Determination of Phytochelatins in Rice by Stable Isotope Labeling

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Determination of Phytochelatins in Rice by Stable Isotope Labeling Liquid Chromatography Mass Spectrometry Ping Liu, Wen-Jing Cai, Lei Yu, Bi-Feng Yuan, and Yuqi Feng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01797 • Publication Date (Web): 13 Jun 2015 Downloaded from http://pubs.acs.org on June 19, 2015

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Journal of Agricultural and Food Chemistry

Determination of Phytochelatins in Rice by Stable Isotope Labeling Liquid Chromatography Mass Spectrometry

Ping Liu, Wen-Jing Cai, Lei Yu, Bi-Feng Yuan, Yu-Qi Feng* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China

*To whom correspondence should be addressed. Tel.: +86-27-68755595; fax: +86-27-68755595. E-mail address: [email protected].

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ABSTRACT

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A highly sensitive method was developed for the detection of phytochelatins (PCs) in rice by

3

stable isotope labeling coupled with liquid chromatography-electrospray ionization-tandem mass

4

spectrometry

5

(ω-bromoacetonylquinolinium bromide, BQB and BQB-d7) were used to label PCs in plant sample

6

and standard PCs, respectively, and then combined prior to LC/MS analysis. The heavy labeled

7

standards were used as the internal standards for quantitation to minimize the matrix and ion

8

suppression effects in MS analysis. In addition, the ionization efficiency of PCs was greatly

9

enhanced through the introduction of a permanent charged moiety of quaternary ammonium of

10

BQB into PCs. The detection sensitivities of PCs upon BQB labeling improved by 14–750 folds

11

and therefore PCs can be quantitated using only 5 mg plant tissue. Furthermore, under cadmium

12

(Cd) stress, we found that the contents of PCs in rice dramatically increased with the increased

13

concentrations and treatment time of Cd. It was worth noting that PC5 was firstly identified and

14

quantitated in rice tissues under Cd stress in current study. Taken together, this IL-LC-ESI-MS/MS

15

method demonstrated to be a promising strategy in detection of PCs in plant with high sensitivity

16

and reliability.

(IL-LC-ESI-MS/MS)

analysis.

A

pair

of

isotopes

labeling

17 18

KEYWORDS: phytochelatins, rice, cadmium stress, isotope labeling, mass spectrometry

19

2


reagents

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INTRODUCTION

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Phytochelatins (PCs) are endogenous detoxification compounds produced by plants, algae, and

22

fungi in response to heavy metal exposure.1 The general structure of PCs is polypeptide with the

23

amino acid sequence of (γ-Glu-Cys)n-Gly, where n=2-11, typically 2-6 in the plant.2, 3 PCs exhibit

24

high affinity for various metals including cadmium (Cd), arsenic (As), copper (Cu), and zinc (Zn)

25

via sulfhydryl and carboxyl residues.1 Cd is a non-essential trace element and its environmental

26

concentrations are approaching toxic levels, especially in some agricultural soils. Cd can be easily

27

absorbed by plants and bioconcentrated through food chain.4 Since rice is the most important cereal

28

crop and provides main food source in the south-east Asia, highly sensitive profiling of PCs in rice

29

is essential to elucidate the mechanisms of PCs in resisting Cd stress.

30

The developed methods to assay PCs mainly include liquid chromatography (LC) with

31

ultraviolet (UV),5-7 fluorescence (FLD)8-11 or electrochemical detection (ED).12-15 However, matrix

32

interferences coeluted could affect the accurate determination of analytes using these methods. For

33

reliable identification and quantitation of PCs, methods based on liquid chromatography/mass

34

spectrometry (LC/MS) were developed.3, 16-19 However, the quantitative profiling of metabolites by

35

MS is still challenging because the MS responses of metabolites fluctuate and ionization efficiencies

36

alter even under the same liquid chromatography conditions.20 The use of isotope internal standards

37

provides high accuracy of quantitative measurement by revising the ion signal differences caused

38

by matrix and ion suppression effects. It is worth noting that previous MS-based studies of PCs

39

often used compounds containing the similar structure with PCs as the internal standards, i.e.,

40

glycine-13C2-GSH17 or methionine sulfon.18 In addition, external standard methods were also

