Asfaha J.B.
      • Ghent C.M.
      • Howard C.J.
      • Hartooni N.
      • Safari M.
      • Frankel A.D.
      • Morgan D.O.
      Phosphoregulation of phase separation by the SARS-CoV-2 N protein suggests a biophysical basis for its dual functions.
      ,
      • Lu S.
      • Ye Q.
      • Singh D.
      • Cao Y.
      • Diedrich J.K.
      • Yates J.R.
      • Villa E.
      • Cleveland D.W.
      • Corbett K.D.
      The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein.
      ,
      • Yang M.
      • He S.
      • Chen X.
      • Huang Z.
      • Zhou Z.
      • Zhou Z.
      • Chen Q.
      • Chen S.
      • Kang S.
      Structural insight into the SARS-CoV-2 nucleocapsid protein C-terminal domain reveals a novel recognition mechanism for viral transcriptional regulatory sequences.
      ) as was previously observed with other coronaviruses. Several sites in the C-terminal tail of M are noted to be present in other viruses suggesting potential functional importance although it does not appear that this has been functionally verified for SARS-CoV-2 yet. Among host cell entry factors, PIKfyve and cathepsin L are phosphorylated (Table S1 in the article by Bouhaddou et al. (
      • Bouhaddou M.
      • Memon D.
      • Meyer B.
      • White K.M.
      • Rezelj V.V.
      • Correa Marrero M.
      • Polacco B.J.
      • Melnyk J.E.
      • Ulferts S.
      • Kaake R.M.
      • Batra J.
      • Richards A.L.
      • Stevenson E.
      • Gordon D.E.
      • Rojc A.
      • et al.
      The global phosphorylation landscape of SARS-CoV-2 infection.
      )), and regulation of the phosphorylation of PIKfyve was further confirmed to be involved in SARS-CoV-2 infection (
      • Bouhaddou M.
      • Memon D.
      • Meyer B.
      • White K.M.
      • Rezelj V.V.
      • Correa Marrero M.
      • Polacco B.J.
      • Melnyk J.E.
      • Ulferts S.
      • Kaake R.M.
      • Batra J.
      • Richards A.L.
      • Stevenson E.
      • Gordon D.E.
      • Rojc A.
      • et al.
      The global phosphorylation landscape of SARS-CoV-2 infection.
      ). Klann et al. (
      • Klann K.
      • Bojkova D.
      • Tascher G.
      • Ciesek S.
      • Münch C.
      • Cinatl J.
      Growth factor receptor signaling inhibition prevents SARS-CoV-2 replication.
      ) used ferric nitrilotriacetate enrichment after in-solution digest and TMT labeling of proteins from whole cell lysate finding 33 sites on six viral proteins (Fig. 1, EJ in the study by Klann et al. (
      • Klann K.
      • Bojkova D.
      • Tascher G.
      • Ciesek S.
      • Münch C.
      • Cinatl J.
      Growth factor receptor signaling inhibition prevents SARS-CoV-2 replication.
      ): pp1ab, nsp6, ORF3a, ORF9b, M, and N). Additional datasets available in the ProteomeXchange (
      • Deutsch E.W.
      • Csordas A.
      • Sun Z.
      • Jarnuczak A.
      • Perez-Riverol Y.
      • Ternent T.
      • Campbell D.S.
      • Bernal-Llinares M.
      • Okuda S.
      • Kawano S.
      • Moritz R.L.
      • Carver J.J.
      • Wang M.
      • Ishihama Y.
      • Bandeira N.
      • et al.
      The ProteomeXchange consortium in 2017: Supporting the cultural change in proteomics public data deposition.
      ) contain various sets of relevant phosphorylation hits and may be found by searching this database.

      Host Cell Proteomics

      PPI

      PPIs play a primary role in the life cycle of animal viruses, from attachment to cells through endosomal compartment escape (for most viruses) and ultimately reorganization of cellular machinery to support viral reproduction and diminish host defense (
      • Marsh M.
      • Helenius A.
      Virus entry: Open sesame.
      ,
      • Mercer J.
      • Schelhaas M.
      • Helenius A.
      Virus entry by endocytosis.
