Rapid Quantification of Neuraminidase Activity by MALDI-TOF MS via On-Target Labeling of Its Substrate and Product

Abstract

Neuraminidase (NA) is a kind of glycoside hydrolase enzyme, functioning to remove terminal sialic acid (Sia) from glycans which are located on the cell surface. NA plays an essential role in cell interactions with ligands, microbes, and other cells during physiological and pathological processes. Additionally, NA is a major target for developing anti-influenza drugs and influenza vaccines. Herein, a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) based method to quantify NA activity is demonstrated for the first time. A reactive matrix 2-hydrazinoquinoline (2-HQ) is used to on-target label the natural substrate (3-sialyllactose, 3′-SL) and its enzymatic product (Sia). The derivatization enhances the ionization efficiency of 3′-SL and Sia, especially in negative ion detection mode. Moreover, the lactose ion signals and noise are significantly suppressed. Consequently, NA activity in influenza vaccines has been successfully quantified by comparing the relative intensity of 2-HQ derivatized Sia and 3′-SL in the absence of an additional internal standard. Moreover, the inhibition efficiencies of NA inhibitors have also been measured. Due to its operating simplicity, high-throughput capacity, and quantification accuracy, the proposed method has potential to be applied for the detection of different kinds of NA activity to reveal the role of NA in immunity, vaccine, and infection processes.