Rapid Differentiation of Seasonal and Pandemic H1N1 Influenza

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Anal. Chem. 2010, 82, 4584–4590

Rapid Differentiation of Seasonal and Pandemic H1N1 Influenza through Proteotyping of Viral Neuraminidase with Mass Spectrometry Alexander B. Schwahn,† Jason W. H. Wong,‡ and Kevin M. Downard*,† School of Molecular Bioscience, University of Sydney, Sydney, New South Wales, Australia, and Prince of Wales Clinical School and Lowy Cancer Research Centre, University of New South Wales, Sydney, New South Wales, Australia 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. The influenza neuraminidase antigen is a surface glycoprotein of the virus that aids the replication of the virus by catalyzing the hydrolysis of sialic acid residues of the host cell receptors that mediate the release of newly formed virions.1 Structural changes and antigenic variability in the neuraminidase2-4 and other antigens, associated with gene replication errors and gene reassortment of multiple strains in a common host, lead to an ongoing need to reassess both vaccine formulations and antiviral inhibitor medications targeted against the virus.5-7 Structural changes in the active site of the neuraminidase have led to as many as 25% of influenza strains in circulation in Europe being resistant to oseltamivir.8 Antiviral-resistant strains of pandemic (H1N1) influenza strains can develop through mutations within the neuraminidase gene or a reassortment event in which genes are exchanged between seasonal and pandemic (H1N1) viruses. Identifying changes in both the primary structure and antigenicity of the influenza neuraminidase is an essential requirement * To whom correspondence should be addressed. Phone: +61 (0)2 9351 4140. E-mail: [email protected]. † University of Sydney. ‡ University of New South Wales. (1) Air, G. M.; Laver, W. G. Proteins 1989, 6, 341–356. (2) Colman, P. M.; Ward, C. W. Curr. Top. Microbiol. Immunol. 1985, 114, 177–255. (3) Colman, P. M. Immunol. Cell Biol. 1992, 70, 209–14. (4) Laver, W. G. Microbiol. Sci. 1984, 1, 37–43. (5) Colman, P. M. Protein Sci. 1994, 3, 1687–1696. (6) von Itzstein, M.; Wu, W. Y.; Jin, B. Carbohydr. Res. 1994, 259, 301–305. (7) Lew, W.; Chen, X.; Kim, C. U. Curr. Med. Chem. 2000, 7, 663–672. (8) Lackenby, A.; Hungnes, O.; Dudman, S. G.; Meijer, A.; Paget, W. J.; Hay, A. J.; Zambon, M. C. Eurosurveillance 2008, 13, 1–2.

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of modern surveillance approaches and strategies to monitor and arrest the levels of infection within human and animal populations. Nine subtypes of influenza neuraminidase within type A strains of the virus are classified N1-N9 accordingly, where N1 and N2 subtypes are responsible for human infections, epidemics, and pandemics. We have developed a direct and rapid approach with which to type and subtype influenza viruses, and assess their lineages and evolution, through the detection of conserved signature peptides within whole virus digests. This proteotyping approach exploits the high mass accuracy attainable with high-resolution mass spectrometers and has been demonstrated for the hemagglutinin,9 nucleoprotein,10 and matrix M1 protein11 antigens across human and animal hosts in a single antigen, whole virus, and mixed virus digests within vaccine formulations.12 In some instances, the detection of a single signature peptide ion can be sufficient to type and subtype the virus providing a far more rapid approach than conventional reverse transcriptase polymerase chain reaction (RT-PCR) approaches.13 In RT-PCR, the viral RNA must first be isolated. Targeted sequences of the neuraminidase gene are amplified using predesigned primers.14 The amplicons are then detected by fluorescence in conjunction with liquid chromatography, or by electrophoretic approaches, and, as necessary, sequenced employing the chain termination method during amplification. Mutations within the target gene sequence can render the approach ineffective as it can prevent both amplification and also the detection of specific gene sequences employing hybridization arrays.15 We demonstrate here that, in the case of the neuraminidase antigen, the virus can be typed and subtyped within whole virus and vaccine digests. Importantly, we show that common circulating human type A (H1N1) strains of the virus can be easily and quickly distinguished from the 2009 type A (H1N1) pandemic strain of the (9) Schwahn, A. B.; Wong, J. W. H.; Downard, K. M. Anal. Chem. 2009, 81, 3500–3506. (10) Schwahn, A. B.; Wong, J. W. H.; Downard, K. M. Analyst 2009, 134, 2253– 2261. (11) Schwahn, A. B.; Wong, J. W. H.; Downard, K. M. J. Virol. Methods 2010, 165, 178-185. (12) Schwahn, A. B.; Wong, J. W. H.; Downard, K. M. Eur. J. Mass Spectrom., in press. (13) Wright, K. E.; Wilson, G. A.; Novosad, D.; Dimock, C.; Tan, D.; Weber, J. M. J. Clin. Microbiol. 1985, 33, 1180–1184. (14) Fereidouni, S. R.; Starick, E.; Grund, C.; Globig, A.; Mettenleiter, T. C.; Beer, M.; Harder, T. Vet. Microbiol. 2009, 135, 253–260. (15) Gall, A.; Hoffmann, B.; Harder, T.; Grund, C.; Ehricht, R.; Beer, M. J. Clin. Microbiol. 2009, 47, 2985–2988. 10.1021/ac100594j  2010 American Chemical Society Published on Web 05/05/2010

