Article pubs.acs.org/ac
Proteotyping of the Parainfluenza Virus with High-Resolution Mass Spectrometry An P. Nguyen and Kevin M. Downard* School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia ABSTRACT: Parainfluenza viruses (PIVs) are one of the most common causes of respiratory tract infections in children and can be life-threatening when the airway becomes obstructed. Infection results in a spectrum of respiratory disease symptoms that makes diagnosis difficult. A new proteotyping approach employing high-resolution mass spectrometry is shown to be able to distinguish common human serotypes of the PIV from the perspective of all surface and internal viral proteins. The detection of signature peptides, conserved in sequence and unique in mass, within the spectra of these protein or whole virus digests enables the parainfluenza virus to be identified and typed and for it to be distinguished from the influenza virus. Given that the approach is more rapid and direct than conventional reverse transcriptase polymerase chain reaction (RT-PCR), and that it can be implemented with high sample throughout at a comparable sensitivity, it affords an effective new means with which to characterize the virus at the molecular level.
A
young children.5 Serological assays have shown that, by the age of 4, 80% of children have antibodies against PIV. This increases to almost 100% by the age of 6. Croup is symptomatic of PIV, and it is estimated that PIV is responsible for 50%−75% of all croup cases6 that often lead to a barking cough and difficulties with breathing.7 PIVs are responsible for more hospitalizations in children under the age of 5 than influenza, and the virus can be life-threatening. However, PIV infection results in a spectrum of upper and lower respiratory disease symptoms that can make its diagnosis difficult. A definitive diagnosis of PIV infection and characterization of the viral strain can only be achieved using laboratory-based methods. Diagnostic approaches used to identify PIV infections include a range of serological methods,5,7 including hemeadsorption inhibition, ELISA8 and complement fixation assays, electron microscopic examinations of nasal secretions or garglings, the use of antisera to screen nasal secretions and cell-cultured viruses by immunofluorescence,9 to molecularbased screens using the reverse transcriptase polymerase chain reaction (RT-PCR).10,11 These represent reliable and sensitive diagnostic methods. However, their success depends on the availability of targeted antibodies in the case of immunoassays and the need for effective primers for RT-PCR. PIVs are prone to unpredictable genetic variation, resulting in antigenic drift that can prevent primers from binding to their target sequences. To perform these methods, many hours to several days may be required. A new molecular-based proteotyping approach12 has recently been developed without these limitations. It has been shown to
lthough both parainfluenza viruses (PIVs) and influenza viruses cause respiratory tract infections that present similar clinical symptoms, the morphologies of the viruses are quite unique. The PIV’s nucleocapsid is much larger than that of influenza. It contains a single-stranded negative sense RNA genome, rather than a segmented one, and it comes from the paramyxoviridae family of viruses.1 The major human viruses of this taxonomic family are measles, mumps, the respiratory syncytial virus (RSV), and parainfluenza virus (PIV). PIV is further categorized into four serotypes (1−4) according to its viral antigenicity. While the common human parainfluenza virus type 1 (hPIV1) and type 2 (hPIV2) serotypes are often associated with croup, human parainfluenza virus type 3 (hPIV3) is second only to the RSV as the most common cause of pneumonia and bronchiolitis in infants and young children. hPIV4 is further subtyped (designated a and b), but both are detected far less often and are associated with upper rather than lower respiratory tract infections. The PIV’s RNA genome contains ∼15 000 nucleotides that encode six functional proteins. The surface hemagglutininneuraminidase (HN) and fusion (F) glycoproteins play a major role in the pathogenesis of the disease.2 Unlike influenza viruses, a single viral protein in PIV has both hemagglutinin and neuraminidase activity.3 The hemagglutinin portion binds to receptors of the host cells and the virus subsequently enters the cell via fusion of the cell membrane.2,3 The most abundant nucleocapsid protein (NP) encapsulates the genomic RNA to form the NP-RNA template.4 The large protein (L), of some 251 kDa, and the phosphoprotein (P) associate with it to form the RNA polymerase complex.1 The matrix (M) protein aids in the assembly and budding of progeny PIVs. Collectively, PIVs account for one of the most common causes of acute lower respiratory tract infections in infants and © 2012 American Chemical Society
