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.

NAction! How Can Neuraminidase-Based Immunity Contribute to Better Influenza Virus Vaccines?

“…A chip-based VaxArray ( 87 ) assay using the same principle is under development (personal communication, Kathy Rowlen, InDevR). In addition, high-performance liquid chromatography (HPLC)- and mass spectrometry-based methods have been developed, but they are more burdensome and do not necessarily indicate protein integrity ( 52 , 88 ). Finally, NA activity assays might be of value as well in analyzing the quality of NAs present in vaccines, since only tetrameric NA has strong sialidase activity ( 53 ).…”

Evaluation of defense strategy against Influenza A in cell line models

“…Unlike human HA that preferentially recognises α (2–6), the viral NA of H1N1 has enzymatic activity to both α(2–6) and α(2–3) types of linkages, however the rate of cleavage of α(2–6) linked SA is lower than α(2–3) ( Solano et al ., 2017). …”

Probing Sialidases or Siglecs with Sialic Acid Analogues, Clusters and Precursors

“…Once sialidases broke the glycoside to produce free sialic acid and glucose, the sialidases could be detected based on the glucose concentration, which was easily detected using glucose meter. In addition, ultra‐high performance liquid chromatography (UPLC) and isotope dilution mass spectrometry (IDMS) were also used to rapid quantification of sialidase activity after enzymatic cleavage of sialic acid from α‐2,3‐sialyllactose, α‐2,6‐sialyllactose and other sugar substrates . Because the 13 C‐labeled‐sialic acid internal standard behaved essentially identical to the released sialic acid analyte and could be distinguished by mass, this method gave good reproducibility and sensitivity.…”