Genotyping of Toxic Pufferfish Based on Specific ... - ACS Publications

Subscriber access provided by University of Otago Library

Article

Genotyping of toxic pufferfish based on specific PCRRFLP products as determined by liquid chromatography/ quadrupole-Orbitrap hybrid mass spectrometry Hajime Miyaguchi, Tadashi Yamamuro, Hikoto Ohta, Hiroaki Nakahara, and Shinichi Suzuki J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03703 • Publication Date (Web): 02 Oct 2015 Downloaded from http://pubs.acs.org on October 2, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

Journal of Agricultural and Food Chemistry

Research Article Genotyping of toxic pufferfish based on specific PCR-RFLP products as determined by liquid chromatography/quadrupole-Orbitrap hybrid mass spectrometry Hajime Miyaguchi*, Tadashi Yamamuro, Hikoto Ohta, Hiroaki Nakahara and Shinichi Suzuki National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan *Corresponding Author. Tel.: +81471358001; Fax: +81471339189; E-mail: [email protected].

1

ACS Paragon Plus Environment


1

ABSTRACT: A method for the discrimination of toxic pufferfish was established

2

based on liquid chromatography/electrospray mass spectrometric analysis of the

3

enzymatically digested amplicons derived from mitochondrial 16S rRNA gene. A

4

MonoBis C18 narrow-bore silica monolith column (Kyoto Monotech) and a Q

5

Exactive mass spectrometer (Thermo Fisher) were employed for separation and

6

detection, respectively. Monoisotopic masses of the oligonucleotides were

7

calculated using Protein Deconvolution 3.0 software (Thermo Fisher). Although

8

a lock mass standard was not used, excellent accuracy (mass error, 0.83 ppm

9

on average) and precision (relative standard deviation, 0.49 ppm on average)

10

were achieved and a mass accuracy of less than 2.8 ppm was maintained for at

11

least 180 hours without additional calibration. The present method was applied

12

to 29 pufferfish samples and results were consistent with Sanger sequencing.

13 14

KEYWORDS: Pufferfish, Fugu, Polymerase chain reaction, Restriction fragment

15

length polymorphism, Electrospray ionization mass spectrometry

16

2


Page 2 of 33

Page 3 of 33


17

INTRODUCTION

18

According to an official bulletin by the Japanese government, 21 species of

19

pufferfish are categorized as edible; however, not all parts (organs/tissues) are

20

edible. The edible parts that are free of tetrodotoxin vary by species. Other

21

pufferfish are prohibited for sale because of the presence of toxins throughout

22

the whole body (e.g., Lagocephalus lunaris1) or because there is a lack of

23

toxicity data for a particular species. Therefore, species-based labeling of

24

pufferfish is officially required in the Japanese market; mislabeling, which could

25

lead to serious food poisoning, is a criminal offence.

26

Differentiation and discrimination of pufferfish is not just a challenge in Japan. In

27

the United States, the distribution of pufferfish is prohibited except for limited

28

importation from Japan of edible parts of Takifugu rubripes. However, a

29

tetrodotoxin poisoning occurred in Chicago in 2007 because of an illegal

30

importation of frozen pufferfish (Lagocephalus family) from China that had been

31

mislabeled as monkfish.2

32

Discrimination of pufferfish species is carried out in the Japanese market

33

morphologically, however, inedible L. lunaris is at risk of misidentification as

34

edible L. wheeleri, owing to the species having similar morphologies.1 Moreover,

35

morphological examination of pufferfish is virtually impossible in the case of

36

filleted or minced fish. A tetrodotoxin poisoning outbreak due to consumption of

37

dried fillets of L. lunaris occurred in Minneapolis in 2014.3 A discrimination

38

capability is also needed in the context of food fraud investigations because

39

Takifugu rubripes is far more expensive than other kinds of pufferfish. 3



40

For DNA-based differentiation of pufferfish species, the genes of cytochrome b,

41

12S rRNA, 16S rRNA and cytochrome c oxidase I in mitochondrial DNA have

42

been analyzed by Sanger sequencing, PCR-restriction fragment length

43

polymorphism (RFLP) analysis and real-time PCR analysis.2,4–8 While Sanger

44

sequencing is accurate and well-established, PCR-RFLP and real-time PCR

45

analysis are simple and rapid for analyzing DNA polymorphism. However,

46

because detection of DNA in the latter two methods is usually carried out by

47

non-sequence-based techniques which typically involve using a DNA-binding

48

fluorescent dye, the reliability of discrimination is inferior to that of Sanger

49

sequencing.

50

Mass spectrometry (MS) is one of the most reliable technologies for identifying

51

and characterizing biomolecules. Although matrix-assisted laser

52

desorption/ionization–time-of-flight MS is frequently used for the analysis of

53

proteins and short oligonucleotides (less than ~25 nucleotides (nt)), it is not

54

applicable to long oligonucleotides owing to the subsequent fragmentation,

55

adduct formation and low ionization efficiency.9–11 Instead, electrospray

56

ionization (ESI) can produce many charge states of multiply charged ions

57

([M-nH]n-) from biomacromolecules and, therefore, the mass of the analyte can

58

exceed the upper limit of the scan range of the spectrometer. This requires

59

transformation of a charge state series into the corresponding mass via

60

deconvolution. In the context of ESI-MS, whereas dissociation of a DNA duplex,

61

purification and desalting are required for direct measurement to overcome

62

ionization issues such as ion suppression and adduct formation,12–17 liquid 4


Page 4 of 33

Page 5 of 33


63

chromatography (LC) provides an on-line separation of the analytes from

64

coexisting substances instead of laborious sample purification and desalting.18,19

65

Whereas LC/ESI-MS has been used for amplicon-based genotyping including

66

differentiation of pathogen species, single nucleotide polymorphism and short

67

tandem repeat,9,12,15,20–24 discrimination of species other than pathogens by

68

LC/ESI-MS has not been reported.

69

In this study, LC/ESI-MS is applied for the first time for PCR-RFLP analysis of

70

fish samples to permit rapid and accurate discrimination of toxic pufferfish

71

species.

72 73

MATERIALS AND METHODS

74

Chemicals and materials

75

1,1,1,3,3,3-Hexafluoro-2-propanol and triethylamine were “eluent additive for

76

LC-MS” grade and supplied by Sigma–Aldrich (St. Louis, MO, USA). Synthetic

77

DNA templates (86–115 nt, sequences are shown in Table 1) were purchased

78

from Eurofins Genomics (Tokyo, Japan).

