title: Quantification of Influenza Neuraminidase Activity by Ultra-High Performance Liquid Chromatography and Isotope Dilution Mass Spectrometry.
abstract: Mounting evidence suggests that neuraminidase's functionality extends beyond its classical role in influenza virus infection and that antineuraminidase antibodies offer protective immunity. Therefore, a renewed interest in the development of neuraminidase (NA)-specific methods to characterize the glycoprotein and evaluate potential advantages for NA standardization in influenza vaccines has emerged. NA displays sialidase activity by cleaving off the terminal N-acetylneuraminic acid on α-2,3 or α-2,6 sialic acid containing receptors of host cells. The type and distribution of these sialic acid containing receptors is considered to be an important factor in transmission efficiency of influenza viruses between and among host species. Changes in hemagglutinin (HA) binding and NA specificity in reassortant viruses may be related to the emergence of new and potentially dangerous strains of influenza. Current methods to investigate neuraminidase activity use small derivatized sugars that are poor models for natural glycoprotein receptors and do not provide information on the linkage specificity. Here, a novel approach for rapid and accurate quantification of influenza neuraminidase activity is achieved utilizing ultra-high performance liquid chromatography (UPLC) and isotope dilution mass spectrometry (IDMS). Direct LC-MS/MS quantification of NA-released sialic acid provides precise measurement of influenza neuraminidase activity over a range of substrates. The method provides exceptional sensitivity and specificity with a limit of detection of 0.38 μM for sialic acid and the capacity to obtain accurate measurements of specific enzyme activity preference toward α-2,3-sialyllactose linkages, α-2,6-sialyllactose linkages, or whole glycosylated proteins such as fetuin.
authors.affiliation: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention , 4770 Buford Highway, Atlanta, Georgia 30341, United States.
authors.forename: Maria I
authors.identifier: 0000-0002-7853-3523
authors.initials: MI
authors.lastname: Solano
authors.affiliation: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention , 4770 Buford Highway, Atlanta, Georgia 30341, United States.
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Woolfitt
authors.affiliation: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention , 4770 Buford Highway, Atlanta, Georgia 30341, United States.
authors.forename: Tracie L
authors.initials: TL
authors.lastname: Williams
authors.affiliation: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention , 4770 Buford Highway, Atlanta, Georgia 30341, United States.
authors.forename: Carrie L
authors.initials: CL
authors.lastname: Pierce
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Infections, Centers for Disease Control and Prevention , Atlanta, Georgia 30329, United States.
authors.forename: Larisa V
authors.initials: LV
authors.lastname: Gubareva
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Infections, Centers for Disease Control and Prevention , Atlanta, Georgia 30329, United States.
authors.forename: Vasiliy
authors.initials: V
authors.lastname: Mishin
authors.affiliation: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention , 4770 Buford Highway, Atlanta, Georgia 30341, United States.
authors.forename: John R
authors.initials: JR
authors.lastname: Barr
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000201:enzymology*; D007252:Influenza Vaccines / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D007785:Lactose / Q000031:analogs & derivatives / Q000032:analysis; D009439:Neuraminidase / Q000378:metabolism*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: C426347:2,6-sialyllactose; D002247:Carbon Isotopes; D007252:Influenza Vaccines; D014764:Viral Proteins; D009439:Neuraminidase; C000615229:Carbon-13; D007785:Lactose