Supplement Article

Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2016, 30 (Suppl. 1), 179–184

(wileyonlinelibrary.com) DOI: 10.1002/rcm.7650

Identification of single amino acid substitutions (SAAS) in

neuraminidase from influenza a virus (H1N1) via mass
spectrometry analysis coupled with de novo peptide sequencing
Qisheng Peng1,2, Zijian Wang2, Donglin Wu3, Xiaoou Li4, Xiaofeng Liu4, Wanchun Sun1 and
Ning Liu2*
1
Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun 130062, China
2
Central Laboratory, Second Hospital, Jilin University, Changchun 130041, China
3
Center for Disease Control and Prevention, Changchun 130025 Jilin, China
4
Tumor Hospital of Jilin Province, Changchun 130022, China

RATIONALE: Amino acid substitutions in the neuraminidase of the influenza virus are the main cause of the emergence of
resistance to zanamivir or oseltamivir during seasonal influenza treatment; they are the result of non-synonymous
mutations in the viral genome that can be successfully detected by polymer chain reaction (PCR)-based approaches. There
is always an urgent need to detect variation in amino acid sequences directly at the protein level. Mass spectrometry
coupled with de novo sequencing has been explored as an alternative and straightforward strategy for detecting amino acid
substitutions, as well – this approach is the primary focus of the present study.
METHODS: Influenza virus (A/Puerto Rico/8/1934 H1N1) propagated in embryonated chicken eggs was purified by
ultracentrifugation, followed by PNGase F treatment. The deglycosylated virion was lysed and separated by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel band corresponding to neuraminidase was
picked up and subjected to liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis.
RESULTS: LC–MS/MS analyses, coupled with manual de novo sequencing, allowed the determination of three amino acid
substitutions: R346K, S349 N, and S370I/L, in the neuraminidase from the influenza virus (A/Puerto Rico/8/1934 H1N1),
which were located in three mutated peptides of the neuraminidase: YGNGVWIGK, TKNHSSR, and PNGWTETDI/LK,
respectively.
CONCLUSIONS: We found that the amino acid substitutions in the proteins of RNA viruses (including influenza A virus)
resulting from non-synonymous gene mutations can indeed be directly analyzed via mass spectrometry, and that manual
interpretation of the MS/MS data may be beneficial. Copyright © 2016 John Wiley & Sons, Ltd.

The circulation of the seasonal influenza virus has posed a acid sequences resulting from any non-synonymous SNPs that
threat to human health across the globe for quite some time. have functional consequences. Proteins have recently become
Owing to fast rates of nucleotide mutation and amino acid recognized proteotyping targets, wherein a variety of proteins
substitution in the major strains such as H3N2 and H1N1, there formed from a single gene can be characterized through
is still concern that the variants will become virulent enough to sophisticated mass spectrometric techniques,[3,4] such as the
cause severe mortality in humans,[1] making close monitoring one explored in the present study. Similarly, the detection of
and strong control techniques highly worthwhile – and the amino acid substitutions at the protein level via mass
urgent – endeavors. Well-established polymer chain reaction spectrometry is now considered a more straightforward strategy
(PCR)-based approaches are widely utilized to genotype than PCR. Mass spectrometry, a key tool in the proteomics
influenza viruses by targeting the viruses’ RNA molecules. field,[5,6] has been used to analyze several mutations in
Because the influenza virus mutates very rapidly, the existing hemoglobin variants,[7,8] for example, as well as transthyretin,[9]
PCR-based methods often fail to detect emerging strains due and one of two allelic wheat glutenin subunits.[10] More
to sequence variations in both the primer and probe.[2] importantly, mass spectrometry coupled with de novo peptide
Certain viral genome mutations can result in amino acid sequencing has already shown that there are at least seven
substitutions in the viral proteins, which are non-synonymous amino acid substitutions in the HA of the influenza A virus.[11]
mutations, and thus able to affect protein functions. Because Neuraminidase, which is located on the surface of the
the protein is the major carrier and functional executor of bodily influenza virus, splits the terminal sialic acid residues from
activities, it is an urgent necessity to detect variations in amino glycan structures on the surface of the infected host cells to
enable release of progeny viruses from the host cells.[12] Two
drugs: zanamivir (Relenza) and oseltamivir (Tamiflu), that
* Correspondence to: Ning Liu, Central Laboratory, Second have been clinically applied to treat influenza infection, are
Hospital, Jilin University, Changchun 130041, China. effective inhibitors of neuraminidase, but resistance to both
179

E-mail: [email protected] in treating seasonal influenza has been increasingly reported

