Mass Spectrometric Characterization of Proteins from the SARS Virus

A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a ∼46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted ∼139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS. A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a ∼46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted ∼139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS. The recent clinical identification of a novel type of atypical pneumonia without a clearly defined etiology, together with epidemiological evidence of high transmissibility, have provoked the World Health Organization to issue a rare travel advisory. The new entity has been called severe acute respiratory syndrome (SARS) 1The abbreviations used are: SARSsevere acute respiratory syndromeNMLNational Microbiology LaboratoryMALDImatrix-assisted laser desorption/ionizationPNGase Fpeptide N-glycosidase FHPLChigh-performance liquid chromatographyMSmass spectrometryMS/MStandem mass spectrometryQqTOFquadrupole/time-of-flightTOFtime-of-flight. ; it apparently began in Guangdong province in China in November of 2002 and has since spread to Hong Kong, Singapore, Vietnam, Canada, the U.S., Taiwan, and several European countries. severe acute respiratory syndrome National Microbiology Laboratory matrix-assisted laser desorption/ionization peptide N-glycosidase F high-performance liquid chromatography mass spectrometry tandem mass spectrometry quadrupole/time-of-flight time-of-flight The outbreak in Canada began in late February 2003 in a traveler returning from Hong Kong whose exposure was to the index case in the Hong Kong epidemic (a physician who had cared for SARS cases in Guangdong province in the People's Republic of China). The Canadian index case died 9 days after the disease onset, and a 43-year-old male relative became ill 2 days after exposure and died of the adult respiratory distress syndrome 15 days after the illness began (1Poutanen S.M. Low D.E. Henry B. Finkelstein S. Rose D. Green K. Tellier R. Draker R. Adachi D. Ayers M. Chan A.K. Skowronski D.M. Salit I. Simor A.E. Slutsky A.S. Doyle P.W. Krajden M. Petric M. Brunham R.C. McGeer A.J. Canada National Microbiology Laboratory the Canadian Severe Acute Respiratory Syndrome Study Team Identification of severe acute respiratory syndrome in Canada.N. Engl. J. Med. 2003; 348: 1995-2005Google Scholar). Subsequently, Canada has faced the largest SARS outbreak outside of Asia, with at least 351 probable or suspected cases and 27 deaths, mostly in the Toronto area (2Booth C.M. Matukas L.M. Tomlinson G.A. Rachlis A.R. Rose D.B. Dwosh H.A. Walmsley S.L. Mazzulli T. Avendano M. Derkach P. Ephtimios I.E. Kitai I. Mederski B.D. Shadowitz S.B. Gold W.L. Hawryluck L.A. Rea E. Chenkin J.S. Cescon D.W. Poutanen S.M. Detsky A.S. Clinical features and short-term outcomes of 144 patients with SARS in the greater Toronto area.J. Am. Med. Assoc. May 6, 2003; 289: 2801-2809Google Scholar, 3Health Canada. Latest Canadian Numbers on SARS.http://www.hc-sc.gc.ca/english/protection/warnings/sars/sars_numbers.htmlGoogle Scholar). Samples from patients with suspected or probable SARS in Canada have been referred to the National Microbiology Laboratory (NML), Health Canada, for laboratory diagnostics. This laboratory, part of the Canadian Science Centre for Human and Animal Health, is Canada's national reference center for infectious diseases and houses the only Class 4 containment facilities in the country. NML has played an active role in an intensive international collaborative effort among 11 laboratories around the world that suggested a distinct coronavirus may be etiologically involved. In particular, the laboratory prepared the nucleotide samples for the first successful effort to determine the genome sequence for the coronavirus (4Marra M.A. Jones S.J. Astell C.R. Holt R.A. Brooks-Wilswon A. Butterfield Y.S. Khattra J. Asano J.K. Barber S.A. Chan S.Y. Cloutier A. Coughlin S.M. Freeman D. Girn N. Griffith O.L. Leach S.R. Mayo M. McDonald H. Montgomery S.B. Pandoh P.K. Petrescu A.S. Robertson A.G. Schein J.E. Siddiqui A. Smailus D.E. Stott J.M. Yang G.S. Plummer F. Andonov A. Artsob H. Bastien N. Bernard K. Booth T.F. Bowness D. Czub M. Drebot M. Fernando L. Flick R. Garbutt M. Gray M. Grolla