      ). Consequently, enumerating host–virus PPIs is crucial to understanding the biology of viruses and developing a starting point for investigation of potential therapeutics. To date, several comprehensive studies of SARS-CoV-2 host–virus PPIs using proteomics technologies have been published, setting the stage for additional studies expanding on this work—studies that may definitively validate or invalidate proposed interactions. An extremely useful resource for SARS-CoV-2 PPIs was brought to our attention during review of this article, and interested readers are directed to it for a more comprehensive up-to-date view of published interactions: https://thebiogrid.org/project/3 (
      • Oughtred R.
      • Rust J.
      • Chang C.
      • Breitkreutz B.J.
      • Stark C.
      • Willems A.
      • Boucher L.
      • Leung G.
      • Kolas N.
      • Zhang F.
      • Dolma S.
      • Coulombe-Huntington J.
      • Chatr-Aryamontri A.
      • Dolinski K.
      • Tyers M.
      The BioGRID database: A comprehensive biomedical resource of curated protein, genetic, and chemical interactions.
      ). The interactions reported in this database and in the research cited later may also be considered in the context of more recently published functional genomics studies for additional perspective (
      • Daniloski Z.
      • Jordan T.X.
      • Wessels H.H.
      • Hoagland D.A.
      • Kasela S.
      • Legut M.
      • Maniatis S.
      • Mimitou E.P.
      • Lu L.
      • Geller E.
      • Danziger O.
      • Rosenberg B.R.
      • Phatnani H.
      • Smibert P.
      • Lappalainen T.
      • et al.
      Identification of required host factors for SARS-CoV-2 infection in human cells.
      ,
      • Wei J.
      • Alfajaro M.M.
      • DeWeirdt P.C.
      • Hanna R.E.
      • Lu-Culligan W.J.
      • Cai W.L.
      • Strine M.S.
      • Zhang S.M.
      • Graziano V.R.
      • Schmitz C.O.
      • Chen J.S.
      • Mankowski M.C.
      • Filler R.B.
      • Ravindra N.G.
      • Gasque V.
      • et al.
      Genome-wide CRISPR screens reveal host factors critical for SARS-CoV-2 infection.
      ,
      • Wang R.
      • Simoneau C.R.
      • Kulsuptrakul J.
      • Bouhaddou M.
      • Travisano K.A.
      • Hayashi J.M.
      • Carlson-Stevermer J.
      • Zengel J.R.
      • Richards C.M.
      • Fozouni P.
      • Oki J.
      • Rodriguez L.
      • Joehnk B.
      • Walcott K.
      • Holden K.
      • et al.
      Genetic screens identify host factors for SARS-CoV-2 and common cold coronaviruses.
      ,
      • Schneider W.M.
      • Luna J.M.
      • Hoffmann H.H.
      • Sánchez-Rivera F.J.
      • Leal A.A.
      • Ashbrook A.W.
      • Le Pen J.
      • Ricardo-Lax I.
      • Michailidis E.
      • Peace A.
      • Stenzel A.F.
      • Lowe S.W.
      • MacDonald M.R.
      • Rice C.M.
      • Poirier J.T.
      Genome-scale identification of SARS-CoV-2 and pan-coronavirus host factor networks.
      ,
      • Zhu Y.
      • Feng F.
      • Hu G.
      • Wang Y.
      • Yu Y.
      • Zhu Y.
      • Xu W.
      • Cai X.
      • Sun Z.
      • Han W.
      • Ye R.
      • Qu D.
      • Ding Q.
      • Huang X.
      • Chen H.
      • et al.
      A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry.
      ).
      A comprehensive interactome was published by Gordon et al. who carried out affinity purification, using transfected viral proteins (human embryonic kidney 293 cells) as baits against human cell proteins in the cells used for viral protein expression, followed by tryptic digestion and LC–MS/MS (
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ). Through this workflow, they were able to map 332 high-confident PPIs and identify 66 druggable human proteins concluding, through additional experiments with compounds targeting interactors, that inhibitors of mRNA translation and regulators of sigma-1 and sigma-2 receptors show potential for SARS-CoV-2 treatment. Noteworthy PPIs involve factors in host mRNA nuclear export and overall mRNA regulation/translation, phosphorylation, secretory pathway targeting of proteins, and protein degradation (Fig. 3, Table 2, and supplemental Table S1). This group has since published a newer study comparing interactors across related coronaviruses SARS-CoV-1 and Middle East respiratory syndrome-CoV and validating or further validating the clinical relevance of three host factor interactors reported in their original study (Tom70 or TOMM70, ILR17RA, and SigmaR1) (
      • Gordon D.E.