Rapid Commun. Mass Spectrom. 2016, 30 (Suppl. 1), 179–184 Copyright © 2016 John Wiley & Sons, Ltd.
 Q. Peng et al.

in recent years, indicating that some influenza strains have polyacrylamide was used to separate the lysed samples in a
evolved to produce amino acid substitutions responsible for Mini-Cell system (Bio-Rad). After electrophoretic separation,
the drug resistance.[13–15] the gels were stained with colloidal Coomassie G250 and
In the present study, we conducted a mass spectrometric scanned with a calibrated densitometer (GS800, Bio-Rad).
analysis of neuraminidase (which was isolated and purified
by sodium dodecyl sulfate polyacrylamide gel electrophoresis In-gel digestion and peptide extraction
(SDS-PAGE)) of the influenza A virus. By manually
interpreting the MS/MS data, we identified three amino acid The protein bands of interest were removed from the gels and
substitutions. The results presented here indicate that mass destained with a solution containing 50 mM NH4HCO3 and
spectrometry coupled with de novo peptide sequencing indeed 50% ACN until the Coomassie blue became invisible. The
reveals the amino acid substitutions in the proteins of RNA destained gel pieces were then dehydrated with 100% ACN,
viruses such as influenza A. reduced in 10 mM DTT/50 mM NH4HCO3 aqueous solution
at 60 °C for 60 min, then alkylated in 50 mM IAA/50 mM
NH4HCO3 aqueous solution at room temperature in the dark
for 30 min. After dehydration with 100% ACN, the gel pieces
EXPERIMENTAL were incubated in freshly prepared digestion buffer (0.1 g/L
TPCK-trypsin and 50 mM NH4HCO3) overnight at 37 °C. The
Chemicals and materials reaction was quenched by the addition of 10% trifluoroacetic acid
Sequencing-grade TPCK-modified trypsin was purchased from (TFA). The tryptic peptides were extracted with extraction buffer
Promega (Madison, WI, USA). PNGase F came from New containing 60% ACN several times as necessary and lyophilized.
England BioLabs (Hitchin, Hertfordshire, UK). Both HPLC-
grade acetonitrile (ACN) and methanol came from Fisher Capillary LC–MS/MS analysis
(Fairlawn, NY, USA). The BCA (bicinchoninic acid) protein
A capillary C18 chromatography column (ChromXP, Eksigent
assay kit came from Pierce (Rockford, IL, USA). Ammonium
Technologies, 150 mm × 75 μm × 3.0 μm) was used for all LC
bicarbonate, dithiothreitol (DTT), and iodoacetamide (IAA)
analyses; the tryptic peptides were dissolved in mobile phase
were purchased from Bio-Rad (Hercules, CA, USA). All other
buffer A (0.1% formic acid) then subjected to capillary
chemicals were purchased from Sigma-Aldrich (St. Louis,
LC–MS/MS analysis. Gradient elution was performed with
MO, USA). Influenza virus (A/Puerto Rico/8/1934 H1N1)
a combination of mobile phase buffer A and mobile phase
was propagated in a biosafety level 2 (BL-2) containment
buffer B (ACN containing 0.1% formic acid) as follows: 0–
facility, and ultra-pure water was prepared with a MilliQ water
60.0 min, 6%–25% B; 60.0–74.0 min, 25%–40% B;
purification system (Millpore, Bedford, MA, USA).
74.0–75.5 min, 40%–90% B; 75.5–84.0 min, 90% B; and
84.0–90.0 min, 5% B. Flow rate was 0.3 mL min 1. We then
Virus propagation and purification analyzed the eluted peptides on an ABI QSTAR spectrometer
Embryonated chicken eggs were inoculated with the influenza in information-dependent acquisition (IDA) mode (Analyst
A virus (A/Puerto Rico/8/1934 H1N1) and incubated at 37 °C QS, Applied Biosystems, Carlsbad, CA, USA). A survey scan
for 72 h; then the virus-containing allantoic fluids in the of 350–1250 Da was first collected for 3 s, followed by 5-s
chicken eggs were harvested and cleared by centrifugation MS/MS scans of 50–1500 Da under rolling collision energy
at 5,000 rpm for 20 min. The collected allantoic fluids were settings. The dynamic exclusion time was set to 1.5 min.
subjected to centrifugation at 40,000 rpm at 4 °C for 1 h, then The raw MS/MS data were converted into MASCOT
the obtained virus pellets were suspended in a buffer generic files with a script embedded in the Analyst QS 2.0
containing 10 mM Tris–HCl (pH 7.4) and 150 mM NaCl, then software (MDS Sciex, South San Francisco, CA, USA), which
loaded onto a 20%–60% (w/w) sucrose gradient cushion and were used to search against the SwissProt protein database
centrifuged in a SW40 Ti rotor (Beckman-Coulter, Fullerton, on a local MASCOT server (version 2.1, Matrix Science,
CA, USA) at 35,000 rpm at 4 °C for 60 min. The virus band London, UK). One missed cleavage was allowed. Cysteine
was carefully collected and suspended in a mixture of carbamidomethylation was specified as a fixed modification,
10 mM Tris–HCl (pH 7.4) and 150 mM NaCl. Aliquots of the whereas methionine oxidation was selected as a variable
purified virus sample were kept at 4 °C until use. modification. The mass tolerance was set to 0.3 and 0.6 Da
for MS and MS/MS ion masses, respectively. We performed
manual de novo sequencing of peptide tandem mass spectra
Virion lysing and PNGase F treatment
with Pepsea (1.1) in the Analyst QS 2.0 software (MDS Sciex).
Prior to PNGase F treatment, the purified influenza virions
were lysed and denatured by boiling at 100 °C for 10 min in PCR-based sequencing of neuraminidase gene
a denaturing buffer containing 0.5% SDS, 40 mM DTT, and
1% NP-40. PNGase F was then added to the reaction mixture. We used a QIAamp MinElute Virus Spin Kit (Qiagen) to extract
Deglycosylation treatment was performed at 37 °C for 5 h, viral RNA from 200 μL of allantoic fluid according to the
followed by heat inactivation at 75 °C for 10 min. manufacturer’s instructions, which was transcribed into cDNA
by superscript III reverse transcriptase (Invitrogen) at 42 °C for
1 h using the Uni12 primer (5′-AGC AAA AGC AGG-3′).[8] The
SDS-PAGE
cDNA was used as a template to amplify the NA gene, of which
The deglycosylated virion lysate preparations were boiled with the sequence was determined by cycle sequencing of amplified
2× Laemmli sample buffer for 5 min. The protein concentration PCR products using an ABI PRISM 3700 DNA Analyzer
180