A. Jones S. Feldmann H. Meyers A. Kabani A. Li Y. Normand S. Stroher U. Tipples G.A. Tyler S. Vogrig R. Ward D. Watson B. Brunham R.C. Krajden M. Petric M. Skowronski D.M. Upton C. Roper R.L. The genome sequence of the SARS-associated coronavirus.Science. May 1, 2003; 300: 1399-1404Google Scholar), a result soon confirmed by several other laboratories (see, for example, Ref. 5Rota P.A. Oberste M.S. Monroe S.S. Nix W.A. Campagnoli R. Icenogle J.P. Penaranda S. Bankamp B. Maher K. Chen M.H. Tong S. Tamin A. Lowe L. Frace M. DeRisi J.L. Chen Q. Wang D. Erdman D.D. Peret T.C. Burns C. Ksiazek T.G. Rollin P.E. Sanchez A. Liffick S. Holloway B. Limor J. McCaustland K. Olsen-Rasmussen M. Fouchier R. Gunther S. Osterhaus A.D. Drosten C. Pallansch M.A. Anderson L.J. Bellini W.J. Characterization of a novel coronavirus associated with severe acute respiratory syndrome.Science. May 1, 2003; 300: 1394-1399Google Scholar). Nevertheless, the genome sequence merely provides a template for the construction of the viral proteins. Thus, an alternative strategy is to examine the proteins themselves, and mass spectrometry has proved to be an efficient tool for this purpose (6Thomas J.J. Bakhtiar R., R. Siuzdak G. Mass spectrometry in viral proteomics.Acc. Chem. Res. 2001; 33: 179-187Google Scholar). The University of Manitoba time-of-flight mass spectrometry laboratory has already been active in characterizing viral proteins (7Seifers D.L. Harvey T.L. Haber S. She Y.-M. Chernushevich I. Ens W. Standing K.G. Natural infection of sorghum by foxtail mosaic disease in Kansas.Plant Dis. 1999; 83: 905-912Google Scholar, 8Seifers D.L. Salomon R. Marie-Jeanne V. Alliot B. Signoret P. Haber S. Loboda A. Ens W. She Y.-M. Standing K.G. Characterization of a novel potyvirus isolated from maize in Israel.Phytopathology. 2000; 90: 505-513Google Scholar, 9She Y.-M Haber S. Seifers D.L. Loboda A. Chernushevich I. Perreault H. Ens W. Standing K.G. Determination of the complete amino acid sequence for the coat protein of brome mosaic virus by time-of-flight mass spectrometry.J. Biol. Chem. 2001; 276: 20039-20047Google Scholar, 10Swanson M.I. She Y.-M Ens W. Brown E.G. Coombs K.M. Mammalian reovirus core protein micro 2 initiates at the first start codon and is acetylated.Rapid Commun. Mass Spectrom. 2002; 16: 2317-2324Google Scholar, 11Mendez, I. I., She, Y. M., Ens, W., and Coombs, K. M. (2003) Digestion pattern of reovirus outer capsid protein σ3 determined by mass spectrometry. Virology, in pressGoogle Scholar), so it was natural for NML to enlist the university laboratory (late in March) as a collaborator in the of the SARS proteins. The first results of this Clinical from the case were for the of and viral (1Poutanen S.M. Low D.E. Henry B. Finkelstein S. Rose D. Green K. Tellier R. Draker R. Adachi D. Ayers M. Chan A.K. Skowronski D.M. Salit I. Simor A.E. Slutsky A.S. Doyle P.W. Krajden M. Petric M. Brunham R.C. McGeer A.J. Canada National Microbiology Laboratory the Canadian Severe Acute Respiratory Syndrome Study Team Identification of severe acute respiratory syndrome in Canada.N. Engl. J. Med. 2003; 348: 1995-2005Google Scholar). and lavage from several of patients were found to be by for and the novel coronavirus (1Poutanen S.M. Low D.E. Henry B. Finkelstein S. Rose D. Green K. Tellier R. Draker R. Adachi D. Ayers M. Chan A.K. Skowronski D.M. Salit I. Simor A.E. Slutsky A.S. Doyle P.W. Krajden M. Petric M. Brunham R.C. McGeer A.J. Canada National Microbiology Laboratory the Canadian Severe Acute Respiratory Syndrome Study Team Identification of severe acute respiratory syndrome in Canada.N. Engl. J. Med. 2003; 348: 1995-2005Google Scholar). of the lavage from the 43-year-old male in cells a on 4 after The second of this viral isolate was used to of the this virus was used to with convalescent samples from SARS The convalescent sera that were found to be for to the virus by in with a ∼46-kDa protein in to the nucleocapsid protein of Scholar). In to this other for digestion, the virus was by of the that 4 with a convalescent from a SARS patient. This was on a in acid and with were from the by one the prominent ∼46-kDa protein and the other a protein with an mass of These were to the university laboratories for with The protein were with one of or from were to the by A. M. M. Mass of proteins from Chem. in or in a A. Chernushevich I. Ens W. Standing K.G. B. M. M. peptide by a of and a quadrupole/time-of-flight mass Commun. Mass Spectrom. Scholar, T. D. A tool for Commun. Mass Spectrom. 