Received: October 11, 2012 Accepted: December 12, 2012 Published: December 12, 2012 1097
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Table 1. Signature Peptides of the Hemagglutinin−Neuraminidase (HN) Protein across Types of the Parainfluenza Virus (PIV) Frequency of Occurrence, Po m/z [M+H]+
residues
sequence
hPIV1 (36)
725.2957 940.5938 1925.1066
62−67 378−385 99−116
QDTCMK QVVNVLIR TINIQSSVQSGIPILLNK
0.94 0.92 0.94
0.00 0.00 0.00
0.56 0.56 0.56
0.00 0.00 0.00
0.00 0.00 0.00
35.27 >50 −41.19
24.45 −26.71 −37.10
593.2347 918.5255 980.4830a 1291.7079 2095.0674 3927.8857
1−5 85−92 257−266 554−564 503−520 222−255
MDGDR ESLTSLIR SCSVVATGTR SLNTLQPMLFK VNPTIMYSNTTNIINMLR SYQVLQLGYISLNSDMFPDLNPVVSHTYDINDNR
0.06 0.06 0.06 0.06 0.06 0.06
0.92 0.96 1.00 1.00 0.96 0.96
0.41 0.43 0.45 0.45 0.43 0.43
0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.09 0.00 0.00
>50 −19.36 7.97 -10.80 17.39 >50
>50 −7.08 -14.91 -5.06 −10.36 15.68
548.3555 648.3311 619.2794 1078.4874 1476.6907 2768.5129
19−22 538−543 498−502 528−537 411−422 544−567
IIFR NTGTQK NESNR AAYTSSTCFK SSSWWSASLFYR IYCLIIIEMGSSLLGEFQIIPFLR
0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00
1.00 0.95 1.00 1.00 1.00 1.00
0.00 0.00 0.00 0.00 0.00 0.00
−27.83 −17.28 −17.28 24.53 30.86 −26.83
>50 23.54 11.95 −12.96 16.56 −15.88
720.3345 756.3385 770.5134 1096.4806
213−219 1−5 412−418 403−411
GCQDIGK MEYWK LLLLGNK QNYWGSEGR
0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
0.97 1.00 0.91 1.00
>50 −5.30 14.65 49.00
15.63 −48.94 −32.61 −7.12
a
Sendai (26)
PIV1* (62)
hPIV2 (19)
hPIV3 (36)
FluGest PIV (ppm)
FluGest Flu (ppm)
Denotes that the peptide was detected with the cysteine residue carbamidomethylated.
h. The viral antigens were stained with Coomassie Brilliant Blue and destained according. The protein-containing bands were individually excised and washed to remove excess strain. Viral antigens were then reduced with 10 mM dithiothreitol at 60 °C for 30 min, followed by alkylation with 50 mM iodoacetamide protected from ambient light for 1 h at room temperature. All proteins were digested in-gel with sequence-grade porcine trypsin (Roche Diagnostics GmbH, Mannheim, Germany) at a ratio of 1:20 and incubated at 37 °C. The resultant peptides were extracted and concentrated in a vacuum concentrator and reconstituted in 25 mM ammonium bicarbonate. PEG Precipitation of Cell-Cultured Virus. Human parainfluenza virus (hPIV) was precipitated from cell culture supernatant by the addition of polyethylene glycol (PEG6000, Merck Millipore, Billerica, MA, USA) at a final concentration of 8% and stored overnight at 4 °C. The virus supernatant was centrifuged at 8500RCF for 30 min and the supernatant then was removed. The remaining pellet was washed with 50 mM ammonium bicarbonate and centrifuged at 13000RCF three times for 5 min each time, and the pellet was reconstituted in 25 μL of 50 mM ammonium bicarbonate. Tryptic Digestion of Whole Parainfluenza Virus. A suspension of viruses (∼30 μg of Sendai and PIV3 viruses) was concentrated to near dryness in a vacuum concentrator, resuspended in 25 μL of digestion buffer (50 mM ammonium bicarbonate in 10% acetonitrile containing 2 mM dithiothreitol, pH 7.8) and digested overnight with modified sequence-grade trypsin (Roche Diagnostics, Sydney, Australia) at 37 °C. Matrix-Assisted Laser Desorption Ionization Fourier Transform−Ion Cyclotron Resonance (MALDI FT-ICR) Mass Spectrometry. An aliquot of the digested whole virus (1 μL) or viral protein sample was diluted at a ratio of 1:5 with α-cyano-4-hydroxycinnamic acid (5 mg/mL matrix in 60% acetonitrile containing 0.1% trifluoroacetic acid), spotted onto a
be able to type and subtype influenza viruses through the detection of signature peptides by high-resolution mass spectrometry.12−15 Signature peptides are proteolysis products of proteins that are conserved in sequence, for a particular viral type or subtype, and which are unique in mass, when considered in terms of the masses of all proteolytic peptides across all known viral proteins and potential protein contaminants. Proteotyping has been employed to type and subtype the major human forms of the influenza virus, establish their lineage,16 monitor the evolution of avian H5N1 strains,17 and distinguish seasonal viruses from pandemic viruses.18 In this study, the proteotyping approach is applied for the first time to another respiratory pathogen in the form of strains of the PIV. The analysis and typing of strains of PIV are presented. In addition, the ability to distinguish the virus from influenza is demonstrated, for the first time, and shows that the proteotyping approach can be used to distinguish among different viral pathogens, not only type and subtype strains of a common virus.