79 80

Preparation of digested amplicons

81

The procedure of sample preparation is summarized in Figure 1. PCR reactions

82

were performed on a 25 µL reaction scale and consisted of 0.625 U of Pfu-X

83

DNA polymerase (Jena Bioscience, Jena, Germany), 0.2 mM of each dNTP

84

(Jena Bioscience), 1× detergent free Phusion HF buffer (New England Biolabs,

85

Ipswich, MA, USA), either 12.5 ng of an extracted fish DNA or 0.5 pmol of the 5



86

synthetic DNA template and 0.4 µM of each primer. Two forward primers

87

(5′-CCATGTGGAATGAAAACACC-3′ for Takifugu and

88

5′-AAAAACAAGAGCCACAGCTCTAA-3′ for Lagocephalus) and one reverse

89

primer (5′-CCCTAGGGTAACTCGGTTCG-3′) were designed with the help of

90

Primer3Plus software (http://www.bioinformatics.nl/primer3plus/) and

91

synthesized by Fasmac (Atsugi, Japan). These primers were used to amplify

92

114–115-bp and 86-bp amplicons of the mitochondrial 16S ribosomal RNA gene

93

of Takifugu species and Lagocephalus species, respectively (Figure 1).25,26

94

Thermocycling was performed in a T100 thermal cycler (Bio-Rad, Hercules, CA,

95

USA) under the following conditions: 95 °C, 2 min, then 30 cycles at 95 °C, 30 s,

96

56 °C, 30 s, 72 °C, 30 s, 72 °C, 7 min and 12 °C hold. After PCR, 2 µL of the 10×

97

universal buffer M (Takara Bio, Otsu, Japan, containing 100 mM Tris-HCl (pH

98

7.5), 100 mM MgCl2, 10 mM dithiothreitol and 500 mM NaCl) and 1 µL of each

99

FastDigest restriction enzyme (Dra I and Msp I, Thermo Fisher Scientific) were

100

added to the tube and incubated for 30 min at 37 °C, followed by additional

101

incubation for 5 min at 72 °C. A portion of the reaction solution was analyzed by

102

polyacrylamide gel electrophoresis (PAGE) using the staining dye Gel Green

103

(Biotium, Hayward, CA, USA), if necessary. The remaining solution was filtered

104

with a Nanosep MF centrifugal device containing a Bio-inert membrane (0.45 µm,

105

Pall Corporation, Port Washington, NY, USA) before LC/ESI-MS analysis. The

106

filtrates were stored at −20 °C until analysis.

107 108

LC/ESI-MS analysis 6


Page 6 of 33

Page 7 of 33


109

The LC/ESI-MS was performed using an Ultimate 3000 liquid chromatograph

110

and a Q Exactive quadrupole–Orbitrap hybrid mass spectrometer (Thermo

111

Fisher Scientific). A reverse-phase silica monolith column (MonoBis C18, Kyoto

112

Monotech, Kyoto, Japan; 2.0×50 mm, mesopore size 30 nm, product number

113

2050H30ODS) was used for the separation at 20 °C with a MonoBis C18 guard

114

column (Kyoto Monotech). The mobile phase, delivered at a flow rate of 0.4

115

mL/min, was 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine

116

(pH 7.9) (A)–methanol (B).27 The proportion of B in the mobile phase was

117

changed over a linear gradient as follows: 0–0.5 min, 5%; 0.5–1 min, 5–30%; 1–

118

3.5 min, 30–40%; 3.5–5 min, 40–98%; 5–6 min, 98%; 6–6.05 min, 98–5% and

119

6.05–8 min, 5%. The sample injection volume was 1.0 µL except for the tests of

120

the stability of mass accuracy and the temperature of the column (0.5 µL each).

121

The post-column flow was connected to the mass spectrometer for 3.5–6 min

122

after sample injection. A heated ESI probe (HESI-II, Thermo Fisher) was used in

123

negative ion mode and with the following parameters: spray voltage, 2.5 kV;

124

vaporizer temperature, 350 °C; sheath gas, 50 arbitrary units (a.u.); auxiliary gas,

125

15 a.u. The parameters for the desolvation at the entrance of the mass

126

spectrometer were as follows: Capillary temperature, 350 °C; sweep gas

127

pressure, 1 a.u.; S-lens radiofrequency level, 100. High resolution mass analysis

128

was performed using the following parameters: scan range, m/z 700–3500;

129

nominal resolution (at m/z 200), 140,000; auto gain control target, 1×106;

130

microscans, 3; maximum ion time, 50 ms; in-source collision induced

131

dissociation, 15 eV. The C-trap pressure, which can be adjusted with the valve 7



132

inside the chassis, was reduced to 0.2 MPa to improve the sensitivity for long

133

oligonucleotides. A lock mass was not assigned. Mass calibration was

134

performed daily using sodium trifluoroacetate clusters (n = 5–24, m/z

135

792.85908–3376.38045).28 The calibration solution was 0.5 mg/mL sodium

136

trifluoroacetate (pH 3.5) in water–acetonitrile (1:1, v/v) and was infused into the

137

mass spectrometer at the flow rate of 10 µL/min. The in-source collision-induced

138

dissociation voltage was set to 60 eV to develop the cluster ions at higher mass

139

range.

140

Peak detection and deconvolution from the raw data using Xtract algorithm29,30

141

were carried out automatically with the Protein Deconvolution version 3.0

142

software (Thermo Fisher). The parameters were as follows: fit factor 90%,

143

charge range 3–50.

144 145 146

Analysis of pufferfish and other fish species

147

Twenty-nine pufferfish samples (1 boiled skin of T. rubripes, 3 raw livers and 1

148

ovary of Takifugu porphyreus, 1 raw muscle tissue of Takifugu vermicularis, 2

149

fresh whole bodies of Takifugu pardalis, 1 dried fillet of Takifugu snyderi, 19

150

fresh whole bodies of Takifugu poecilonotus and 1 dried fillet of Lagocephalus

151

wheeleri) and eight raw muscle tissues of other fish (Atlantic mackerel (Scomber

152

scombrus), Japanese horse mackerel (Trachurus japonicus), Japanese pilchard

153

(Sardinops melanostictus), Skipjack tuna (Katsuwonus pelamis), Yellowfin tuna

154

(Thunnus albacares), Japanese amberjack (Seriola quinqueradiata), Atlantic 8


Page 8 of 33

Page 9 of 33


155

salmon (Salmo salar) and Flying fish (Cypselurus pinnatibarbatus japonicus)

156

were obtained from fish wholesalers and retail markets in Japan. Total genomic

157

DNA in fish tissue (~50 mg) was extracted with a DNeasy Blood and Tissue Kit

158

(Qiagen, Hilden, Germany) in accordance with the protocol of the manufacturer.