      • Hiatt J.
      • Bouhaddou M.
      • Rezelj V.V.
      • Ulferts S.
      • Braberg H.
      • Jureka A.S.
      • Obernier K.
      • Guo J.Z.
      • Batra J.
      • Kaake R.M.
      • Weckstein A.R.
      • Owens T.W.
      • Gupta M.
      • Pourmal S.
      • et al.
      Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms.
      ). Li et al. (
      • Li J.
      • Guo M.
      • Tian X.
      • Wang X.
      • Yang X.
      • Wu P.
      • Liu C.
      • Xiao Z.
      • Qu Y.
      • Yin Y.
      • Wang C.
      • Zhang Y.
      • Zhu Z.
      • Liu Z.
      • Peng C.
      • et al.
      Virus-host interactome and proteomic survey reveal potential virulence factors influencing SARS-CoV-2 pathogenesis.
      ) more recently published another comprehensive interactome using similar affinity-purification LC–MS/MS methods and found 45 targets shared with the article by Gordon et al. (
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ) (~16% of their interactome, including, e.g., SigmaR1) while uncovering many unreported PPIs. In particular, Li et al. found many immune system–related interactions (
      • Li J.
      • Guo M.
      • Tian X.
      • Wang X.
      • Yang X.
      • Wu P.
      • Liu C.
      • Xiao Z.
      • Qu Y.
      • Yin Y.
      • Wang C.
      • Zhang Y.
      • Zhu Z.
      • Liu Z.
      • Peng C.
      • et al.
      Virus-host interactome and proteomic survey reveal potential virulence factors influencing SARS-CoV-2 pathogenesis.
      ) (Table 2 and supplemental Table S1).
      Figure thumbnail gr3
      Fig. 3The SARS-CoV-2 viral life cycle and selected host proteins involved. The viral life cycle is displayed proceeding from host cell entry through new virion synthesis, packaging, and export. Host cell proteins are labeled in green, and SARS-CoV-2 proteins are labeled in blue. Red arrows (→) indicate protease cleavage. The representation of virus shows ribonucleoproteins (RNPs) (consisting of five dimers of N) in the tetrahedral geometry recently reported (Yao et al. (
      • Yao H.
      • Song Y.
      • Chen Y.
      • Wu N.
      • Xu J.
      • Sun C.
      • Zhang J.
      • Weng T.
      • Zhang Z.
      • Wu Z.
      • Cheng L.
      • Shi D.
      • Lu X.
      • Lei J.
      • Crispin M.
      • et al.
      Molecular architecture of the SARS-CoV-2 virus.
      )). This article reported an average of 26 ± 15 copies of prefusion S per virion and 26 ± RNPs per virion. The life cycle in a given cell begins with host cell entry mediated by ACE2 (the receptor), TMPRSS2 (or alternatively CatB/L—CSTB/CTSL—fusion priming enzymes), and proceeds with trafficking through endosomes. Endosomal maturation required for viral–host–cell membrane fusion involves the proteins PIKfyve and TPC2. After fusion and uncoating of the viral RNA, the replication-transcription complex is expressed, and new viral genomic RNAs (gRNAs, + and − sense) and subgenomic RNAs (sgRNAs, + and − sense) are produced. The translation of viral proteins and modulation of host protein translation is affected by protein–protein interactions (Nsp2-eIFE2/GIGYF2, Nsp9-eIF4H, and N-LARP1 are shown) and signaling. New virion structural protein N is phosphorylated (CK2, PKC, and CDK), forms RNPs, winds gRNAs, and collects at the ERGIC membrane for envelopment. Viral proteins E, M, and S traffic through the secretory pathway for further processing including addition of glycans. Filopodia formation is enhanced (proposed to be CK2 driven by Bouhaddou et al. (
      • Bouhaddou M.
      • Memon D.
      • Meyer B.
      • White K.M.
      • Rezelj V.V.
      • Correa Marrero M.
      • Polacco B.J.
      • Melnyk J.E.
      • Ulferts S.
      • Kaake R.M.
      • Batra J.
      • Richards A.L.
      • Stevenson E.
      • Gordon D.E.
      • Rojc A.
      • et al.
      The global phosphorylation landscape of SARS-CoV-2 infection.