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EXPERIMENTAL SECTION Commerically Sourced Virus Strains. Murine parainfluenza virus type 1 (PIV1) (Sendai virus) and human parainfluenza virus type 3 (hPIV3) were purchased from Advanced ImmunoChemicals, Inc. (Long Beach, CA, USA). The PIV1 Sendai virus was grown in the allantoic fluid of embryonated chicken eggs while parainfluenza virus type 3 (PIV3) was grown in MA cells. All viruses were inactivated and used without further purification. The viruses contain ∼3000 (M), 2500−2700 (NP), 1000 (HN), 600−1600 (F), 300 (P), and 40−50 (L) protein molecules per virion.19 SDS-PAGE Separation of Viral Proteins. Viral proteins of PIV1 (Sendai virus) and PIV3 were separated by SDS-PAGE, using 12.5% polyacrylamide gel ran at a constant 120 V for 1.5 1098
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Figure 1. High-resolution MALDI mass spectrum of the tryptic digest of gel-separated hemagglutinin−neuraminidase (HN) protein of a strain of Sendai virus.
each of the PIV serotypes. The alignments for PIV1 were further subdivided according to the nature of the host, with separate alignments performed for type 1 strains originating from human (hPIV) and mouse hosts. Sendai virus (mouse PIV-1) is the closest known homologue of hPIV1.20 Tryptic peptide segments of conserved sequence were identified using the FluAlign algorithm and the uniqueness of their mass established using the FluGest algorithm12 against a library of masses of all peptides derived from the in silico tryptic digest of all viral proteins across all characterized strains of both parainfluenza (PIV) and influenza (flu) viruses. The results are summarized in Tables 1−3. This table contains all signature peptides with a frequency of occurrences Po that exceeds 90% (Po > 0.9) and are unique in mass from those of the theoretical masses for all tryptic peptides derived from all antigens of known sequence of both PIV and influenza. To be a signature peptide, the ΔPo value should exceed 0.9 across types PIV1−PIV3 or among hPIV1 or Sendai virus. Nine HN peptides are signatures of either hPIV (3) or Sendai viruses (6) with ΔPo values of >0.9. All have sequences and masses unique to PIV1 strains and are not detected in any strains of the PIV2 or PIV3 serotype, with the exception of one signature peptide at m/z 1291.7079, which is comprised of HN residues 554−564 detected in only 9% of hPIV3 strains (Table 1). Figure 1 shows the tryptic digest of the hemagglutinin− neuraminidase (HN) antigen derived from the murine PIV strain after its partial separation on a SDS-PAGE gel. The spectrum exhibits 11 HN peptide ions spanning 15% of the protein sequence. Some ions associated with the nucleoprotein are also detected due to the similar molecular weight of these proteins (60−70 kDa, subject to glycosylation levels). A signature peptide is observed at m/z 1037.5041 (theoretical
384 Anchor chip MALDI target plate and dried in air. Viral protein digests were analyzed by high-resolution MALDI-MS on a Bruker FT-ICR APEX-Qe mass spectrometer, as previously described.12−18 The spectra were processed with the Data Analysis software (Bruker Daltonics, Melbourne, Australia). In Silico Identification of Signature Peptides. Translated full-length, nonredundant, gene sequences for each antigen of PIV1, PIV2, and PIV3 were downloaded from the NCBI protein database. PIV1 sequences were further subdivided into human (hPIV1) and Sendai (mouse) virus strains. These sequences were aligned individually using ClustalW2 to generate a consensus sequence. Consensus sequences were digested in silico with trypsin. The FluAlign algorithm12 was used to determine the frequency of occurrence Po of all possible tryptic peptides. Signature peptides were defined as tryptic peptides that had a ΔPo value of ≥0.9 across type PIV1−PIV3 and between hPIV1 and Sendai viruses. The uniqueness of the masses of these peptides was assessed using the FluGest algorithm.12 Peptide masses that differ by greater than 5 ppm from any other peptide derived from the in silico tryptic digest of all available PIV and influenza (flu) viral proteins across 10 or more sequence entries were designated signatures. These mass differences are shown in the last two columns of all tables in this work.