2001; Scholar, C. C. of to of peptide Res. 2003; prepared from and other were from The the peptide were and in of in of the was with acid in and on the of a matrix-assisted laser desorption/ionization The was by liquid chromatography and the were on a target for mass were an and were used for the of Samples were a and with a of in The was with acid and by a a target at Y. V. Standing K.G. Ens W. and for Mass on Mass and for Mass Scholar). The of the were the so were was to of the mass protein to a to this effort in the present the only that had were samples from of the protein one from a and one from a The from the was by and used for of the The from the was first by peptide N-glycosidase F to the glycosylation C.M. Plummer of by Scholar), by to in The on the were by mass spectrometry and by tandem mass spectrometry in the mass as by M. Ens W. Standing K.G. A tandem quadrupole/time-of-flight mass with a matrix-assisted laser desorption/ionization and Commun. Mass Spectrom. 2000; Scholar). In this by of the target with from a laser with of from the the a mass and a in the in MS and as as the is the of the of from of the ∼46-kDa protein in that a amino acid to in 2 and in and A of this is in and an of the same is in the most in has to a of the is that peptide to after of the and have a by a or to the protein on the peptide to that it was a novel peptide was in to this samples were in the of a of and as the of or and the from the by A. Chernushevich I. Ens W. Standing K.G. B. M. M. peptide by a of and a quadrupole/time-of-flight mass Commun. Mass Spectrom. Scholar, T. D. A tool for Commun. Mass Spectrom. 2001; Scholar, C. C. of to of peptide Res. 2003; Scholar). In to determine the amino acid sequence of the clearly peptide was in as a by the of the and to in the example, the from the is in the of the for the and clearly The the the on the the the have a A the and the from the sequence is in and for and from of the in a new in the the from of the of the and of the a from and at a of the the the one peptide that A of and for the of the in is in In and most and the for the amino acid to the the high for a in to of and of found in of the in a new The MS and were first to the from of the in and to the of a F. T.L. and A new of protein Res. Scholar, A. Loboda A. A. S. A. Ens P.W. Standing K.G. the of with by time-of-flight mass spectrometry and Chem. 2001; of the protein with was in to for The most was by the In that the results of the in coronavirus nucleocapsid and of to with of the in the that is a coronavirus protein (a in this had a of only and a high of Thus, the ∼46-kDa protein is clearly a coronavirus nucleocapsid is complete the first and found by in a of the that is the other only of the with of the other so the SARS virus is from of the other with the other to in particular, the evidence for the SARS coronavirus and other coronavirus in the had been and most of almost complete sequence on the as in The of together the was were a of in this an had also been on the and and were as soon as was to be at that a nucleotide sequence of infectious prepared by was by a at the Centre in (4Marra M.A. Jones S.J. Astell C.R. Holt R.A. Brooks-Wilswon A. Butterfield Y.S. Khattra J. Asano J.K. Barber S.A. Chan S.Y. Cloutier A. Coughlin S.M. Freeman D. Girn N. Griffith O.L. Leach S.R. Mayo M. McDonald H. Montgomery S.B. Pandoh P.K. Petrescu A.S. Robertson A.G. Schein J.E. Siddiqui A. Smailus D.E. Stott J.M. Yang G.S. Plummer F. Andonov A. Artsob H. Bastien N. Bernard K. Booth T.F. Bowness D. Czub M. Drebot M. Fernando L. Flick R. Garbutt M. Gray M. Grolla A. Jones S. Feldmann H. Meyers A. Kabani A. Li Y. Normand S. Stroher U. Tipples G.A. Tyler S. Vogrig R. Ward D. Watson B. Brunham R.C. Krajden M. Petric M. Skowronski D.M. Upton C. Roper R.L. The genome sequence of the SARS-associated coronavirus.Science. May 1, 2003; 300: 1399-1404Google soon by results from several other laboratories for Ref. 5Rota P.A. Oberste M.S. Monroe S.S. Nix W.A. Campagnoli R. Icenogle J.P. Penaranda S. Bankamp B. Maher K. Chen M.H. Tong S. Tamin