41

developed for the PCs quantitation since the isotope standards of PC were not commercially

42

available,3, 16, 19 which, however, may compromise the accuracy and precision of the quantitation. 3



43

To resolve these problems, stable isotope labeling strategy has been developed for quantitative

44

profiling of targeted molecules with the MS-based platform.21 The typical method of differential

45

isotope labeling normally uses chemical labeling to introduce a light isotope tag to the analytes in

46

sample and a heavy isotope tag to standards, followed by mixing the light labeled sample and heavy

47

labeled standards for MS analysis. The heavy labeled standards can be used as the internal standards

48

to solve the problems of inaccurate or imprecise quantitation caused by the matrix and ion

49

suppression effects. In addition, isotope labeling can also improve the ionization efficiencies of

50

metabolites due to the addition of ionizable functional groups. For example, Ogawa and co-workers

51

reported the use of 4-(4’-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) and its

52

deuterated analog 2H4-DAPTAD for the quantitation of vitamin D3 metabolites in urine samples.22

53

Zhang et al. described an LC/MS method for analysis of steroid hormones by chemical labeling

54

with 4-(dimethylamino)-benzoic acid (DMBA) and d4-DMBA, and the detection sensitivity could

55

be enhanced by more than 103- to 104-folds compared to the underivatized counterparts.23 Recently,

56

our group have also used the stable isotope labeling strategy coupled with LC/MS analysis for the

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determination of phytohormones, carboxyl- and thiol-containing metabolites in plant and human

58

urine samples.20, 24, 25

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In this work, we developed a novel strategy by combing stable isotope labeling with LC/MS

60

analysis to improve the identification and quantitation of PCs in rice samples. The schematic

61

diagram of the principle of this method was shown in the Figure 1. A pair of isotopes labeling

62

reagents of BQB and BQB-d7 were used to label the analytes in sample and PC standards,

63

respectively, and then combined before LC/MS analysis. In this respect, the heavy labeled standards

64

were used as the internal standards for quantitation to minimize the matrix and ion suppression

65

effect in MS analysis. In addition, the ionization efficiency of PCs was also greatly enhanced 4


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through the introduction of a permanent charged moiety of quaternary ammonium of BQB into the

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analytes. The established method was successfully applied to the sensitive quantitation of

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endogenous PCs in rice under Cd stress with different Cd concentrations and treatment time.

69 70

MATERIALS AND METHODS

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Chemicals and reagents

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Phytochelatins (PC2, PC3, PC4, PC5 and PC6) were purchased from Anaspec (California, USA).

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Glycine and tris(2-carboxyethyl)phosphine hydrochloride salt (TCEP) were purchased from Sigma

74

(St. Louis, MO, USA). Formic acid and ethylenediaminetetraacetic acid (EDTA) were purchased

75

from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Chromatographic grade

76

acetonitrile (ACN) was purchased from TEDIA Co. Inc. (Ohio, USA). All other solvents and

77

chemicals used were of analytical grade. The water used throughout the study was purified by a

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Milli-Q apparatus (Millipore, Bedford, MA). Stock standard solutions of PCs were prepared in 1.0

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mM EDTA solution containing 0.05% formic acid at a concentration of 1.0 mM.

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Rice culture and treatments

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Rice seedings (Oryza sativa L. cv. Zhenghan No. 2) seeds were germinated and grown by

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hydroponic culture in Hoagland’s nutrient solution in a growth chamber with 70-80% relative

83

humidity under a 16 h light (28 oC) / 8 h dark (25 oC) photoperiods according to previously

84

described method with some modifications.26 The Hoagland´s nutrient solution contains NH4NO3

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1.425 mM, NaH2PO4 0.323 mM, K2SO4 0.513 mM, CaCl2 0.998 mM, MgSO4 1.643 mM, MnCl2

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0.008 mM, (NH4)6Mo7O24 0.062 µM, H3BO3 0.015 mM, ZnSO4 0.122 µM, CuSO4 0.124 µM, FeCl2

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0.038 mM and citric acid 0.062 mM. After 10-days growing, the seedings with uniform size were

88

subjected to Cd stress with different concentrations of CdNO3 (0, 10, 100, 500 and 1000 µM). Rice 5



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seedings were harvested after Cd stress for 3 hours, 3 days and 7 days, and then weighted,

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immediately frozen in liquid nitrogen, and stored at -80 oC.