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RESULTS AND DISCUSSION Identification of Subtype-Specific Signature Peptides of Surface Viral Proteins. The translated gene sequences for the surface hemagglutinin−neuraminidase (numbering 117 sequences in total), fusion (89 sequences), and matrix (63 sequences) viral proteins were aligned using ClustalW across 1099
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Table 2. Signature Peptides of the Fusion (F) Protein across Types of the Parainfluenza Virus Frequency of Occurrence, Po hPIV1 (11)
Sendai (51)
PIV1* (62)
hPIV2 (15)
hPIV3 (12)
FluGest PIV (ppm)
FluGest Flu (ppm)
m/z [M+H]+
residues
672.4290
146−151
DIALIK
1.00
0.98
0.98
0.00
0.00
−13.58
>50
1088.5120 1240.7511 1293.7524 1750.8721 1883.8593 1941.8726 3072.9302
542−551 83−93 290−301 41−56 339−354 466−482 486−514
NNHGNIYGIS LLTPLIENLSK VIDLIAISANHK SLMYYTDGGASFIVVK YNEGSPIPESQYQCLR SAEDWIADSNFFANQAR TLYSLSAIALILSVITLVVVGLLIAYIIK
0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00
1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00
>50 >50 −19.38 >50 >50 23.84 >50
9.88 −19.18 −7.59 −6.04 −5.61 5.10 >50
438.2347 603.3249 611.3875 712.4716 807.3730 884.5200 952.4734 1020.5836 1059.6813 1436.6322 1614.8348 1619.8203 1727.0061 2548.1741
70−72 472−475 411−415 282−287 465−471 182−189 174−181 228−236 48−56 57−69 307−319 251−263 157−173 376−400
QYK EWIR IITHK LPLLTR SDLEESK EIVPSIAR SVQDYVNK LQGIASLYR YLILSLIPK IEDSNSCGDQQIK EWYIPLPSHIMTK YDIYDLLFTESIK AVQSVQSSIGNLIVAIK YAFVNGGVVANCITTTCTCNGIGNR
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
25.63 35.07 >50 >50 6.65 >50 −11.82 −10.98 −16.96 >50 33.14 35.93 >50 41.19
25.63 −6.62 >50 >50 10.82 >50 −10.40 −13.60 −27.56 10.62 −7.35 18.32 −16.75 −5.71
sequence
Table 3. Signature Peptides of the Matrix (M) Protein across Types of the Parainfluenza Virus Frequency of Occurrence, Po hPIV1 (8)
Sendai (39)
PIV1* (47)
hPIV2 (5)
hPIV3 (11)
FluGest PIV (ppm)
FluGest Flu (ppm)
HGVR TGPDK AIPHIR YYPNVVAK VALAPQCLPVDK
1.00 1.00 1.00 1.00 1.00
1.00 0.95 0.92 0.97 0.97
1.00 0.96 0.94 0.98 0.98
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
>50 21.71 −15.88 >50 −22.78
−47.96 21.71 −37.47 −11.06 −13.83
361−365 347−350 209−212 137−142 281−287 313−325 181−192 197−208 260−280
FSPFK IAIR LLCK EHALCK SLGHIPK SLWSVGCEIESAK NLNYQVARPILK FVYSIHLELILR CMSMQLQVSIADLWGPTIIIK
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
>50 >50 −15.49 −20.86 −48.44 5.18 −18.54 −21.05 −6.27
−47.38 >50 −15.49 >50 −14.96 −13.17 −17.60 −26.86 −5.62