159

In the case of whole body samples, fresh fins were collected for extraction. The

160

species of the pufferfish samples being studied were verified using the official

161

method of the Office of Import Food Safety, Inspection and Safety Division,

162

Department of Food Safety, Ministry of Health, Labour and Welfare, Japan

163

(2011), which was based on Sanger sequencing of the 16S ribosomal RNA

164

gene.6 The same method was also used to confirm the common names of other

165

fish samples with the aid of the BLAST search (DNA Data Bank of Japan).

166

Amplification and LC/ESI-MS analysis were performed as described above.

167 168

RESULTS AND DISCUSSION

169

Optimization of LC/ESI-MS conditions

170

Past studies dealing with LC/ESI-MS analysis of long oligonucleotides have

171

mostly been carried out using a poly(styrene-divinylbenzene) (PS-DVB) capillary

172

monolith column (0.2 mm i.d.) at low flow rate (2 µL/min).18–24,31 Instead, we

173

tested a narrow-bore type (2.0 mm i.d.) reverse-phase silica monolith column for

174

the separation of long oligonucleotides employing a much higher flow rate (400

175

µL/min), which enabled the use of a heated sprayer at the interface. Accordingly,

176

the DNA duplexes were denatured, regardless of column temperature, when the

9



177

mobile phase was sprayed into the ion source with the aid of heated nitrogen

178

(350 °C).

179

Column temperature was optimized (Figure 2). Signals for the three pairs of

180

DNA duplexes (26, 37 and 53 bp) and the primers were separated from one

181

another at 20 °C and 60 °C, whereas distorted peaks were observed at 40 °C.

182

These differences may be related to the formation of the double helix structure

183

during chromatographic separation. Oberacher et al reported that a DNA duplex

184

longer than 25–50 bp could be denatured in the analytical column at a

185

temperature between 50–80 °C.24 Accordingly, possible explanations for this

186

behavior are that the DNA duplexes maintained their rigid structure at 20 °C,

187

denatured partially at 40 °C, and denatured completely at 60 °C. Taking the

188

separation behavior and the maximum column temperature recommended by

189

the manufacturer (40 °C) into consideration, the column temperature was

190

adjusted to 20 °C. Despite the low column temperature, the maximum back

191

pressure of the system (12.5 MPa) was below the upper limit of the column (30

192

MPa) recommended by the manufacturer, owing to the inherently large channel

193

size of the silica monolith.32

194 195

Discrimination of synthetic DNA by PCR-RFLP coupled with LC/ESI-MS

196

The PCR-RFLP method was used to discriminate 11 synthetic DNA (range, 86

197

to 115 bp) samples (Table 1), which served as the positive control templates for

198

the pufferfish. The list for the target pufferfish was compatible with the published

199

method by the Japanese government based on the Sanger sequencing. The 10


Page 10 of 33

Page 11 of 33


200

exceptions to this were T. pardalis and T. snyderi and, given that the target

201

sequences for these two species are identical, T. pardalis was selected as being

202

a representative example. The result obtained by PAGE is shown in Figure 3a,

203

and that obtained by LC/ESI-MS is given in Figure 3b and Table 2. Using PAGE,

204

the band pattern could only be divided into three groups: Takifugu chrysops,

205

another species of genus Takifugu and genus Lagocephalus (Figure 3a). Each

206

band, which corresponded to the DNA duplex detected by PAGE, was also

207

detected by LC/ESI-MS as a peak (Figure 3b), and where each peak in the

208

chromatogram represented the mass spectra of the forward and reverse

209

oligonucleotides. Because the digestion with the endonucleases was performed

210

just after PCR amplification without purification, the sticky end generated by the

211

Msp I endonuclease was filled up with the remaining DNA polymerase during

212

incubation (Figure 1).12

213 214

Method validation

215

Accuracy and precision data for the LC/ESI-MS analyses are presented in Table

216

2. Although an internal mass standard was not used, excellent accuracy (mass

217

error, 0.83 ppm on average) and precision (standard deviation, 0.49 ppm on

218

average) were achieved. Although this performance reflects the inherent

219

performance of the Orbitrap analyzer, deconvolution also contributed to an

220

improved accuracy and precision. Because dozens of peaks having different

221

charge states and isotopic compositions are taken into account for the

222

calculation of a monoisotopic mass, the monoisotopic mass calculated by 11



223

deconvolution has a reduced observational error and must have been more

224

accurate than any molecular mass information determined from an individual

225

peak.30,33 In addition, the use of the monoisotopic mass for mass

226

characterization is preferable to that of the average mass for achieving

227

enhanced accuracy and precision.33 Because the monoisotopic mass is, for all

228

practical purposes, independent of the isotope ratio,34 the acquired data can be

229

directly compared with the theoretical monoisotopic mass calculated from the

230

molecular formula. An alternative mass spectrometry metric for a biomolecule is

231

the average mass, which is defined as the centroid of the isotopic distribution of

232

the biomolecule. However, according to a calculation by Zubarev et al, the

233

accuracy of the average mass measurement is limited to 10 ppm for

234

biomolecules larger than 10,000 Da because of natural variations of isotopic

235

abundances of the elements. Furthermore, the non-symmetric shape of the

236

isotopic distribution causes negative bias.33 Manduzio et al reported an average

237

mass deviation of 9.15 ± 7.11 ppm as a result of analyzing a 114-bp PCR

238

product despite an Orbitrap analyzer being used.12 Kullolli et al used an

239

LTQ-Orbitrap Veros for analyzing microRNA ranging from 22 to 25 nt.35 A mass

240

accuracy of 1.3 ppm on average was reported; however, these studies

241

compared the monoisotopic peaks of the multiply charged ions with the

242

corresponding theoretical ones instead of the deconvoluted monoisotopic mass

243

of the molecule. Because the monoisotopic peak disappears from a mass

244

spectrum with increasing molecular mass,33 the approach involving the

245

monoisotopic peaks is only applicable for short oligonucleotides. 12


Page 12 of 33

Page 13 of 33


246

Periodic analyses were carried out to evaluate the stability of mass accuracy.

