High resolution, high mass accuracy mass spectra of hemagglutinin and whole virus digests of influenza are shown to be able to be used to type and subtype the major circulating forms of the virus in humans. Conserved residues and peptide segments of the hemagglutinin antigen have been identified across type A and B strains, and for type B strains of the Yamagata 16/88 and Victoria 2/87 lineages. The theoretical masses for the protonated peptide ions for tryptic peptides of conserved sequence were subsequently shown to be unique in mass when compared to in silico generated peptides from all influenza viral protein sequences and those proteins known to contaminate virus preparations. The approach represents a more rapid and direct approach with which to type and subtype the virus that is of critical need to prepare strategies and treatments in the event of a local epidemic or global pandemic.

The use of high resolution mass spectrometry to record the accurate mass of signature peptides within proteolytic digests of the nucleoprotein antigen, and whole influenza virus, is shown to be able to rapidly type and subtype the virus. Conserved sequences for predicted tryptic peptides were identified through alignments of those for the nucleoprotein across all influenza types and subtypes. Peptides with unique theoretical masses from those generated in silico for all influenza antigen sequences, and from those proteins known to contaminate virus preparations in laboratory grown samples, were identified using a purpose built algorithm (FluGest). The frequency of occurrence of such conserved peptide signatures was assessed across all nucleoprotein sequences to subsequently type and subtype human strains of the virus. The application of the approach is illustrated for both type A H1N1 and H3N2, and type B strains of human influenza virus.

Signature peptides of the neuraminidase antigen across all common circulating human subtypes of type A and B influenza are identified through the bioinformatic alignment of translated gene sequences. The detection of these peptides within the high-resolution mass spectra of whole antigen, virus, and vaccine digests enables the strains to be rapidly and directly typed and subtyped. Importantly, unique signature peptides for pandemic (H1N1) 2009 influenza are identified and detected that enable pandemic strains to be rapidly and directly differentiated from seasonal type A (H1N1) influenza strains. The detection of these peptides can enable the origins of the neuraminidase gene to be monitored in the case of reassorted strains.