351−353 44−49 35−40 113−118 212−219 148−155 287−295 249−256 156−167 99−112 297−322
QWN IGNPPK AVPHIR LDIEVR TGVQTDSK GMLFDANK TLASQLVFK MYSVEYCK VALAPQCLPLDR YTGNDQELLQAATK EICYPLMDLNPHLNLVIWASSVEITR
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.91 0.91 1.00 1.00 0.91 1.00 0.91 1.00 1.00 0.91 1.00
>50 >50 −47.69 15.15 >50 25.10 −24.98 41.83 −35.70 −34.32 >50
47.22 18.00 −11.36 15.15 −7.81 23.61 −21.62 −20.81 −28.06 5.64 −10.77
m/z [M+H]+
residues
468.2677 517.2617 706.4359 953.5091 1253.6923
46−49 25−29 31−36 333−340 152−163
625.3344 472.3242 476.2901 700.3447 751.4461 1408.6776 1428.8322 1502.8729 2347.2222 447.1987 625.3668 692.4202 744.4250 835.4156 895.4342 1006.5931 1022.4321 1295.7140 1551.7649 3026.5479
sequence
1100
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Table 4. Signature Peptides of the Nucleoprotein (NP) Protein across Types of the Parainfluenza Virus Frequency of Occurrence, Po hPIV1 (14)
Sendai (12)
PIV1 (26)
hPIV2 (5)
hPIV3 (12)
FluGest PIV (ppm)
FluGest Flu (ppm)
m/z [M+H]+
residue
647.3359 842.3348
201−206 124−129
QDGTVK DMEYER
1.00 1.00
1.00 0.96
1.00 0.97
0.00 0.00
0.08 0.00
−14.16 −27.89
7.35 20.08
2208.1362a
225−244
SQQSLVSLMVETLVTMNTAR
0.08
1.00
0.67
0.00
0.00
46.39
6.53
581.2712 591.3283 743.3471 779.3795 887.4330 913.5465 934.4199 1116.5394 1348.4923 1699.8512 1769.7798 2131.9172
120−124 225−229 348−353 198−203 371−378 400−407 510−516 249−258 444−454 259−273 468−483 492−509
STMSR SMVVR NYAYGR YQQQGR QQGAVDNR LSLSQLPR AMHEQYR YYAMVGDIGK YDNYDSDGEDR YIEHSGMGGFFLTLK GEPGQPNNQTSDNQQR TSGMSSEEFQHSMNQYIR
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
26.24 36.81 >50 >50 >50 −35.39 10.63 44.87 >50 −12.90 24.71 >50
−19.34 −5.74 −5.40 −10.06 −28.16 24.63 7.02 −9.46 16.37 −6.18 6.42 18.62
639.3322 769.4243 888.4534 998.5629 1067.5806 1311.6692 1671.9210 1799.7719 1831.9623 2252.1624 2460.2397 3059.4832 3059.5393
64−68 112−118 469−475 19−29 297−305 262−273 280−294 500−515 48−63 224−243 200−223 317−344 69−97
QHAQR YGGFVVK TEQQNIR SAGGAIIPGQK ALMELYLSK DAGLASFFNTIR MAALTLSTLRPDINR TNQDEIDDLFNAFGSN MTLALLFLSHSLDNEK SQQSLVTLMVETLITMNTSR QDGTVQAGLVLSGDTVDQIGSIMR DPIHGEFAPGNYPAIWSYAMGVAVVQNR AGFLVSLLSMAYANPELYLTTNGSNADVK
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
1.00 0.92 0.92 0.92 0.92 1.00 0.92 0.92 1.00 0.92 0.92 0.92 0.92
>50 −32.72 40.96 −25.19 −34.11 −13.55 3.92 20.21 −9.81 17.61 11.84 18.38 −18.33
4.15 9.35 12.65 −21.17 −6.95 2.56 −5.90 22.09 8.30 6.44 −8.54 −8.64 −26.99
a
sequence
Denotes that the peptide was detected in its mono-oxidized form.
