High resolution mass spectrometry could provide a new strategy in preparations for influenza outbreaks

Researchers in Australia have shown that exquisitely accurate mass spectrometry can be used to distinguish between different sub-types of the influenza virus - a key issue for health agencies when monitoring outbreaks of the disease and the possible emergence of dangerous new mutants.

Each year influenza causes severe illness in up to five million people, of whom between 250,000 and 500,000 die. To provide protection against the most commonly circulating strains, vaccines - made from inactivated forms of the virus - must be reassessed and reformulated annually.

This requires the strains to be characterised, based on two ’antigenic’ proteins that are found on the surface of the virus, each of which occur in a variety of sub-types. Currently the identity of these proteins is confirmed by a genetic technique called reverse transcriptase polymerase chain reaction, or RT-PCR, where the gene sequences corresponding to the protein sub-types are amplified and analysed. A potential problem with the method is that mutations in parts of the gene sequence targeted by the PCR ’primer’ could prevent the primer from binding to its target.

Now, a group led by Kevin Downard of the University of Sydney has shown that it is possible to directly sub-type the proteins rather than rely on their genetic code by using a technique called Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry.

Here, the whole virus or the relevant antigenic protein is digested by an enzyme into peptide fragments. These fragments are then analysed by FT-ICR mass spectrometry. The technique has such a high resolution that it can unambiguously assign a peptide from mass alone, even when it is part of a highly complex mixture, such as a whole-virus digest. The resulting ’fingerprint’ of peptides allows the virus to be typed and sub-typed, with different forms of the antigen yielding different fingerprints.

’We have developed a very powerful and rapid influenza surveillance method that has significant advantages in terms of speed and sample throughput over traditional screens,’ Downard says.

John McCauley, an expert on the influenza virus from the National Institute for Medical Research in London, says, ’Anything that gives us new or better information on the influenza virus is to be welcomed and mass spectrometry will be used more in the future for influenza virus and vaccine assessment.’ However, McCauley says that current PCR-based methods are generally robust and have the advantage of being able to analyse low-volume samples directly from the clinic without the need for culturing the virus or extracting the proteins. ’This is clearly a useful discovery, but I suspect it will have more downstream applications, for example in the analysis of vaccines, where we do have problems in quantifying the amount of antigen in the preparations.’

Simon Hadlington