virus16 using the proteotyping method. The approach complements the proteomic-based methods developed in our laboratory to determine the antigenicity of strains of the virus by localizing determinants within viral antigens,17-20 where results were in accord with traditional hemagglutination inhibition assays.21 EXPERIMENTAL SECTION Influenza Virus and Vaccine Strains. Influenza virus strains A/Brisbane/10/07(H3N2) and B/Tokyo/53/99 were purchased from Advanced ImmunoChemicals Inc. (Long Beach, CA). The inactivated viruses, prepared from allantoic fluid of embryonated eggs, were used without further purification. The inactivated influenza split virion vaccines Fluvax, formulated for the 2009 southern hemisphere influenza season, and PanVax H1N1 were purchased from CSL Limited (Parkville, Victoria, Australia). The trivalent Fluvax vaccine contains the following influenza strains: A/Brisbane/59/2007(H1N1), A/Uruguay/716/2007(H3N2), and B/Florida/4/2006, each at a dose corresponding to 30 ng · mL-1 of hemagglutinin. The PanVax H1N1 vaccine contains strain NYMC X-181, a reassortment strain composed of genomic segments coding for the HA, NA, and PB1 antigens derived from the pandemic (H1N1) 2009 strain A/California/7/2009. The remaining segments are derived from strain PR8. The vaccine is formulated at a dose corresponding to 30 ng · mL-1 of hemagglutinin. Neuraminidase Separation and Digestion. The neuraminidase antigen sourced from 20 µg of virus for the strain B/Tokyo/ 53/99 and from ∼35 µg of total viral protein from the Fluvax vaccine or ∼12 µg of total viral protein of the PanVax vaccine, respectively, was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12.5% separation gel) and visualized with a Coomassie brilliant blue stain. Neuraminidase-containing bands were excised and destained (25 mM ammonium bicarbonate in 50% v/v acetonitrile) at 37 °C. Cysteine residues were reduced with dithiothreitol (10 mM in 50 mM ammonium bicarbonate) for 30 min at 56 °C and alkylated with iodoacetamide (55 mM iodoacetamide in 50 mM ammonium bicarbonate) for 20 min at room temperature in the dark. Excess iodoacetamide was removed by washing with acetonitrile (3 × 350 µL), and the gel pieces were dried in a vacuum concentrator. Ingel tryptic digestion of the neuraminidase using 13 ng · µL-1 of modified trypsin (Roche Diagnostics GmbH, Mannheim, Germany) was performed overnight at 37 °C in a digestion buffer containing 25 mM ammonium bicarbonate, 10% acetonitrile, and 3.4 mM octyl-β-D-glucopyranoside. Cleaved peptides were extracted by repeated sonication in 60% acetonitrile containing 0.1% trifluoroacetic acid. Extracted peptides were dried completely in a vacuum concentrator and dissolved in 25 mM ammonium bicarbonate. Tryptic Digestion of Whole Influenza Virus and Vaccine. An amount of 5 µL of a suspension containing 100 ng · µL-1 of the A/Brisbane/10/07(H3N2) virus strain was irradiated in Neumann, G.; Noda, T.; Kawaoka, Y. Nature 2009, 459, 931–939. Kiselar, J. G.; Downard, K. M. Biochemistry 1999, 43, 14185–14191. Morrissey, B.; Downard, K. M. Proteomics 2006, 6, 2034–2041. Downard, K. M.; Morrissey, B. Analyst 2007, 132, 611–614. Morrissey, B.; Streamer, M.; Downard, K. M. J. Virol. Methods 2007, 145, 106–114. (21) Schwahn, A. B.; Downard, K. M. J. Immunochem. Immunoassay 2009, 30, 245–261. (16) (17) (18) (19) (20)