was assayed with a BCA protein assay kit. SDS-PAGE with 12% (Applied Biosystems, Foster City, CA, USA).

wileyonlinelibrary.com/journal/rcm Copyright © 2016 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2016, 30 (Suppl. 1), 179–184
Mapping single amino acid substitutions in neuraminidase

RESULTS AND DISCUSSION nominal mass shift ( 28 Da) was obtained for the detected
doubly charged ion peak in comparison with the molecular
Mass spectrometric analysis of NA protein from the weight of the theoretical sequence of YGNGVWIGR (338–
influenza A virus (H1N1) 346) in NA, which may have resulted from the amino acid
substitution of one of five residues in the theoretical sequence:
The influenza A virus grown in embryonated chicken eggs served
R346 → Q, V342 → A, and R346 → K. The detection of fragment
as the neuraminidase source; the allantoic fluids were collected
ions at m/z 486.2 and 503.3 eliminated the possibility of amino
from the eggs, from which influenza virions were purified by a
acid substitution of V342 → A, of which the corresponding
series of chromatographic fractionations as discussed above.
fragment ions are supposed to be at m/z 514.2 and 531.3,
Because neuraminidase is glycosylated, the influenza virions
respectively. The detection of both y2 (m/z 204.1) and y3
were treated with PNGase F prior to further analysis. The
(m/z 317.2) ions, as well as some of the a and b series ions
deglycosylated virus virions were then lysed and separated on
(e.g., a2, a3, a4, a5, b2, and b3), confirmed that V342 was not
12% SDS-PAGE. Staining with colloidal Coomassie G250 revealed
subject to amino acid substitution; accordingly, the mass shift
two major bands at 15 and 56 kDa, which respectively refer to
of 28 Da can be derived from the amino acid substitution of
matrix protein 1 (M1) and nucleoprotein (NP) from the virus
either R346 → Q or R346 → K, which we further confirmed after
virion, , as indicated in Fig. 1. Beneath the NP band, there was a
detecting the immonium ion of either K or Q at m/z 129. It is
faint band that was cut off and subjected to in-gel tryptic
worth mentioning that Q is not a typical C-terminal residue of
digestion. Mass spectrometry of the tryptic peptides coupled with
tryptic peptides; our observation of the diagnostic y1 ion of
protein database searching identified a total of 12 unique peptides
m/z 147.1 for K, as well as the absence of the y1 ion of 175.1
from the neuraminidase (A/Puerto Rico/8/1934 H1N1) (Table 1).
for R, in the MS/MS spectrum suggests that the tryptic
We identified three N-linked glycosylation sites in the
peptide very likely has a K residue at the C-terminus. Precise
neuraminidase from the influenza virus (A/Puerto Rico/8/
mass data of the immonium ion were detected at m/z 129.11,
1934 H1N1), wherein the glycosylated asparagine (Asn, N)
which was much closer to the theoretic mass data of the
in each peptide was converted into an aspartic acid residue
immonium ion of K (129.1022) than that of Q (129.0659). To
(Asp, D) upon treatment with PNGase F. In addition, manual
this effect, the site upon amino acid substitution was
interpretation of some of the available MS/MS data that were
confidently determined to be R346 (R → K).
not identified by database searching allowed us to assign three
amino acid (AA) substitutions (R346K, S349 N, and S370I/L)
within the three peptide sequences. Identification of AA substitution S349 N
De novo sequencing of the MS/MS spectrum of a doubly charged
ion peak at m/z 415.21 identified a partial sequence of KNHS
Identification of AA substitution of R346K with y series ions at m/z 728.3, 710.3, 600.3, 582.3, 486.2, 349.2,