247

Digested amplicons derived from the synthetic DNA of Takifugu chrysops were

248

analyzed every 10 h for 180 h. As a result, all the monoisotopic masses of the

249

digested PCR amplicons were successfully determined within a mass error of

250

2.8 ppm without any additional mass calibration. Consequently, a daily

251

calibration can be replaced by a weekly calibration.

252

As a result of the above findings and observations, a flow chart for the

253

discrimination of the pufferfish is proposed (Figure 4). The target pufferfish

254

always yields two or three peaks in the TIC chromatogram at a retention time of

255

4–5.5 min; otherwise the sample is not the target pufferfish. If two peaks are

256

observed in the range, a pair of the monoisotopic masses of the DNA duplex

257

corresponding to the latter peak will be compared to the theoretical values to

258

determine if any one of the species is T. chrysops, L. inermis, L. gloveri, L.

259

lunaris or L. wheeleri. The mass tolerance for the determination is 3 ppm which

260

does not cause overlap. If three peaks are observed in the range, a similar

261

matching procedure is required for the second peak in some cases as well as for

262

the third peak (Figure 4).

263 264

Mixed template

265

The synthetic DNA template of Lagocephalus lunaris was mixed with that of L.

266

wheeleri at ratios of 1:1, 1:4 and 1:19, and the mixtures were analyzed as

267

described above. As the result of the preparation, four pairs of DNA duplexes

268

would have existed in the sample solution and the success of the discrimination 13



269

relies on the difference of the masses between each oligonucleotide. The results

270

are shown in Figure 5. In the case when the mass difference was about 25 Da

271

(Figure 5a, single base substitution) and 7 Da (Figure 5b, triple base

272

substitution), each strand could be analyzed independently for the mixing ratio of

273

1:1; however, minor constituents could not be calculated properly. When the

274

difference was about 6 Da (Figure 5c, the complementary sequence of Figure 5b)

275

because of an overlapping of clusters of peaks, the isotopic mass could not

276

calculated even for a mixing ratio of 1:1 although this could be compensated for

277

by the results for the complementary strand. These observations are basically

278

consistent with the study of Manduzio et al who investigated the discrimination

279

ability for an equal mixture of templates with single base substitution.12 They

280

reported that a mass difference of 16 Da (A G) could be discriminated,

281

although that of 9 Da (T A) could not be discriminated for analysis of 114 bp

282

DNA. This was overcome by emphasizing the difference of the m/z values using

283

endonuclease digestion, which is consistent with the present strategy.

284

As an alternative to calculating the monoisotopic mass, the extracted ion

285

chromatograms (XICs) of the most abundant isotopic ions were examined as a

286

means to discriminate T. rubripes from T. pardalis. However, it was impossible to

287

discriminate the two species using XIC because of the overlapping of the peaks

288

(Figure 6), although they could be discriminated using a deconvolution approach

289

(Table 2). Therefore, deconvolution was the preferred approach for species

290

identification, especially when the sample was considered to be derived from a

291

single source. 14


Page 14 of 33

Page 15 of 33


292 293

Application for small samples

294

A small amount of pufferfish alone or its presence in another fish was analyzed

295

by the present methods. As a result of the analysis of a raw muscle tissue

296

homogenate of T. vermicularis (0.1 mg equivalent, n = 2), all the expected DNA

297

fragments were successfully identified with an average mass error of 1.50 ppm.

298

In addition, raw muscle tissue of T. vermicularis mixed with that of Korean black

299

scraper (Thamnaconus modestus) (2.5 wt % of T. vermicularis in 50 mg mixture)

300

could be identified with an average mass error of 1.15 ppm (n = 2). Therefore,

301

the present method is considered applicable to small sample amounts as long as

302

PCR amplification proceeds successfully.

303 304

Discrimination of pufferfish and other fish samples

305

The present method was applied to 29 pufferfish samples and 8 other fish

306

species (Figure 7). The results for discrimination based on the present criteria

307

(Figure 4) were consistent with the official method based on Sanger sequencing,

308

although the present method could not discriminate T. pardalis from T. snyderi

309

because these species contain the same amplicon sequence (data not shown).

310

Additionally, no deconvolution value was calculated from the chromatograms of

311

fish samples other than pufferfish.

312

As mentioned above, the use of LC/ESI-MS for fish genotyping has been

313

demonstrated for the first time. A remaining problem is the interpretation of data

314

obtained from a mixed template at a closer ratio as mentioned above. Using 15



315

multiplex PCR with another pair of pufferfish-specific or species-specific primers

316

may be an effective way for resolving analytes in a mixture.

317 318

Funding

319

This work was supported by a Grant-in-Aid for Scientific Research by the Japan

320

Society for the Promotion of Science (15K08060).

321

16


Page 16 of 33

Page 17 of 33


322

REFERENCES

323

(1)

324

Distribution of tetrodotoxin in pufferfish collected from coastal waters of

325

Sihanouk Ville, Cambodia. Shokuhin Eiseigaku Zasshi 2008, 49, 361–365.

326

(2)

327

White, K. D.; Thompson, T. M.; Wahl, M.; Pham, T. D.; Guichard, F. M.; Huh, I.;

328

Austin, C.; Dizikes, G.; Gerber, S. I., Public health response to puffer fish

329

(tetrodotoxin) poisoning from mislabeled product. J. Food Prot. 2009, 72, 810–

330

817.

331

(3)

332

M.; Centers for Disease, C.; Prevention, Tetrodotoxin poisoning outbreak from

333

imported dried puffer fish — Minneapolis, Minnesota, 2014. MMWR Morb. Mortal.

334

Wkly. Rep. 2015, 63, 1222–1225.

335

(4)

336

Hwang, D.-F., Puffer fish-based commercial fraud identification in a segment of

337

cytochrome b region by PCR–RFLP analysis. Food Chem. 2010, 121, 1305–

338

1311.

339

(5)

340

puffer fish inferred from partial sequences of cytochrome b gene and restriction

341

fragment length polymorphism analysis. J. Agric. Food Chem. 2004, 52, 4159–

342

4165.