a microwave (Samsung MX245) at 900 W of power for 4 × 20 s. Then 15 µL of a 2.6 mM dithiothreitol solution was added to reduce disulfide bridges. The sample was sonicated for 10 min in a sonicator bath and incubated at 60 °C in an Eppendorf thermomixer for 40 min. The suspension was evaporated to dryness in a vacuum concentrator, and viral protein was reconstituted in 4 µL of digestion buffer (31.3 mM ammonium bicarbonate, 12.5% acetonitrile, 4.3 mM octyl-β-D-glucopyranoside) and sonicated. An amount of 1.0 µL of modified trypsin (65 ng · µL-1; Roche Diagnostics, Mannheim, Germany) was added, and the digest solution was incubated overnight at 37 °C. The digestion mixture was concentrated to dryness, and the tryptic cleavage products were dissolved directly in 3 µL of matrix solution (1.5 mg · mL-1 R-cyano-4-hydroxycinnaminic acid, 6.3 mM ammonium bicarbonate, 45% acetonitrile, 0.075% trifluoroacetic acid), vortexed, and sonicated for 5 min. An amount of 185 µL of the Fluvax vaccine (consisting of ∼50 µg of total viral protein) was diluted with 200 µL of wash buffer (10 mM Tris-Cl pH 8.0, 140 mM NaCl, 0.1% CHAPS) and passed through a Nanosep 300K molecular weight cutoff filter (Pall Corporation, Ann Arbor, MI) at 8000 rpm. Retained virus particles were washed twice with 400 µL of wash buffer and twice with 400 µL of 50 mM ammonium bicarbonate and resuspended in three fractions of 150 µL of 50 mM ammonium bicarbonate. The pooled virus fractions were evaporated to near dryness in a centrifugal concentrator, resuspended in 50 µL of digestion buffer (50 mM ammonium bicarbonate, 10% acetonitrile, 2 mM dithiothreitol), and incubated at 37 °C for 4 h. An amount of 1.5 µL of modified trypsin (1 mg · mL-1; Roche Diagnostics, Mannheim, Germany) was added, and the digestion was carried out overnight at 37 °C. The digestion buffer was evaporated, and the tryptic cleavage products were dissolved in 25 µL of 25 mM ammonium bicarbonate. An amount of 60 µL of the Panvax vaccine (consisting of ∼5 µg of total viral protein) was passed through a Nanosep 300K molecular weight cutoff filter (Pall Corporation, Ann Arbor, MI) at 8000 rpm. Retained virus particles were washed once with 300 µL of wash buffer and twice with 300 µL of 50 mM ammonium bicarbonate and resuspended in two fractions of 75 µL of 50 mM ammonium bicarbonate. The pooled virus fractions were evaporated to near dryness in a centrifugal concentrator and resuspended in 5 µL of 50 mM ammonium bicarbonate. The virus suspension was sonicated for 10 min and irradiated in a microwave (Samsung, model MX245) at 900 W power for 2 × 20 s. For predigestion reduction of cysteine residues 15 µL of 2.6 mM dithiotheitol was added, and the suspension was incubated at 60 °C in an Eppendorf thermomixer for 30 min. Viral proteins were concentrated to dryness and resuspended in 5.5 µL of digestion buffer (31.3 mM ammonium bicarbonate, 12.5% acetonitrile, 4.3 mM octyl-β-Dglucopyranoside) and sonicated for 5 min. An amount of 2.0 µL of modified trypsin (75 ng · µL-1; Roche Diagnostics, Mannheim, Germany) was added, and the digest solution was incubated overnight at 37 °C. The digestion mixture was concentrated to dryness, and tryptic cleavage products were dissolved in 20 µL of 10 mM dithiothreitol in 50 mM ammonium bicarbonate for the postdigestion reduction of Analytical Chemistry, Vol. 82, No. 11, June 1, 2010