1037.5044) associated with HN residues 257−266 in which the cysteine residue at position 258 is carbamidomethylated. A further signature associated with HN residues 554−564 is detected at m/z 1291.7077, in addition to its mono-oxidized form at m/z 1307.7022. Both are labeled in bold. While these peptides are detected in 45% of all PIV1 strains, they are not detected in any PIV2 strains and are present in 50 >50 >50 >50 17.36 17.02 >50 15.92 >50 38.32 −8.73 −15.24 40.26
>50 −27.24 21.94 >50 11.42 −9.83 −5.25 −15.86 14.02 31.60 −5.74 9.17 5.26
537−541 499−503 84−89 483−487 10−16 492−498 395−404 554−568 416−434
AAYVK DTEPR SGEESR EDEFR EDSEVER NPVYQER QIQESVESFR AVMELVEEDIESLTN EQNSLLMSNLSTLHIITDR
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.96 0.93 0.93 1.00 1.00 0.93 0.96 0.93 1.00
0.70 0.68 0.68 0.73 0.73 0.68 0.70 0.68 0.73
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
>50 >50 45.02 −5.79 >50 43.89 −2.02 −37.91 22.08
−38.35 −10.58 −15.53 >50 >50 >50 >50 >50 >50
724.3876 857.5342 1206.7317 1257.7776 2158.2859 3309.6821
368−373 224−230 58−69 246−257 91−110 190−223
TEFVTK EIIELLK SKPVAAGPVKPR ILATSATIINLK LPINTPIPNPLLPLARPHGK SSSGVIPGVPQSRPQLASSPAHADPAPASAENVK
0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00
1.00 1.00 1.00 1.00 1.00 1.00
0.00 0.00 0.00 0.00 0.00 0.00
11.81 >50 −45.95 >50 >50 38.23
−34.71 −11.53 −9.28 −48.86 −39.13 −8.47
680.2919 685.3012 772.4312 774.3199 850.3537 1364.5747 1558.9050 1685.9796 1907.9783 3550.7395
1−6 185−190 448−453 441−446 355−361 84−96 454−467 366−381 406−423 22−53
MESDAK NSDHGR LIENQR MDESHR DTEESNR QSGSSHECTTEAK EQLSLITSLISNLK AITLLQNLGVIQSTSK IDFLAGLVIGVSMDNDTK STNISSALNIIEFILSTDPQEDLSENDTINTR
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
1.00 1.00 1.00 1.00 1.00 0.93 1.00 1.00 1.00 1.00
42.67 >50 −32.53 >50 >50 43.59 >50 5.18 −9.06 21.63
−26.05 >50 −47.07 32.52 39.86 11.17 −29.67 19.30 −6.49 −7.41
m/z [M+H]+
residues
sequence
440.2365 510.2922 512.2827 582.2882 646.3155 661.3151 765.4141 791.3655 801.3777 1074.4697 1127.6418 1223.5321 2150.0586
146−148 542−545 405−408 361−365 386−390 483−487 447−453 285−290 410−415 136−144 524−533 197−207 464−482
551.3188 617.2889 664.2896 695.2995 863.3741 905.4476 1222.6062 1691.8043 2185.1282a
a
Denotes that the peptide was detected in its mono-oxidized form.
the weight of the larger PIV particle is estimated based on that reported for influenza23 at 10 × 10−16 g, and given that only 1 /100th or less of the loaded sample is consumed during the analysis, approximately 1.2 × 107 copies of the virus are needed to produce the proteotyping data of whole virus digests. This compares favorably with the sample requirements for RT-PCR for this virus.
The whole virus digest of the hPIV3 strain is shown in Figure 4. Although the sample is heavily contaminated with cellular actin, which remains from its production in culture, two NP signature peptides are detected at m/z 1311.6690 and 1671.9201. Both of these signature peptides were also detected in the spectrum of the digest of the partially gel-separated protein (see Figure 2b), as described above. The ability to easily mass resolve signature peptide ions from any contaminants demonstrates the power of the approach. Sensitivity of the Approach versus Real-Time PCR. Recent real-time PCR studies have reported that some 5 × 107 copies of the virus per milliliter were required to be able to detect PIVs via both PCR and fluorescent-antibody assays.21,22 In the proteotyping results described here, 1.2 μg (30/25 μg) total weight of virus was prepared for MALDI MS analysis. If
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CONCLUSIONS A thorough analysis of all known sequences of the viral proteins of parainfluenza viruses (PIVs) has enabled signature peptides to be identified across all human serotypes and Sendai viruses. Their detection in the high-resolution mass spectra of protein (or, more simply, whole virus) digests enables the virus to be identified and typed, and distinguished from influenza that 1103
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Figure 3. High-resolution MALDI mass spectrum of the tryptic whole virus digest of a strain of the Sendai virus.