343

(6)

344

Shiomi, K.; Watabe, S., Molecular identification of pufferfish species using PCR

Ngy, L.; Taniyama, S.; Shibano, K.; Yu, C. F.; Takatani, T.; Arakawa, O.,

Cohen, N. J.; Deeds, J. R.; Wong, E. S.; Hanner, R. H.; Yancy, H. F.;

Cole, J. B.; Heegaard, W. G.; Deeds, J. R.; McGrath, S. C.; Handy, S.

Hsieh, C.-H.; Chang, W.-T.; Chang, H. C.; Hsieh, H.-S.; Chung, Y.-L.;

Hsieh, Y. W.; Hwang, D. F., Molecular phylogenetic relationships of

Ishizaki, S.; Yokoyama, Y.; Oshiro, N.; Teruya, N.; Nagashima, Y.;

17



Page 18 of 33

345

amplification and restriction analysis of a segment of the 16s rRNA gene. Comp.

346

Biochem. Physiol. Part D Genomics Proteomics 2006, 1, 139–144.

347

(7)

348

assay for the detection of pufferfish products. J. Food Prot. 2010, 73, 1698–

349

1702.

350

(8)

351

of the genus Lagocephalus: L. lunaris, L. spadiceus and L. inermis, using

352

multiplex PCR. Food Biotechnol. 2014, 28, 216–231.

353

(9)

354

Overview of recent developments in the mass spectrometry of nucleic acid and

355

constituents. In Mass spectrometry of nucleosides and nucleic acids, Banoub, J.

356

H.; Limbach, P. A., Eds. CRC Press: Boca Raton, FL, 2010; pp 1–90.

357

(10)

358

for characterization of sequence variability in genomic DNA. Anal. Bioanal.

359

Chem. 2008, 391, 135–149.

360

(11)

361

analysis

362

charge-tagging. Nucleic Acids Res. 2003, 31, e63.

363

(12)

364

mass spectrometer for the analysis of polymerase chain reaction products.

365

Rapid Commun. Mass Spectrom. 2010, 24, 3501–3509.

366

(13)

367

approaches for purifying and desalting polymerase chain reaction products prior

Jones, Y. L.; Oliver, H. F.; Deeds, J. R.; Yancy, H. F., Real-time PCR

Sangthong, P.; Ngernsiri, L.; Sangthong, D., Identification of puffer fish

Banoub, J. H.; Miller-Banoub, J.; Jahouh, F.; Joly, N.; Martin, P.,

Oberacher, H., On the use of different mass spectrometric techniques

Sauer, S.; Lehrach, H.; Reinhardt, R., MALDI mass spectrometry of

single

nucleotide

polymorphisms

by

photocleavage

and

Manduzio, H.; Ezan, E.; Fenaille, F., Evaluation of the LTQ-Orbitrap

Manduzio, H.; Martelet, A.; Ezan, E.; Fenaille, F., Comparison of

18


Page 19 of 33


368

to electrospray ionization mass spectrometry. Anal. Biochem. 2010, 398, 272–

369

274.

370

(14)

371

thermal denaturation for the production of single-stranded PCR amplicons:

372

Piperidine-induced destabilization of the DNA duplex? J. Am. Soc. Mass

373

Spectrom. 2002, 13, 232–240.

374

(15)

375

characterization of nucleic acids by electrospray ionization mass spectrometry.

376

Rapid Commun. Mass Spectrom. 2003, 17, 2699–2706.

377

(16)

378

preparation techniques for mass measurements of PCR products using

379

ESI-FT-ICR mass spectrometry. J. Am. Soc. Mass Spectrom. 2002, 13, 338–

380

344.

381

(17)

382

synthetic nucleic acids by electrospray ionization quadrupole time-of-flight mass

383

spectrometry. J. Mass Spectrom. 2005, 40, 932–945.

384

(18)

385

chromatography–mass spectrometry. Mass Spectrom. Rev. 2001, 20, 310–343.

386

(19)

387

chromatography and mass spectrometry. In Handbook of analysis of

388

oligonucleotides and related products, Bonilla, J. V.; Srivatsa, G. S., Eds. CRC

389

Press: Boca Raton, FL, 2011; pp 137–166.

390

(20)

Mangrum, J. B.; Flora, J. W.; Muddiman, D. C., Solution composition and

Null, A. P.; Benson, L. M.; Muddiman, D. C., Enzymatic strategies for the

Null, A. P.; George, L. T.; Muddiman, D. C., Evaluation of sample

Oberacher, H.; Niederstatter, H.; Parson, W., Characterization of

Huber, C. G.; Oberacher, H., Analysis of nucleic acids by on-line liquid

Pourshahian, S.; McCarthy, S. M., Analysis of oligonucleotides by liquid

Beer, B.; Erb, R.; Pitterl, F.; Niederstatter, H.; Maroñas, O.; Gesteira, A.; 19



391

Carracedo, A.; Piatkov, I.; Oberacher, H., CYP2D6 genotyping by liquid

392

chromatography-electrospray ionization mass spectrometry. Anal. Bioanal.

393

Chem. 2011, 400, 2361–2370.

394

(21)

395

Identification of bacteria by polymerase chain reaction followed by liquid

396

chromatography–mass spectrometry. Anal. Chem. 2005, 77, 4563–4570.

397

(22)

398

chromatography mass spectrometry: A useful tool for the detection of DNA

399

sequence variation. Angew. Chem. Int. Ed. Engl. 2001, 40, 3828–3830.

400

(23)

401

Optimized suppression of adducts in polymerase chain reaction products for

402

semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry.

403

J. Am. Soc. Mass Spectrom. 2004, 15, 1897–1906.

404

(24)

405

polymerase chain reaction products by on-line liquid chromatography–mass

406

spectrometry for genotyping of polymorphic short tandem repeat loci. Anal.

407

Chem. 2001, 73, 5109–5115.

408

(25)

409

M., Toxicity and molecular identification of green toadfish Lagocephalus lunaris

410

collected from Kyushu coast, Japan. J. Toxicol. 2011, 2011, 801285.

411

(26)

412

Doi, H.; Takahashi, H.; Mabuchi, K.; Nishida, M.; Sakai, H., Explosive speciation

413

of Takifugu: Another use of fugu as a model system for evolutionary biology. Mol.