A. Lowe L. Frace M. DeRisi J.L. Chen Q. Wang D. Erdman D.D. Peret T.C. Burns C. Ksiazek T.G. Rollin P.E. Sanchez A. Liffick S. Holloway B. Limor J. McCaustland K. Olsen-Rasmussen M. Fouchier R. Gunther S. Osterhaus A.D. Drosten C. Pallansch M.A. Anderson L.J. Bellini W.J. Characterization of a novel coronavirus associated with severe acute respiratory syndrome.Science. May 1, 2003; 300: 1394-1399Google Scholar). soon became to that the open reading frame identified by the as the coronavirus nucleocapsid protein in the amino acid sequence of the ∼46-kDa protein that were as be from the were to the in the in A of results with the predicted sequence is in the mass of the predicted sequence and and The mass also of the and of other as as of the as in The and as a result of R. F. and Scholar), of be predicted by the nucleotide results the predicted sequence a result with the samples from the same infectious at In to the almost defined ∼46-kDa have a protein that as a at an mass of in the the in the were found to to the ∼139-kDa predicted by the nucleotide sequence and of were for confirmed the identification coverage). A of the and the for this protein is in This protein is to spike proteins in other a of glycosylation sites or Thus, to be and to as proteins. the predicted sequence of the spike protein of the SARS coronavirus of of identified as sites by the at may also be has been examined glycosylation in the spike a was with F to the as This to the corresponding of mass of from the from the predicted amino acid This identified from of and the sequence to on the confirmed the predicted sites and that and had glycosylation sites example, distinct for with at and on the that the was on on the that and were in this is in MS and of the peptide this on the and of the We that in were sites that were that of the other sites may also be by found in of spike with after of in a new The of the spike protein without F of four of that were for as in the for the from to the in were several by in were be of a of and with the from the of the was in four and was an and were found to have the other and to the pattern in the coronavirus protein of the coronavirus Med. Biol. Scholar). These one in We that may result from be by the of on the E. J. A. of the of protein by high-performance liquid mass spectrometry and 1999; Scholar, Identification of novel sites of of time-of-flight mass 2003; the results suggest that is an in the present case found in of spike protein without with in a new the mass of the This is in as one or glycosylation and one glycosylation only one to it is that and with with on only one of as by an in In MS of the clearly the of the of the of that is is the tandem mass of with a This of one to of the of the one and of the a of and confirmed the of from of was also on of and with results with the suggested in in this that the was of the of one at a were in were of the of may be as at of the of it has been that has a on at least in the case of laser desorption/ionization mass spectrometry of Spectrom. 1999; Scholar, S.M. Jones in glycosylation of for the and and for glycoprotein 2000; Scholar, J.P. Ens Standing K.G. M. Perreault H. A of glycosylation in and by and matrix-assisted laser desorption/ionization mass 2002; Scholar). the several found in this suggest the of the to in The glycosylation is only and be by and and A also of the E. J. A. of the of protein by high-performance liquid mass spectrometry and 1999; have already that of may be on of from proteins by a of and of amino acid of coronavirus proteins only the coronavirus and from the coronavirus the the of sequence sequence and Res. of the amino acid that the virus is only to of the other The the on this it to the of the SARS recent in the have implicated that used for in particularly the whose genome was in the the of the SARS protein with other S.A. S.M. nucleocapsid protein and sequence of virus 2000; Scholar). The of SARS protein that of and in a of it and The amino acid sequence in this is among for a core for or a among coronavirus proteins. This has been as the of the of SARS protein is that it amino acid sequence in that to be this the of a the The of a of the protein late in infection has been with infectious and coronavirus to in has been that coronavirus for this