Interpretation of the MS/MS spectrum of the doubly charged and 262.1 (Fig. 3), which was not found in the theoretical
ion peak at m/z 497.26 (Fig. 2) allowed us to identify a partial sequence of NA. That said, further investigation of the
sequence of GNGV, considering the ion series of m/z 830.4, theoretical sequence of NA revealed a sequence of KSHS
773.4, 659.3, 602.3 at the high mass end of the spectrum were (348–351), which was identical to the deduced sequence apart
y-type fragment ions. The GNGV sequence was easily located from the S349 residue. This allowed us to identify amino acid
in one of the tryptic peptides of NA: YGNGVWIGR (338–346) substitution of S349 → N that had resulted in a mass shift of
with theoretical m/z value of 511.26 for its doubly charged ion +27.01 Da. The KSHS (348–351) sequence was contained in
(Supplementary Fig. S1, Supporting Information). Thus, a a tryptic peptide of NA, TKSHSSR (347–353), of which the
calculated m/z value of the doubly-charged ion was 401.71
(Supplementary Fig. S1, Supporting Information). Therefore,
a nominal mass shift of +27 Da was observed for the peak at
m/z 415.21 as compared to the theoretical sequence of
TKSHSSR (347–353), which was identical to the mass shift
resulted from amino acid substitution of S349 → N identified
through de novo sequencing. It should be noted that, in
addition to S349, another amino acid residing in the sequence
of TKSHSSR (347–353), T347, results in a mass shift of +27 Da
upon substitution as T347 → K. The detection of both a2 and b2
ions, m/z 202.2 and 230.1, respectively, indicated that the first
two residues in the peptide were TK but not KK, however,
thus eliminating the possibility of substitution of T347 → K.
The peak at m/z 415.21 was accordingly identified as the
tryptic peptide in the residues from 347 to 353 with amino
Figure 1. Identification of neuraminidase from purified acid substitution of S349 → N.
influenza virions (A/Puerto Rico/8/1934 H1N1). The
purified influenza virions were deglycosylated by PNGase F,
and then lysed and separated on 12% SDS-PAGE. The faint Identification of AA substitution S370I/L
band beneath the NP band was cut off and subjected to
in-gel digestion, followed by mass spectrometric analysis. 12 By interpreting the MS/MS spectrum of the doubly charged
peptide sequences from neuraminidase of influenza A virus ion peak at m/z 580.78 (Fig. 4), we readily deduced a partial
181

were identified by database searching. sequence of GWT, considering the ion series of m/z 949.4,

Rapid Commun. Mass Spectrom. 2016, 30 (Suppl. 1), 179–184 Copyright © 2016 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm
 Q. Peng et al.

Table 1. Summary of tryptic peptides identified in neuraminidase from influenza virus (A/Puerto Rico/8/1934 H1N1) by
database searching

Calculated m/z Measured m/z

Peptide No. Peptide sequence Charge status (monoisotopic) (monoisotopic) Residues

P1 N*STWVK 2 368.19 368.19 58–63

P2 HSN*GTVKDR 2 507.75 507.75 129–137
P3 GDVFVIR 2 403.23 403.23 97–103
P4 IGSKGDVFVIR 2 595.85 595.85 93–103
P5 GWAIYSKDNSIR 2 705.36 705.36 81–92
3 470.58 470.58 81–92
P6 GRPKEK 2 357.72 357.72 414–419
P7 YNGIITETIK 2 576.32 576.32 193–202
P8 YNGIITETIKSWR 2 790.93 790.92 193–205
3 527.62 527.62 193–205
P9 TFFLTQGALLNDK 2 734.40 734.40 116–128
3 489.94 489.94 116–128
P10 DTTSVILTGN*SSLC*PIR 2 917.96 917.96 64–80
3 612.31 612.31 64–80
P11 ALM*SC*PVGEAPSPYNSR 2 926.42 926.42 142–158
3 617.95 617.95 142–158
P12 ALMSC*PVGEAPSPYNSR 2 918.42 918.42 142–158
3 612.62 612.62 142–158
M*: mono-oxidized methionine.
N*: glycosylated asparagine upon PNGase F treatment.
C*: cysteine modified by iodoacetamide.