Mayr, B. M.; Kobold, U.; Moczko, M.; Nyeki, A.; Koch, T.; Huber, C. G.,

Oberacher, H.; Oefner, P. J.; Parson, W.; Huber, C. G., On-line liquid

Oberacher, H.; Parson, W.; Hölzl, G.; Oefner, P. J.; Huber, C. G.,

Oberacher, H.; Parson, W.; Muhlmann, R.; Huber, C. G., Analysis of

Nagashima, Y.; Matsumoto, T.; Kadoyama, K.; Ishizaki, S.; Terayama,

Yamanoue, Y.; Miya, M.; Matsuura, K.; Miyazawa, S.; Tsukamoto, N.;

20


Page 20 of 33

Page 21 of 33


414

Biol. Evol. 2009, 26, 623–629.

415

(27)

416

modified oligonucleotides by tandem ion-pair reversed-phase high-performance

417

liquid chromatography/electrospray ionization mass spectrometry. Rapid

418

Commun. Mass Spectrom. 2003, 17, 646–653.

419

(28)

420

as a tune/calibration compound for positive- and negative-ion electrospray

421

ionization mass spectrometry in the mass range of 100–4000 Da. J. Am. Soc.

422

Mass Spectrom. 1998, 9, 977–980.

423

(29)

424

interpretation of high resolution electrospray mass spectra of large molecules. J.

425

Am. Soc. Mass Spectrom. 2000, 11, 320–332.

426

(30)

427

monoisotopic masses and ion populations for large biomolecules from resolved

428

isotopic distributions. J. Am. Soc. Mass Spectrom. 1995, 6, 229–233.

429

(31)

430

chromatography–electrospray ionization mass spectrometry of single- and

431

double-stranded nucleic acids using monolithic capillary columns. Anal. Chem.

432

2000, 72, 4386–4393.

433

(32)

434

nucleic

435

ionization mass spectrometry. Trends Anal. Chem. 2002, 21, 166–174.

436

(33)

Fountain, K. J.; Gilar, M.; Gebler, J. C., Analysis of native and chemically

Moini, M.; Jones, B. L.; Rogers, R. M.; Jiang, L., Sodium trifluoroacetate

Horn, D. M.; Zubarev, R. A.; McLafferty, F. W., Automated reduction and

Senko, M. W.; Beu, S. C.; McLafferty, F. W., Determination of

Premstaller, A.; Oberacher, H.; Huber, C. G., High-performance liquid

Oberacher, H.; Huber, C. G., Capillary monoliths for the analysis of acids

by

high-performance

liquid

chromatography–electrospray

Zubarev, R. A.; Demirev, P. A.; Håkansson P.; Sundqvist B. U. R., 21



437

Approaches and limits for accurate mass characterization of large biomolecules.

438

Anal. Chem. 1995, 67, 3793–3798.

439

(34)

440

distributions in mass spectra of large molecules. Anal. Chem. 1983, 55, 353–

441

356.

442

(35)

443

microRNA analysis using high resolution mass spectrometry. J. Am. Soc. Mass

444

Spectrom. 2014, 25, 80–87.

Yergey, J.; Heller, D.; Hansen, G.; Cotter, R. J.; Fenselau, C., Isotopic

Kullolli, M.; Knouf, E.; Arampatzidou, M.; Tewari, M.; Pitteri, S. J., Intact

445

22


Page 22 of 33

Page 23 of 33


446

FIGURE CAPTIONS

447

Figure 1. Method summary. Bold indicates the nucleotides filled up with the

448

remaining DNA polymerase. p indicates a phosphate group.

449

Figure 2. Chromatograms of the PCR products of the synthetic DNA of Takifugu

450

rubripes for comparison of temperatures for the MonoBis C18 column. TIC, total

451

ion current.

452

Figure 3. Results for PCR-RFLP analysis of synthetic pufferfish DNA. a, PAGE;

453

b, LC/ESI-MS.

454

Figure 4. Flow chart for the discrimination of pufferfish. TIC, total ion current. RT,

455

retention time.

456

Figure 5. Total ion current chromatograms obtained from the mixed templates.

457

nt, nucleotides.

458

Figure 6. An example of failure of discrimination between T. rubripes and T.

459

pardalis using individual isotopic peaks. nt, nucleotides.

460

Figure 7. Total ion current chromatograms derived from actual fish samples.

461

23



Table 1

Page 24 of 33

DNA Sequences Used for the PCR Templates.

Species

DNA sequencesa

Takifugu rubripes Takifugu porphyreus

CCATGTGGAATGAAAACACCCTTTTTAAACCCAAGAGTCACCACTCTAGGATACAGAACATCTGACCAAT-AATGATCCGGCT-AAAGCCGATTAACGAACCGAGTTACCCTAGGG

Length (nt)b 114

Refer encec (26)

Accession No. AP006045

CCATGTGGAATGAAAACACCCTTTTTAAAACCAAGAGTCACCACTCTAGGATACAGAACATCTGACCAAT-AATGATCCGGCT-AAAGCCGATTAACGAACCGAGTTACCCTAGGG

114

(26)

AP009529

Takifugu chrysops

CCATGTGGAATGAAGACACCCTCTCTAAAACCAAGAGTCACCACTCTAAGTTACAGAACATCTGACCAAT-AATGATCCGGCT-AAAGCCGATTAACGAACCGAGTTACCCTAGGG

114

(26)

AP009525

Takifugu vermicularis

CCATGTGGAATGAAAACACCCTTTTTAAAACCAAGAGTCACCACTCTAAGTTACAGAACATCTGACCAATTAATGATCCGGCT-AAAGCCGATTAACGAACCGAGTTACCCTAGGG

115

–

AB741999

Takifugu pardalis

CCATGTGGAATGAAAACACCCTTTTTAAAACCAAGAGTCACCACTCTAAGTTACAGAACATCTGACCAAT-AATGATCCGGCT-AAAGCCGATTAACGAACCGAGTTACCCTAGGG

114

(26)

AP009528

Takifugu niphobles

CCATGTGGAATGAAAACACCCTTTTTAAAACCAAGAGTCACCACTCTAAGTTACAGAACATCTGACCAAT-AATGATCCGGCTTAAAGCCGATTAACGAACCGAGTTACCCTAGGG

115

(26)