F. N. D. C. S.J. The viral nucleocapsid protein of coronavirus is by and 2000; Scholar). A is present in of the coronavirus proteins. the of the protein was with a in virus by a of These suggest that of viral nucleocapsid protein by be a by cells is present in the coronavirus The spike protein is a major target of the to and an role in the of the of the virus to the and the of the viral and The of of the proteins has been in in several of and A that of the proteins have on and with F. W. H. J.J. of the glycoprotein of Scholar). The have been to with acid on the and it is that the of A and J.S. S. S. A. P. R. of epidemic and with and 2003; have been to the and on of coat proteins to infectious virus A. R. T. T. that glycosylation is for virus Scholar, A. R.A. The role of in the of Med. Biol. Scholar). and the of an and found that it after of The proteins were to at viral by A. R.A. A strategy for the of to of viral Scholar). in that had a on glycosylation glycosylation to 2001; 83: Scholar), and as this as for of B. virus may also to N. A.S. S. J. R.A. sugars the and of a of for the of virus U. S. A. 1999; Scholar). on has to A. N. R.A. as and the of coronavirus spike proteins in cells and found that had an on virus and example, of spike by the of in the of R. H. The viral spike protein is in the of in Scholar). The same also the of of proteins of R. H. The viral spike protein is in the of in Scholar). We that the SARS-associated coronavirus genome sequence a or from another virus or (4Marra M.A. Jones S.J. Astell C.R. Holt R.A. Brooks-Wilswon A. Butterfield Y.S. Khattra J. Asano J.K. Barber S.A. Chan S.Y. Cloutier A. Coughlin S.M. Freeman D. Girn N. Griffith O.L. Leach S.R. Mayo M. McDonald H. Montgomery S.B. Pandoh P.K. Petrescu A.S. Robertson A.G. Schein J.E. Siddiqui A. Smailus D.E. Stott J.M. Yang G.S. Plummer F. Andonov A. Artsob H. Bastien N. Bernard K. Booth T.F. Bowness D. Czub M. Drebot M. Fernando L. Flick R. Garbutt M. Gray M. Grolla A. Jones S. Feldmann H. Meyers A. Kabani A. Li Y. Normand S. Stroher U. Tipples G.A. Tyler S. Vogrig R. Ward D. Watson B. Brunham R.C. Krajden M. Petric M. Skowronski D.M. Upton C. Roper R.L. The genome sequence of the SARS-associated coronavirus.Science. May 1, 2003; 300: 1399-1404Google Scholar), is that and of be by the the proteins Scholar, Y. W. J. Chan SARS as a of severe acute respiratory 2003; Scholar). has been that acid in We to the of spike after by the acid of a glycoprotein be determined in an isolated The of by MS is (see, for example, Ref. Identification of by mass 2001; Scholar), and to The present the first of the proteins from the novel coronavirus to be the agent of SARS. to the pattern with G. N. A. V. A. C. of to coronavirus infection in by 2002; Scholar), the to be the major as it was the only viral protein by acute and early convalescent sera from several patients from SARS. the to the as an early for it is that an to this protein it is an protein and to target the proteins Scholar). it has been for other that of the protein be on the of cells by cells Scholar). The spike glycoprotein is a so it may an attractive with for new example, of the or of the viral the glycoprotein or the SARS coronavirus also to and the of glycosylation that have on cells be an A. R.A. A strategy for the of to of viral Scholar). an by a for on of the viral called or the of the coronavirus J. R., and R. for of Science Scholar). for of SARS also the of or the spike glycoprotein together with other viral proteins. The of by MS provides an alternative to the for protein has the of the proteins from that or predicted by the nucleotide may also be useful to that of be by protein in the complete of genome the results of the and the that the protein that NML had isolated was a nucleocapsid protein to an were at a of the in this on a the nucleotide sequence became the nucleotide sequence is of the proteins is in that provides particularly on R. F. and Scholar). The in the nucleocapsid protein particularly that the result is useful in as an of the of the glycosylation in the SARS spike first is it is to a role in of the virus to and may have as We to the Canadian SARS Health Canada, as as Canadian Health and for the and We and for and and Loboda for with

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.