      )) and may improve transmission of egressing virus between cells. ACE2, angiotensin-converting enzyme 2; CTSL, cathepsin L; ERGIC, endoplasmic reticulum golgi intermediate compartment; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TMPRSS2, transmembrane serine protease 2.
      Table 2Selected host proteins in infection
      Primary processGene/complex/familyProtein namePPI?Abundance?Phosphorylation?Act.?Function in infection (known and/or hypothesized)Cell locationSelected SARS-CoV-2 proteomics references
      Host cell entryACE2Angiotensin-converting enzyme 2S+/−Virus receptorPM(
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ,
      • Hikmet F.
      • Méar L.
      • Edvinsson Å.
      • Micke P.
      • Uhlén M.
      • Lindskog C.
      The protein expression profile of ACE2 in human tissues.
      ,
      • Wang Y.
      • Wang Y.
      • Luo W.
      • Huang L.
      • Xiao J.
      • Li F.
      • Qin S.
      • Song X.
      • Wu Y.
      • Zeng Q.
      • Jin F.
      • Wang Y.
      A comprehensive investigation of the mRNA and protein level of ACE2, the putative receptor of SARS-CoV-2, in human tissues and blood cells.
      ,
      • Feng L.
      • Yin Y.Y.
      • Liu C.H.
      • Xu K.R.
      • Li Q.R.
      • Wu J.R.
      • Zeng R.
      Proteome-wide data analysis reveals tissue-specific network associated with SARS-CoV-2 infection.
      ,
      • Stanley K.E.
      • Thomas E.
      • Leaver M.
      • Wells D.
      Coronavirus disease-19 and fertility: Viral host entry protein expression in male and female reproductive tissues.
      ,
      • Bojkova D.
      • Klann K.
      • Koch B.
      • Widera M.
      • Krause D.
      • Ciesek S.
      • Cinatl J.
      • Münch C.
      Proteomics of SARS-CoV-2-infected host cells reveals therapy targets.
      ,
      • Aguiar J.A.
      • Tremblay B.J.
      • Mansfield M.J.
      • Woody O.
      • Lobb B.
      • Banerjee A.
      • Chandiramohan A.
      • Tiessen N.
      • Cao Q.
      • Dvorkin-Gheva A.
      • Revill S.
      • Miller M.S.
      • Carlsten C.
      • Organ L.
      • Joseph C.
      • et al.
      Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.
      ,
      • Sun Z.
      • Ren K.
      • Zhang X.
      • Chen J.
      • Jiang Z.
      • Jiang J.
      • Ji F.
      • Ouyang X.
      • Li L.
      Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications.
      ,
      • Zhao P.
      • Praissman J.L.
      • Grant O.C.
      • Cai Y.
      • Xiao T.
      • Rosenbalm K.E.
      • Aoki K.
      • Kellman B.P.
      • Bridger R.
      • Barouch D.H.
      • Brindley M.A.
      • Lewis N.E.
      • Tiemeyer M.
      • Chen B.
      • Woods R.J.
      • et al.
      Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.
      )
      TMPRSS2Transmembrane protease serine 2SCleaves S (“priming”), especially at S2' sitePM(
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ,
      • Feng L.
      • Yin Y.Y.
      • Liu C.H.
      • Xu K.R.
      • Li Q.R.
      • Wu J.R.
      • Zeng R.
      Proteome-wide data analysis reveals tissue-specific network associated with SARS-CoV-2 infection.
      ,
      • Stanley K.E.
      • Thomas E.
      • Leaver M.
      • Wells D.
      Coronavirus disease-19 and fertility: Viral host entry protein expression in male and female reproductive tissues.
      ,
      • Aguiar J.A.
      • Tremblay B.J.
      • Mansfield M.J.
      • Woody O.
      • Lobb B.
      • Banerjee A.
      • Chandiramohan A.
      • Tiessen N.
      • Cao Q.
      • Dvorkin-Gheva A.
      • Revill S.
      • Miller M.S.
      • Carlsten C.
      • Organ L.
      • Joseph C.
      • et al.
      Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.
      )
      CTSBCathepsin BS?Cleaves S, alternative to TMPRSS2EN(
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ,
      • Hikmet F.
      • Méar L.
      • Edvinsson Å.
      • Micke P.
      • Uhlén M.
      • Lindskog C.
      The protein expression profile of ACE2 in human tissues.