AP009526

Takifugu poecilonotus

CCATGTGGAATGAAAACACCCTTTTTAAAACCAAGAGTCACCACTCTAAGTTACAGAACATCTGACCAGT-AATGATCCGGCTT-AAGCCGATTAACGAACCGAGTTACCCTAGGG

114

(26)

AP009539 AP009538

Lagocephalus wheeleri

AAAAAC-AAGAGCCACAGCTCTAATGAGCAGAACATCTGACCTACCA--GATCCGGC--ATAGCCGATCAACGAACCGAGTTACCCTAGGG

86

(26)

Lagocephalus gloveri

AAAAAC-AAGAGCCACAGCTCTAATGAACAGAACATCTGACCTACCA--GATCCGGC--ATAGCCGATCAACGAACCGAGTTACCCTAGGG

86

–

AB742032

Lagocephalus lunaris

AAAAAC-AAGAGCCACAGCTCTAATAAACAGAACATCTGACCAGCCA--GATCCGGC--CTAGCCGATCAACGAACCGAGTTACCCTAGGG

86

(25)

Not available

Lagocephalus inermis

AAAAAC-AAGAGCCACAGCTCTAATAAACAGAACATCTGACCCACCA--GATCCGGC--ACAGCCGATCAACGAACCGAGTTACCCTAGGG

86

–

AB742030

a

Underline indicates primer-binding site and bold indicates the recognition sites of the restriction enzymes. bnt, nucleotide. c–, unpublished.

24


Page 25 of 33


Table 2

LC/ESI-MS Analysis of the Amplicons Digested with the Endonucleases.

Species

T. rubripes

T. porphyreus

T. chrysops

T. vermicularis

T. pardalis

T. niphobles

T. poecilonotus

L. wheeleri

L. gloveri

L. lunaris

L. inermis

F/Ra

F F F R R R F F F R R R F F R R F F F R R R F F F R R R F F F R R R F F F R R R F F R R F F R R F F R R F F R R

Length (nt)b

26 53 37 37 53 26 26 53 37 37 53 26 79 37 37 79 26 54 37 37 54 26 26 53 37 37 53 26 26 53 38 38 53 26 26 53 37 37 53 26 52 36 52 36 52 36 52 36 52 36 52 36 52 36 52 36

Expected molecular Monoisotopic mass, n = 5 (interday) formula Theoretical Measured value Relative value (Da) (Da, average ± standard standard deviation) deviation (ppm) C254H321O154N94P25 7925.362 7925.366 ± 0.003 0.37 C515H647O307N208P53 16296.750 16296.763 ± 0.006 0.34 11466.909 11466.920 ± 0.002 0.18 C360H452O218N147P37 C360H456O225N132P36 11341.885 11341.890 ± 0.003 0.25 C522H659O331N186P53 16468.655 16468.661 ± 0.004 0.25 8085.340 8085.352 ± 0.001 0.13 C256H322O157N98P26 C254H321O154N94P25 7925.362 7925.367 ± 0.004 0.44 C516H647O306N210P53 16320.762 16320.764 ± 0.017 1.05 C360H452O218N147P37 11466.909 11466.919 ± 0.006 0.50 C360H456O225N132P36 11341.885 11341.892 ± 0.004 0.33 C522H660O332N183P53 16443.648 16443.651 ± 0.010 0.62 C256H322O157N98P26 8085.340 8085.350 ± 0.002 0.28 C768H965O458N303P78 24173.107 24173.129 ± 0.017 0.70 C360H452O218N147P37 11466.909 11466.918 ± 0.006 0.56 C360H456O225N132P36 11341.885 11341.891 ± 0.006 0.52 C779H980O489N283P79 24566.979 24567.021 ± 0.016 0.64 C254H321O154N94P25 7925.362 7925.367 ± 0.004 0.46 C526H661O314N209P54 16599.801 16599.821 ± 0.009 0.56 11466.909 11466.922 ± 0.006 0.49 C360H452O218N147P37 C360H456O225N132P36 11341.885 11341.890 ± 0.005 0.48 C533H672O336N190P54 16780.717 16780.724 ± 0.004 0.21 C256H322O157N98P26 8085.340 8085.349 ± 0.005 0.65 7925.362 7925.367 ± 0.002 0.30 C254H321O154N94P25 C516H648O307N207P53 16295.755 16295.764 ± 0.020 1.21 C360H452O218N147P37 11466.909 11466.922 ± 0.007 0.60 11341.885 11341.889 ± 0.006 0.49 C360H456O225N132P36 C523H660O331N185P53 16467.659 16467.671 ± 0.011 0.66 8085.340 8085.350 ± 0.003 0.36 C256H322O157N98P26 C254H321O154N94P25 7925.362 7925.367 ± 0.005 0.64 C516H648O307N207P53 16295.755 16295.764 ± 0.012 0.76 C370H465O225N149P38 11770.955 11770.971 ± 0.005 0.41 11654.943 11654.951 ± 0.004 0.30 C370H468O230N137P37 16467.659 16467.666 ± 0.006 0.34 C523H660O331N185P53 C256H322O157N98P26 8085.340 8085.353 ± 0.003 0.38 C254H321O154N94P25 7925.362 7925.369 ± 0.005 0.61 16311.750 16311.730 ± 0.014 0.84 C516H648O308N207P53 C360H453O220N144P37 11457.898 11457.910 ± 0.003 0.23 C360H455O223N135P36 11350.897 11350.892 ± 0.007 0.62 16452.660 16452.664 ± 0.007 0.45 C522H659O330N186P53 C256H322O157N98P26 8085.340 8085.349 ± 0.003 0.33 15937.744 15937.761 ± 0.008 0.48 C505H632O296N209P51 11138.852 11138.844 ± 0.010 0.87 C349H439O212N143P36 C511H647O327N179P52 16131.586 16131.595 ± 0.006 0.39 C350H443O219N130P35 11053.834 11053.844 ± 0.004 0.38 C505H632O295N209P51 15921.750 15921.765 ± 0.007 0.46 C349H439O212N143P36 11138.852 11138.848 ± 0.006 0.50 C512H648O328N178P52 16146.585 16146.580 ± 0.009 0.54 C350H443O219N130P35 11053.834 11053.849 ± 0.010 0.89 C505H631O293N212P51 15930.761 15930.776 ± 0.006 0.36 C348H439O213N141P36 11114.841 11114.846 ± 0.006 0.58 C512H649O330N175P52 16137.574 16137.583 ± 0.007 0.42 C350H442O218N133P35 11078.841 11078.836 ± 0.003 0.24 C504H631O293N210P51 15890.755 15890.773 ± 0.006 0.38 C348H438O211N144P36 11123.852 11123.851 ± 0.004 0.35 C513H649O330N177P52 16177.580 16177.590 ± 0.005 0.33 C350H443O220N130P35 11069.829 11069.836 ± 0.008 0.71