Figure 2. MS/MS spectrum of the doubly charged ion at m/z 497.26.The mutated peptide
(YGNGVWIGK) of a normal sequence (residues 338–346) from tryptic digestion of neuraminidase is
identified, in which the R346 was substituted with K.

892.4, 706.3, 605.3 at the high mass end of the spectrum were y- Considering that the calculated molecular weight (M) from
type fragment ions. We also easily located the sequence of the doubly charged ion peak at m/z 580.78 was 1159.6 Da,
GWT in the residues from 364 to 366 in the expected sequence the most likely sequence of this peak in NA should have been
of NA (Supplementary Fig. S1, Supporting Information). a mutated peptide of PNGWTETDSK (362–371), of which the
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wileyonlinelibrary.com/journal/rcm Copyright © 2016 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2016, 30 (Suppl. 1), 179–184
Mapping single amino acid substitutions in neuraminidase

Figure 3. MS/MS spectrum of the doubly charged ion at m/z 415.21. The mutated peptide (TKNHSSR)
of a normal sequence (residues 347–353) from tryptic digestion of neuraminidase is identified, in which
the S349 was substituted with N.

Figure 4. MS/MS spectrum of the doubly charged ion at m/z 580.78. The mutated peptide
(PNGWTETDI/LK) of a normal sequence (residues 362–371) from tryptic digestion of
neuraminidase is identified, in which the S370 was substituted with I/L.

theoretical molecular weight was 1133.5 Da. This indicated a readily identified, because three kinds of residues (H, A, C)
nominal mass shift of +26 Da, which may have been the result were not contained in the theoretical sequence
of the AA substitution of either H → Y, A → P, C → E, or (PNGWTETDSK). Additionally, detection of y1, y2-NH3 and
S → I/L. Obviously, amino acid substitution of S370I/L was
183

y2 ions at m/z 147.1, 243.2 and 260.2, respectively, in the

Rapid Commun. Mass Spectrom. 2016, 30 (Suppl. 1), 179–184 Copyright © 2016 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm
 Q. Peng et al.

MS/MS spectrum of the ion peak provided solid evidence that [4] W. Li, Q. Wang, J. Chen, J. Zhou, X. Zhou, P. Xie. A
the peak sequence ended with I/LK and not SK, thus multiple-reaction-monitoring mass spectrometric method
confirming the identification of S370 → I/L. for simultaneous quantitative analysis of five plasma
apolipoproteins. SCIENCE CHINA-Chemistry 2014, 57, 723.
[5] D. Wan, Q. Chen, M. Gao, X. Zhang, P. Yang.
Confirmation of the three SAAS by PCR-based sequencing Combination of extraction tip and MALDI-TOF-MS for
efficient separation and analysis of cysteine-containing
The sequence of the open reading frame (ORF) of the peptides. SCIENCE CHINA-Chemistry 2014, 57, 703.
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Fig. S2, Supporting Information). The PCR-based gene tryptic digests of dried-blood spot extracts detects HbS,
sequencing allowed determination of mutations at K346, N349 HbC, HbD, HbE, HbO-Arab, and HbG-Philadelphia
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[8] R. Théberge, G. Infusini, W. Tong, M.E. McComb, C.E. Costello.
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[9] A. I. Nepomuceno, C. J. Mason, D. C. Muddiman,
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We identified three amino acid substitutions altogether, R346K,
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S349 N, and S370I/L, in the neuraminidase from the influenza ionization fourier-transform ion-cyclotron-resonance mass
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been introduced during the synthesis of progeny viral RNA high-performance liquid chromatography/electrospray
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substitutions relies heavily on manual interpretation of MS/MS an avian influenza virus (H1N1). FASEB J. 2009, 23, 3377.
data, which may benefit from the specificity of tandem mass [12] I.C. Huang, W. Li, J. Sui, W. Marasco, H. Choe, M. Farzan.
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[13] N. Dia, M.N. Niang, S.A. Diadhiou, D.G. Goudiaby, A. Faye,
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