      ,
      • Li J.
      • Guo M.
      • Tian X.
      • Wang X.
      • Yang X.
      • Wu P.
      • Liu C.
      • Xiao Z.
      • Qu Y.
      • Yin Y.
      • Wang C.
      • Zhang Y.
      • Zhu Z.
      • Liu Z.
      • Peng C.
      • et al.
      Virus-host interactome and proteomic survey reveal potential virulence factors influencing SARS-CoV-2 pathogenesis.
      ,
      • Perrin-Cocon L.
      • Diaz O.
      • Jacquemin C.
      • Barthel V.
      • Ogire E.
      • Ramière C.
      • André P.
      • Lotteau V.
      • Vidalain P.O.
      The current landscape of coronavirus-host protein-protein interactions.
      )
      CTSLCathepsin LSCleaves S, alternative to TMPRSS2EN(
      • Zecha J.
      • Lee C.-Y.
      • Bayer F.P.
      • Meng C.
      • Grass V.
      • Zerweck J.
      • Schnatbaum K.
      • Michler T.
      • Pichlmair A.
      • Ludwig C.
      • Kuster B.
      Data, reagents, assays and merits of proteomics for SARS-CoV-2 research and testing.
      ,
      • Hikmet F.
      • Méar L.
      • Edvinsson Å.
      • Micke P.
      • Uhlén M.
      • Lindskog C.
      The protein expression profile of ACE2 in human tissues.
      ,
      • Stanley K.E.
      • Thomas E.
      • Leaver M.
      • Wells D.
      Coronavirus disease-19 and fertility: Viral host entry protein expression in male and female reproductive tissues.
      ,
      • Aguiar J.A.
      • Tremblay B.J.
      • Mansfield M.J.
      • Woody O.
      • Lobb B.
      • Banerjee A.
      • Chandiramohan A.
      • Tiessen N.
      • Cao Q.
      • Dvorkin-Gheva A.
      • Revill S.
      • Miller M.S.
      • Carlsten C.
      • Organ L.
      • Joseph C.
      • et al.
      Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.
      ,
      • Perrin-Cocon L.
      • Diaz O.
      • Jacquemin C.
      • Barthel V.
      • Ogire E.
      • Ramière C.
      • André P.
      • Lotteau V.
      • Vidalain P.O.
      The current landscape of coronavirus-host protein-protein interactions.
      )
      Endosomal releasePIKFYVE1-phosphatidylinositol 3-phosphate 5-kinaseEndosome maturation, with TPC2EN(
      • Bouhaddou M.
      • Memon D.
      • Meyer B.
      • White K.M.
      • Rezelj V.V.
      • Correa Marrero M.
      • Polacco B.J.
      • Melnyk J.E.
      • Ulferts S.
      • Kaake R.M.
      • Batra J.
      • Richards A.L.
      • Stevenson E.
      • Gordon D.E.
      • Rojc A.
      • et al.
      The global phosphorylation landscape of SARS-CoV-2 infection.
      )
      Protein expressionNUP98Nuclear pore complex protein Nup98Orf6+Prevent host nuclear mRNA exportNM(
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ,
      • Bouhaddou M.
      • Memon D.
      • Meyer B.
      • White K.M.
      • Rezelj V.V.
      • Correa Marrero M.
      • Polacco B.J.
      • Melnyk J.E.
      • Ulferts S.
      • Kaake R.M.
      • Batra J.
      • Richards A.L.
      • Stevenson E.
      • Gordon D.E.
      • Rojc A.
      • et al.
      The global phosphorylation landscape of SARS-CoV-2 infection.
      )
      LARP1La-related protein 1N-Prioritize virus protein expressionCP, NU(
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ,
      • Bouhaddou M.
      • Memon D.
      • Meyer B.
      • White K.M.
      • Rezelj V.V.
      • Correa Marrero M.
      • Polacco B.J.
      • Melnyk J.E.
      • Ulferts S.
      • Kaake R.M.
      • Batra J.
      • Richards A.L.
      • Stevenson E.
      • Gordon D.E.
      • Rojc A.
      • et al.
      The global phosphorylation landscape of SARS-CoV-2 infection.
      ,
      • Perrin-Cocon L.
      • Diaz O.
      • Jacquemin C.
      • Barthel V.
      • Ogire E.
      • Ramière C.
      • André P.