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Table 1. Signature Peptides of Neuraminidase Derived from Type B Human Influenza Virus

a

monoisotopic mass of [M + H]+ ions

residues

sequence

PO,modal

uniqueness of mass (in ppm)

466.23297 611.29364 646.40350a 795.34875 798.36279 800.41620 940.55743 1260.64294 1672.74915 2124.00464

368-371 364-367 300-304 286-292 293-299 350-356 108-116 305-315 420-435 129-147

TMSK WYSR RPFVK TIECACR DNSYTAK GGFVHQR GNSAPLIIR LNVETDTAEIR CDVPCIGIEMVHDGGK HFALTHYAAQPGGYYNGTR

0.9830 1.0000 0.9962 0.9887 0.9680 0.9962 0.9890 0.9679 0.9715 0.9759

>50 16.19 15.31 22.33 28.38 -1.67 11.95 8.91 15.31 1.59

Also common to type A N1 neuraminidase.

cysteine-bridged peptides (60 °C, 30 min). The reduction buffer was evaporated, and peptides were dissolved in 20 µL of alkylation buffer (20 mM iodoacetamide in 50 mM ammonium bicarbonate). Cysteine residues were alkylated for 30 min in the dark. The alkylation buffer was evaporated, and peptides were dissolved in 5 µL of 25 mM ammonium bicarbonate. Matrix-Assisted Laser Desorption Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. An amount of 1 µL of solution of the gel-separated neuraminidase, the whole virus or whole vaccine tryptic digestion, was diluted with 3 µL of matrix solution (2 mg · mL-1 R-cyano-4-hydroxycinnaminic acid, 50% acetonitrile, 0.1% TFA). An amount of 0.75-2 µL of the analyte and matrix solution was spotted onto a MALDI target (MTP AnchorChip 400/384 TF, Bruker Daltonics, Billerica, MA) and dried by evaporation in air. Matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance (MALDI FTICR) mass spectra were recorded on a 7T Bruker APEX-Qe instrument (Bruker Daltonics, Billerica, MA) in the positive ion mode. Ions produced from 25-200 laser shots (at 5% laser power) from the MALDI plate, held at 400 V, were accumulated and then transferred to the FTICR cell. Ions were accumulated above the plate in the ion source for 0.2 s, stored in the hexapole for 1.0 s, and then passed through the transfer optics in 1.0 ms using a side kick voltage of 0 V and an offset of -1.5 V. A total of 15-32 scans were acquired and averaged into a single mass spectrum. Spectra were acquired with 1 M data points using a broad-band excitation across a mass range of m/z 404-4000. A mass resolution of over 100 000 (fwhm) at m/z 1296 was typically achieved. The instrument was mass calibrated with an external mixture of peptides comprising angiotensin I, adrenocorticotropic hormone (ACTH) fragments comprising residues 1-17, 7-38, and 18-39, and a synthetic hemagglutinin antigen-derived peptide. Mass spectra were processed using the Data Analysis v3.4 software (Billerica, MA) and internally mass calibrated using identified peptide ions in each spectrum derived from the viral proteins or tryptic autolysis products. Mass accuracies of between 0.1 and 1 ppm were routinely achieved for all ions detected. This mass accuracy enables peptides to be confidently assigned without need for tandem mass spectra to sequence peptides or generate mass tags that may be required in the case of organisms with a more complex proteome. 4586

