Mass Spectrometric Characterization of Proteins from the SARS Virus

Abstract

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