      • Lotteau V.
      • Vidalain P.O.
      The current landscape of coronavirus-host protein-protein interactions.
      )
      UPF1Regulator of nonsense transcripts 1NBinding by N represses NMD?CP, NU(
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ,
      • Perrin-Cocon L.
      • Diaz O.
      • Jacquemin C.
      • Barthel V.
      • Ogire E.
      • Ramière C.
      • André P.
      • Lotteau V.
      • Vidalain P.O.
      The current landscape of coronavirus-host protein-protein interactions.
      )
      EIF4HEukaryotic translation initiation factor 4HNsp9Cap-dependent mRNA translationPN(
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      )
      EIF4E2Eukaryotic translation initiation factor 4E type 2Nsp2Represses cap-dependent translationCP(
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ,
      • Perrin-Cocon L.
      • Diaz O.
      • Jacquemin C.
      • Barthel V.
      • Ogire E.
      • Ramière C.
      • André P.
      • Lotteau V.
      • Vidalain P.O.
      The current landscape of coronavirus-host protein-protein interactions.
      )
      Sec61 complexSEC61 channel-forming translocon complexNsp8Protein entry into endoplasmic reticulumCP, ERM(
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      BRD4Bromodomain-containing protein 4E+Interference with antiviral response?NU(
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      Protein processingFURINFurinSCleaves S (“priming”), especially at S1/S2 siteGolgi
      Protein degradationCUL2Cullin 2Orf10+Increase degradation of restriction factors?CP, NU(
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      Cell signalingCDKCyclin-dependent kinaseCell cycle arrest, S/G2NU, MT, CP(
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      • Memon D.
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      • Rezelj V.V.
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      MAPKMitogen-activated protein kinase++Viral replication+, stress responseNU, CP, MT(
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      AKTRAC-alpha serine/threonine-protein kinase++−/+Viral replication+, cell proliferation & apoptosis regulationNU, CP (PM)(
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      • Gupta S.
      • Svensson Akusjärvi S.
      • Ambikan A.T.
      • Mikaeloff F.
      • Saccon E.
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      Cell structurePHB complexProhibitin complexnsp2Signaling interference, mitochondrial antiviral signaling, apoptosis−MT, NU, CP, PM(
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      • Jang G.M.
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      • Obernier K.
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      CK2 complexCasein kinase IIN+Cytoskeleton changes, filopodia+CP, NU(
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      Stress, immunityHSPA5Endoplasmic reticulum chaperone BiPUnfolded protein response, virus receptor?CP, PM(
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      NKRFNF-kappaB–repressing factor(nsp10)IL-8 inductionNO, NU, CP(
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      CFBComplement factor B−/++Alternative complement pathway factorSecreted(
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      • Michalick L.
      • White M.
      • Freiwald A.
      • Textoris-Taube K.
      • Vernardis S.I.
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      CFDComplement factor DActivates complement-dependent killingSecreted(
      • Messner C.B.
      • Demichev V.
      • Wendisch D.
      • Michalick L.
      • White M.
      • Freiwald A.
      • Textoris-Taube K.
      • Vernardis S.I.
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      CFIComplement factor I++Prevented from modulating complementSecreted(
      • Messner C.B.
      • Demichev V.
      • Wendisch D.
      • Michalick L.
      • White M.
      • Freiwald A.
      • Textoris-Taube K.
      • Vernardis S.I.
      • Egger A.S.
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      Serum proteomics in COVID-19 patients: Altered coagulation and complement status as a function of IL-6 level.
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      CFHComplement factor H+/−+Prevented from modulating complementSecreted(
      • Messner C.B.
      • Demichev V.
      • Wendisch D.
      • Michalick L.
      • White M.
      • Freiwald A.
      • Textoris-Taube K.
      • Vernardis S.I.
      • Egger A.S.
      • Kreidl M.
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      • Wang H.
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      • Dai J.
      • Duan H.
      • Xu Y.
      • Yu X.
      • Li Y.
      Serum protein profiling reveals a landscape of inflammation and immune signaling in early-stage COVID-19 infection.
      ,
      • D’Alessandro A.
      • Thomas T.
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      • Hill R.
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      • Hudson K.
      • Zimring J.
      • Hod E.
      • Spitalnik S.
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      Serum proteomics in COVID-19 patients: Altered coagulation and complement status as a function of IL-6 level.