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Alignment of Translated Neuraminidase Sequences. Translated protein sequences for gene sequences of neuraminidase derived from influenza type A and B viruses were obtained from the NCBI Influenza Virus Resource22 using nonredundant data derived from the NIAID Influenza Genome Sequencing Project and GenBank.23 Multiple sequence alignments for specified subsets for different neuraminidase types (A and B) and subtypes (H1N1, pandemic 2009 H1N1, H3N2, H5N1, HxN1, HxN2) were generated using the ClustalW algorithm.24 Identification of Signature Peptides Using the FluAlign Algorithm. The sequence alignments produced by the ClustalW algorithm were further analyzed with the aid of a purpose built algorithm (FluAlign v2.5.2) written in this laboratory. The algorithm calculates the theoretical monoisotopic mass for all protonated peptide ions [M + H]+ from the in silico digest of all antigen sequences used in the alignment to determine the frequency of occurrence PO for each predicted peptide. In addition, the algorithm calculates the statistical frequency of occurrence for each consensus peptide (the peptide that resembles the consensus sequence calculated from the multiple sequence alignments) as previously described.9 The modal peptide, that is the peptide with the highest frequency of occurrence PO,modal, created at common cleavage sites, was used for prediction of signature peptides together with the corresponding consensus peptide and its frequency of occurrence PO,cons. Peptides yielding a PO value above a given threshold (PO > 0.90) were further assessed as candidates for signature peptides. In the majority of cases, the modal peptide was identical to the consensus peptide with similar PO values. The modal peptide and its frequency of occurrence were used in cases were sequence differences existed. Establishment of the Uniqueness of Signature Peptide Masses Using the FluGest Algorithm. The candidate signature peptide masses for a particular type and subtype of the virus were compared against the masses of all possible tryptic fragments derived from the in silico digest of all nonredundant influenza antigen sequences from all strains and hosts, and several proteins known to contaminate egg-grown virus preparations in addition those from human keratins and the autolysis of trypsin and human (22) Bao, Y.; Bolotov, P.; Dernovoy, D.; Kiryutin, B.; Zaslavsky, L.; Tatusova, T.; Ostell, J.; Lipman, D. J. Virol. 2008, 28, 596–601. (23) Benson, D. A.; Karsch-Mizrachi, I.; Lipman, D. J.; Ostell, J.; Sayers, E. W. Nucleic Acids Res. 2008, 36, D25–D30. (24) Larkin, M. A.; Blackshields, G.; Brown, N. P.; Chenna, R.; McGettigan, P. A.; McWilliam, H.; Valentin, F.; Wallace, I. M.; Wilm, A.; Lopez, R.; Thompson, J. D.; Gibson, T. J.; Higgins, D. G. Bioinformatics 2007, 23, 2947–2948.

Figure 1. High-resolution MALDI mass spectrum of the tryptic digest of the neuraminidase antigen of the B/Toyko/53/1999 strain.

Table 2. Signature Peptides of N1 Neuraminidase Derived from Type A H1N1, H5N1, and 2009 Pandemic H1N1 Human Influenza Virus

* Detected as ions with cysteine residues carbamidomethylated at theoretical monoisotopic masses of 1534.67771 (uniqueness +9.52 ppm), 1494.67516 (uniqueness +14.45 ppm), and 1907.89900 (uniqueness +2.01 ppm), respectively.

keratin, using the FluGest v1.2.2 computer algorithm.9-11 The mass difference to the next closest tryptic peptide (in ppm) derived from any influenza antigen with 10 or more entries, or that from a possible contaminant, was used to establish the uniqueness of the signature peptide masses.

RESULTS AND DISCUSSION The alignment of translated neuraminidase antigen (NA) sequences across all human influenza type B strains of the virus has allowed 10 conserved signature peptides to be identified whose masses are unique within a specified mass deviation. These Analytical Chemistry, Vol. 82, No. 11, June 1, 2010

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Figure 2. High-resolution MALDI mass spectrum of the whole virus digest of the Fluvax 2009 vaccine targeted against seasonal influenza. Greek letters designate ion peaks associated in an isotopic cluster.