a

Average mass errorc (ppm) 0.57 0.80 0.93 0.45 0.42 1.44 0.73 0.78 0.85 0.60 0.53 1.19 0.92 0.84 0.58 1.69 0.69 1.21 1.11 0.42 0.40 1.06 0.64 1.11 1.08 0.51 0.76 1.21 0.82 0.80 1.36 0.74 0.39 1.57 0.91 1.21 1.10 0.53 0.42 1.07 1.04 0.90 0.61 0.88 0.94 0.49 0.51 1.33 0.96 0.51 0.59 0.37 1.10 0.26 0.64 0.84

F, forward strand; R, reverse strand (see Figure 1). bnt, nucleotide. cMass error is defined as the absolute deviation of a measured monoisotopic mass from the theoretical one.

25



Fish sample

DNA extraction

PCR

Enzymatic digestion

LC/ ESI-MS

Data analysis

Genus Takifugu (except for Takifugu chrysops) Forward primer for Takifugu 5ʹ 3ʹ 26 bp

Dra I digestion

Msp I digestion

TTT pAAA AAAp TTT

CCG pCGG GGCp GCC

53–54 bp

37–38 bp

3ʹ 5ʹ

Reverse primer

Takifugu chrysops Forward primer for Takifugu 5ʹ 3ʹ

Msp I digestion CCG pCGG GGCp GCC

TCTAAA AGATTT

37 bp

3ʹ 5ʹ

Reverse primer

79 bp

Genus Lagocephalus Forward primer for Lagocephalus

Msp I digestion CCG pCGG GGCp GCC

5ʹ 3ʹ 52 bp

36 bp

3ʹ 5ʹ

Reverse primer

Figure 1

26


Page 26 of 33

Page 27 of 33


Relative intensity

20 °C

60 °C

40 °C

1.04 ×107

4.53 ×106

1.08 ×107

0 1.06 ×105

0 2.43 ×104

0 7.86 ×104

26 bp

0 6.80 ×104 37 bp 0 7.43 ×104

53 bp

0 6.50 ×104

m/z = 2835.720 + 2866.976

37 bp

37 bp 0 2.52 ×104

0 8.45 ×104 53 bp

53 bp 0 6

m/z = 2641.785 + 2695.111

26 bp

26 bp 0 2.04 ×104

0 3

TIC

m/z = 1163.548 + 1175.827

0 3

6

3

6

Time / min

Figure 2

27



Figure 3

28


Page 28 of 33

Page 29 of 33


Draw TIC chromatogram

≤1

Peak Number at RT = 4–5.5 min

3

Negative

(genus Takifugu) 2 (Takifugu chrysops or genus Lagocephalus)

Monoisotopic mass of the 3rd peak(Da)

Monoisotopic mass of the 2nd peak (Da)

16443.599–16443.697 16452.610–16452.709 16467.610–16467.709 16468.605–16468.704

T. poecilonotus T. exascurusa

16400

6

16500 16599.751–16599.851 16780.667–16780.767

16200

T. vermicularis

16131.537–16131.634 16137.525–16137.622 16146.537–16146.634 16177.531–16177.629

T. chrysops

24173.034–24173.179 24566.905–24567.053

T. porphyreus T. obscurusa

L. lunaris

L. gloveri

L. wheeleri

15890.707–15890.803 15921.702–15921.797 15930.713–15930.809 15937.697–15937.792

16000

16100

16300

6

L. inermis

15900

5 Time / min

5 Time / min

T. rubripes

4

4

16295.706–16295.804 16296.701–16296.799 16311.701–16311.799 16320.713–16320.811

Monoisotopic mass of the 2nd peak (Da)

4

11300

5 Time / min

11466.875–11466.944 11500 11600

11770.920–11770.991 11800

Figure 4

29


T. niphobles

11654.908–11654.978 11700

T. pardalis T. snyderi T. ocellatusa T. xanthopterusa T. stictonotusa

11341.851–11341.919 11400

6


a 36 nt, reverse strand [M-4H]4-, ∆ = 25.006 (Da)

L. wheeleri : L. lunaris

L. wheeleri 0.48 ppm

b 52 nt, forward strand [M-5H]5- , ∆ = 6.983 (Da) L. lunaris 0.29 ppm


L. lunaris 0.70 ppm

4:1


19:1

2760

L. wheeleri NA


L. lunaris NA



L. lunaris NA

2770

c 52 nt, reverse strand [M-5H]5- , ∆ = 5.988 (Da)


L. lunaris 0.44 ppm

1:1


L. lunaris NA

3180

3190

Figure 5

30


Page 30 of 33

3220

L. lunaris NA

L. lunaris NA

L. lunaris NA

3230

m/z

Page 31 of 33


Figure 6

31


Relative intensity


2.39×106

Negative control

0 3.60×107

T. rubripes

0 4.11×107

T. porphyreus

0 3.95×107

T. vermicularis

0 3.65×107

T. pardalis

0 1.63×107

T. snyderi

0 3.55×107

T. poecilonotus

0 3.07×107

L. wheeleri

0 6.17×106

Atlantic mackerel (Scomber scombrus)

0 4.15×106 0 4.63×106 0 2.09×106 0 1.86×106

0 2.67×106

Japanese horse mackerel (Trachurus japonicus)

Japanese pilchard (Sardinops melanostictus)

Skipjack tuna (Katsuwonus pelamis)

Yellowfin tuna (Thunnus albacares)

Japanese amberjack (Seriola quinqueradiata)

0 6.51×106

Atlantic salmon (Salmo salar)

0 2.81×106

Flying fish (Cypselurus pinnatibarbatus japonicus)

0

3.5

4.0

4.5 5.0 Time / min

5.5

6.0

Figure 7

32


Page 32 of 33

Page 33 of 33


Graphic for TOC

33


Genotyping of Toxic Pufferfish Based on Specific ... - ACS Publications

Recommend Documents