Analysis of viral glycoproteins by MALDI-TOF mass spectrometry.
Membrane glycoproteins were shown to be useful biomarkers of enveloped viruses using on-target deglycosylation and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Sindbis virus, the prototype alpha-virus, was used as a model system. The glycoproteins and the capsid protein of the Sindbis virus were successfully detected by MALDI-TOF MS using two solvent systems. One of them is 0.5% n-octyl glucoside/0.5% trifluoroacetic acid. The two components of this solvent acted synergistically on the virus to help release and solubilize the structural proteins. The other is 70% acetonitrile/30% formic acid. This solvent solubilized the integral membrane glycoproteins very effectively even after serious aggregation. On-target deglycosylation was performed to confirm the detection of the glycoprotein peak and to produce protein moieties that can be used as biomarkers. After a simple and fast incubation using peptide-N-glycosidase F on target, sequential mass shifts were observed, which proved that the proteins detected at 51 000 Da have N-linked carbohydrate moieties at two sites. Observation of this mass shift could provide confirmatory evidence for viral identification.
Kim
Y J
YJ
Department of Biochemistry and Chemistry, University of Maryland, College Park 20742, USA.
Freas
A
A
Fenselau
C
C
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003902:Detergents; D005960:Glucosides; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000032:analysis*; D012845:Sindbis Virus / Q000737:chemistry*; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014269:Trifluoroacetic Acid; D014759:Viral Envelope Proteins / Q000032:analysis*
D003902:Detergents; D005960:Glucosides; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; C022012:glycoprotein E1, Sindbis virus; C064950:glycoprotein E2, Sindbis virus; C018619:octyl-beta-D-glucoside; D014269:Trifluoroacetic Acid