values are shown in Table 1 together with the peptide sequences and their PO,modal values. Only peptides whose PO,modal values exceed 0.90 are shown. The spectrum of the gel-separated neuraminidase antigen of the B/Tokyo/53/1999 strain after tryptic digestion is shown in Figure 1. Consistent with the data derived in Table 1, three signature peptides at m/z 800.41656, 940.55739, and 1260.642992 are detected in addition to two signature peptides associated with type B nucleoprotein previously identified10 at m/z 1418.79947 and 1610.874907 due to the co-migration of the two proteins in the gel. The NA-derived peptide ions alone are sufficient to confidently type the strain as a type B virus. Signature peptides derived from the neuraminidase of type A influenza derived from all human H1N1, H5N1, and (H1N1) pandemic 2009 (denoted P2009) strains are shown, respectively, in Table 2. It is evident from the data that relatively few ions are unique to neuraminidase N1 (shaded) and that the peptide ions at m/z 805.45667 associated with residues 112-118 of the N1 antigen are common across all subtypes. The pandemic 2009 strain contains three ions unique to this strain, at m/z values of 549.29907, 1380.62866, and 2543.12964, that if detected enable this strain to be unambiguously differentiated from all other H1N1 and H5N1 strains. Figure 2 shows the products derived from a tryptic digest of the Fluvax 2009 vaccine following reduction and alkylation of cysteine residues with iodoacetamide. This split virion vaccine formulated for the southern hemisphere contains equal quantities (based on hemagglutinin antigen levels) of the influenza strains A/Brisbane/59/2007 (H1N1), A/Uruguay/ 716/2007 (H3N2), and B/Florida/4/2006. The presence of the 4588

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latter type B strain is indicated by the detection of a type B NA signature peptide ions at m/z 800.41634 and 1260.64471 (Table 1) together with a further two NA-derived peptide ions of unique mass whose PO,modal values are below 0.90 at m/z 1426.57751 (PO,modal ) 0.71) and 1770.97029 (PO,modal ) 0.66). Four type A neuraminidase N2 signature peptides comprising residues 421-428, 108-118, and 351-364 are detected at m/z 1099.56120 (carbamidomethyl-cysteine derivative), 1174.62127, 1625.68063, and 1721.73816 (biscarbamidomethyl-cysteine derivative) (Table 3). Two additional N2specific signature peptides with PO,modal values below 0.90 are detected at m/z 1325.75350 (PO,modal ) 0.49) and 1769.96331 (PO,modal ) 0.78). N1 signature peptides (Table 2) corresponding to residues 112-124 and 131-143 are detected at m/z 1467.78149 and 1534.67771, respectively, the latter representing the biscarbamidomethyl-cysteine alkylated form of the peptide. The detection of the isoform-specific NA-derived signature peptides confirms the presence of the three different virus strains in the vaccine. Further evidence is given by the detection of type A and B signature peptides of other antigens. These include a type B signature peptide derived from the matrix M1 protein11 at m/z 1441.80159, due to the high copies numbers of this antigen per virion, and those specific to nucleoprotein antigen for type B virus including peptides at m/z 996.55178 and 1610.85565.10 M1 signature peptides are also detected at m/z 1125.69929 and 1635.91115 associated with type A strains within the formulation. Despite the complexity of the sample in which peptides derived from multiple antigens of all three strains are present, type A and type B neuraminidase signature peptides, including

Table 3. Signature Peptides of the N2 Neuraminidase Derived from All HxN2 and H3N2-Only Strains of Human Influenza monoisotopic mass of [M + H]+ ions

residues

sequence

all HxN2 PO,modal

H3N2-only PO,modal

uniqueness of mass (in ppm)

562.26196 589.30139 591.32489 592.28717 948.54004 1042.53894a 1174.62146 1607.69409a 1625.68018

293-296 77-80 284-288 416-420 254-261 421-428 108-118 173-177 351-364

DNWK EICPK YPGVR SCINR ILFIEEGK CFYVELIR LSAGGDIWVTR QVCIAWSSSSCHDGK GWAFDDGNDVWMGR

0.9913 0.9156 0.9366 0.9792 0.9517 0.9945 0.9851 0.9606 0.9681

0.9904 0.9461 0.9746 0.9832 0.9778 0.9940 0.9870 0.9865 0.9750

25.98 6.51 5.71 11.02 -14.67 -7.09 2.87 >50 -18.23

subtype N2 N2 N2 N2 N2 N2 N2 N2 N2

and N3, N6, N8 only only only only only only only only

a Detected with cysteine residues carbamidomethylated of theoretical monoisotopic masses 1099.56048 (uniqueness -6.73 ppm) and 1724.73702 (uniqueness +37.57 ppm).

