Label‐free mass spectrometry‐based quantification of hemagglutinin and neuraminidase in influenza virus preparations and vaccines

Abstract

Please cite this paper as: Getie‐Kebtie et al. (2012) Label‐free mass spectrometry‐based quantification of hemagglutinin and neuraminidase in influenza virus preparations and vaccines. Influenza and Other Respiratory Viruses 7(4), 521–530. Background Influenza vaccination is the primary method for preventing influenza and its severe complications. An accurate rapid method to determine hemagglutinin (HA) concentration would facilitate reference antigen preparation and consequently expedite availability of seasonal as well as pandemic vaccines. Objective The goal of this study was to develop a label‐free mass spectrometry (MS) based method that enables simultaneous identification and quantification of HA, neuraminidase (NA), and other viral proteins and protein contaminations in influenza vaccine or virus preparations. Methods The method presented is based on LC/MSE analysis of vaccine or virus preparations tryptic digests spiked with a known amount of protein standard from which a universal response factor is generated and applied to calculate the concentration of proteins identified in the mixture. Results We show that, with the use of an appropriate internal standard, the label‐free MS‐based protein quantification method is applicable for simultaneous identification and absolute quantification of HA and identification and relative quantification of other influenza proteins as well as protein impurities in influenza vaccines and virus preparations. We show that different subtype recombinant HA is preferred internal standard that provides the most accurate results in absolute quantification of HAs and other influenza proteins. We applied this method to measure the absolute quantity of HA as well as relative quantities of other viral proteins and impurities in preparations of whole virus and monovalent vaccine, providing data to demonstrate strain‐dependent differences in the amount of NA. Conclusion The label‐free MS method presented here is ideally suited for timely preparation of reference material needed for potency testing of seasonal and pandemic vaccines.

Different Propionibacterium acnes Phylotypes Induce Distinct Immune Responses and Express Unique Surface and Secreted Proteomes

“…Samples were centrifuged at 20,000g for 10 minutes twice, with the supernatant retained. Samples were processed in a nano Acquity 2D UHPLC system (Waters, Milford, MA) and analyzed with a Synapt G2 mass spectrometer (Waters, Milford, MA) using MS E , which is accurate for label-free protein quantification (Bond et al, 2013;Getie-Kebtie et al, 2013). The allowed mass range was 50-2,000 Da.…”

The expanding role of mass spectrometry in the field of vaccine development

“…Additionally, a direct Ultra‐Performance Liquid Chromatography (UPLC)‐IDMS method was reported for the rapid and accurate quantification of influenza NA . Label‐free MS‐based methods have also been reported for the simultaneous identification and quantification of HA and NA in influenza vaccine with samples analyzed by LC‐MS E on a Waters Synapt G2 mass spectrometer . Quantification of proteins by label‐free LC‐MS E is a powerful tool, in this method, alternating scans of low collision energy and elevated collision energy during LC‐MS analysis to obtain both protein identity and quantity in a single experiment.…”

Ultrasensitive Fluorogenic Reagents for Neuraminidase Titration

“…Increasing the concentration of 3 to 10 mm significantly sped up the reaction, (Figure S5 C), revealing at otal NA concentration of 253 nm (equivalent to 12.6 mgmL À1 )i nt he vaccine,w hich is consistent with previous reports. [47,48] Titration of the NA content of other commercial vaccines is currently underway. In summary,w eh ave developed aN At itration reagent that can precisely and conveniently measure concentrations of active NA down to sub-nanomolar levels.The utility of this reagent was demonstrated by measuring, for the first time, true kinetic parameters for neuraminidases within virus samples.T he reagent also proved valuable in titrating NA levels within vaccine samples.W ith these convenient, rapid, precise,and ultrasensitive properties,weexpect that reagent 3 will find wide application in influenza science.…”