Figure 4. High-resolution MALDI mass spectrum of the tryptic whole virus digest of a strain of the PIV3 virus.
presents similar clinical symptoms. This is the first time that the proteotyping approach has been shown to be able to distinguish among different biopathogens, which, in the case of PIV and influenza viruses, present similar clinical symptoms. Since proteotyping is more rapid and direct than PCR, and avoids the
need to develop and reassess primers, it provides an effective new approach with which to characterize the virus at a comparable sensitivity. It can be implemented with high sample throughout by means of parallel sample processing and deposition, with the 1104
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acquisition of hundreds of mass spectra within a few minutes from a large-format MALDI sample target.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +61 (0)2 9351 4140. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Vainionpäa,̈ R.; Hyypiä, T. Clin. Microbiol. Rev. 1994, 7, 265−275. (2) Yao, Q.; Hu, X.; Compans, R. W. J. Virol. 1997, 71, 650−656. (3) Lawrence, M. C.; Borg, N. A.; Streltsov, V. A.; Pilling, P. A.; Epa, V. C.; Varghese, J. N.; McKimm-Breschkin, J. L.; Colman, P. M. J. Mol. Biol.2004, 335, 1343−1357. (4) Lyn, D.; Gill, D. S.; Scroggs, R. A.; Portner, A. J. Gen. Virology 1991, 72, 983−987. (5) Wong, D. T.; Welliver, R. C.; Riddlesberger, K. R.; Sun, M. S.; Ogra, P. L. J. Clin. Microbiol. 1982, 16, 164−167. (6) Zoorob, R.; Sidani, M.; Murray, J. Am. Fam. Physician 2011, 83, 1067−1073. (7) Munoz, F. M. Up to Date 2011, F33 (1), 33832. (8) Julkunen, I. J. Med. Virology 1984, 14, 177−187. (9) Downham, M. A. P. S.; McQuillin, J.; Gardner, P. S. Arch. Dis. Childhood 1974, 49, 8. (10) Corne, J. M.; Green, S.; Sanderson, G.; Caul, E. O.; Johnston, S. L. J. Virol. Methods 1999, 82, 9−18. (11) Aguilar, J. C.; Pérez-Breña, M. P.; García, M. L.; Cruz, N.; Erdman, D. D.; Echevarría, J. E. J. Clin. Microbiol. 2000, 38, 1191− 1195. (12) Schwahn, A. B.; Wong, J. W. H.; Downard, K. M. Anal. Chem. 2009, 81, 3500−3506. (13) Schwahn, A. B.; Wong, J. W. H.; Downard, K. M. Analyst 2009, 134, 2253−2261. (14) Schwahn, A. B.; Wong, J. W. H.; Downard, K. M. J. Virol. Methods 2010, 165, 178−185. (15) Schwahn, A. B.; Wong, J. W. H.; Downard, K. M. Eur. J. Mass Spectrom. 2010, 16, 321−329. (16) Schwahn, A. B.; Downard, K. M. J. Virol. Methods 2011, 171, 117−122. (17) Ha, J. W.; Downard, K. M. Analyst 2011, 36, 3259−3267. (18) Schwahn, A. B.; Wong, J. W. H.; Downard, K. M. Anal. Chem. 2010, 82, 4584−4590. (19) Lamb, R. A.; Mahy, B. W.; Choppin, P. W. Virology 1976, 69, 116−131. (20) Sangster, M.; Smith, F. S.; Coleclough, C.; Hurwitz, J. L. Virology 1995, 10, 13−19. (21) Kuypers., J.; Wright, N.; Ferrenberg, J.; Huang, M. L.; Cent, A.; Corey, L.; Morrow, R. J. Clin. Microbiol. 2006, 44, 2382−2388. (22) Campbell, A. P.; Chien, J. W.; Kuypers, J.; Englund, J. A.; Wald, A.; Guthrie, K. A.; Corey, L.; Boeckh, M. J. Infect Dis. 2010, 201, 1404−1413. (23) Vollmer, F.; Arnold, S.; Kengb, D. Proc. Natl. Acad. Sci. USA 2008, 105, 20701−20704.
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NOTE ADDED AFTER ASAP PUBLICATION This paper published ASAP on January 2, 2013. Due to a production error, Table 4 contained erroneous data. The correct version was reposted to the Web on January 15, 2013.
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