Figure 3. High-resolution MALDI mass spectrum of the whole virus digest of the Panvax 2009 vaccine targeted against 2009 pandemic (H1N1) influenza virus (responsible for so-called “swine flu”). Greek letters designate ion peaks associated in an isotopic cluster.

those that signify the presence of N2 neuraminidase (see discussion below), are easily resolved and their masses measured. The total sequence coverage for the neuraminidase antigen across all three strains is 15.9% for type A N1, 37.7% for type A N2, and 19.0% for type B neuraminidase. The combined data across all antigens allows viral strains to be typed and subtyped with even greater confidence. Figure 3 exhibits the MALDI mass spectrum derived from a whole virus digest of the Panvax 2009 vaccine targeted to 2009 pandemic type A (H1N1) strains. H1N1 virus unique to this vaccine is easily distinguished from seasonal type A (H1N1) strains. Peptides ions at m/z 1494.67023 are associated with residues 119-130 of the N1 neuraminidase antigen (Table 2) in which both cysteine residues are carbamidomethylated. This peptide is unique to pandemic (H1N1) 2009 strains

(PO,modal ) 0.97) and not detected in seasonal H1N1 influenza (PO,modal ) 0.00). A further peptide with ions at m/z 1907.89915 is also highly conserved in pandemic H1N1 influenza (PO,modal ) 0.99) associated with residues 157-173 of the N1 neuraminidase antigen with a carbamidomethylated cysteine at position 161. The spectrum also exhibits a signature peptide of the H1 hemagglutinin antigen at m/z 1268.61171 as well as a highly correlated (PO,modal ) 0.98, but nonzero in other H1N1 strains) H1 peptide at m/z 1416.78491.9 Signature peptides for N2 neuraminidase for all type A HxN2 and H3N2-only strains isolated from a human host are shown in Table 3. All peptides are unique to the N2 antigen with the exception of the peptide ions at m/z 562.26196 which share a Analytical Chemistry, Vol. 82, No. 11, June 1, 2010

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Figure 4. High-resolution MALDI mass spectrum of the tryptic digest of the neuraminidase antigen of the A/Brisbane/10/2007 (H3N2) strain. Greek letters designate ion peaks associated in an isotopic cluster.

common mass to peptides derived from the N3, N6, and N8 antigens where the mass error exceeds some 25 ppm. The MALDI mass spectrum of the whole virus digest of the A/Brisbane/10/2007 (H3N2) strain is shown in Figure 4. N2 signature peptides are detected at m/z 1174.61687 and 1625.68396 in addition to ions at m/z 880.43294 associated with previously reported signature peptide of type A H3 hemagglutinin that enables the strain to be both typed and subtyped from the simple detection of all three ions. The same two N2 signature peptides also appear in spectrum of the Fluvax 2009 vaccine virus digest (Figure 2) at m/z 1174.62127 and 1625.68396, associated with residues 108-118 and 351-364 of the N2 antigen of the A/Uruguay/716/2007 (H3N2) strain. CONCLUSIONS Signature peptides derived from translated gene sequences of the influenza neuraminidase antigen across all common circulating human subtypes of type A and B influenza have been identified. These enable human strains of the virus to be typed

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and subtyped directly upon their detection within the highresolution mass spectra of whole antigen, virus, and vaccine digests. Unique signature peptides for 2009 pandemic (H1N1) virus enable pandemic strains to be differentiated from seasonal type A (H1N1) influenza virus. The ability to directly and expeditiously distinguish viral strains, employing the proteotyping surveillance approach described, should greatly aid in the response to this rapidly evolving virus. ACKNOWLEDGMENT A. B. Schwahn was supported by an Australian Research Council Discovery Project Grant (DP0770619). The FTICR mass spectrometer used in these investigations was purchased with funds provided by an Australian Research Council Discovery LinkageInfrastructureEquipmentFacility(LIEF)Grant(LE0668439) and the University of Sydney. Received for review March 4, 2010. Accepted April 20, 2010. AC100594J