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Sample preparation

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Sample extraction was carried out according to a previously described method with slight

93

modifications.7 Plant tissues (leafs and stems) were frozen in liquid nitrogen and grounded into fine

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powder with a mortar and pestle. Then 5 mg of plant powder was transferred into a 0.6-mL

95

centrifuge tube and extracted with 50 µL of 0.1 M cold HCl (4 oC) containing 1 mM EDTA for 30

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min. The exacted sample was centrifuged at 12,000 rpm for 30 min under 4 °C. Then 30 µL of

97

supernatant was added with 1 M NaOH (2 µL) to adjust the pH to 2 and then treated with 10 nmol

98

of TCEP (10 mM, 1 µL) under 45 °C for 60 min.

99

Chemical labeling

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The chemical labeling procedure was performed according to our previous work.27 Briefly, 20

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nmol of BQB (1 mM, 20 µL) was added into a 1.5 mL tube and dried under nitrogen gas.

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Subsequently, 90 µL of Gly-HCl buffer solution (5.0 mmol/L, pH 3.5) and 10 µL of treated plant

103

extracts were added. The mixture was incubated at 60 °C for 60 min with shaking at 1,500 rpm. The

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BQB-d7 labeled PCs were added to serve as internal standards for the quantitation. Then 20 µL of

105

samples were subjected to LC/MS analysis.

106

LC-ESI-MS/MS analysis

107

Analysis of samples was performed on the LC-ESI-MS/MS system consisting of an AB 3200

108

QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA) with an electrospray

109

ionization source (Turbo Ionspray) and a Shimadzu LC-20AD HPLC (Tokyo, Japan) with two

110

LC-20AD pumps, a SIL-20A auto sampler, a CTO-20AC thermostated column compartment and a

111

DGU-20A3 degasser. Data acquisition and processing were performed using AB SCIEX Analyst 6


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1.5 Software (Applied Biosystems, Foster City, CA, USA). The HPLC separation was performed on

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a Shimadzu VP-ODS column (150 mm × 2.0 mm i.d., 5 µm, Tokyo, Japan) with a flow rate of 0.2

114

mL/min at 30 oC. Formic acid in water (0.1%, v/v, solvent A) and acetonitrile (solvent B) were

115

employed as mobile phase. A gradient of 0-20 min 5% to 40% B, 20-25 min 40% to 60% B, 25-26

116

min 60% to 5% B, and 26-40 min 5% B was used.

117

All BQB labeled PCs were quantitated by multiple reaction monitoring (MRM) mode in

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positive mode. The optimal ESI source conditions were as follows: turbo heater temperature (TEM)

119

550°C, ion spray voltage 4500 V, curtain gas 30 psi, nebulizing gas (gas 1) 40 psi and heated gas

120

(gas 2) 60 psi.

121

Method validation

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To evaluate the linearity of the method, standards of PCs-BQB at concentrations of 1, 2, 5, 10,

123

20, 50, 100, 200, 500 nM fixed amounts of internal standards (PC2,

3, 4, 6-BQB-d7,

50 nM;

124

PC5-BQB-d7, 10 nM) were used to construct calibration curves by plotting the peak area ratio

125

(analyte/IS) against the PCs-BQB concentrations with triplicate measurements. The residual plots

126

were also considered and the Durbin–Watson test was applied to prove linearity. The limits of

127

detection (LOD) and limits of quantitation (LOQ) were determined at a concentration where the

128

S/N ratios were 3 and 10, respectively.

129

The accuracy and precision of the developed method were assessed by the recoveries and by

130

the intra- and inter-day relative standard deviations (RSDs). Both recoveries and intra- and inter-day

131

RSDs were calculated with PC standards spiked in rice tissue samples at three different

132

concentrations. For each concentration, triplicate measurements were performed. The intra-day

133

variation was evaluated by repeating the process for three times within one day, and the inter-day

134

variation was investigated on three successive days. 7



135 136

RESULTS AND DISCUSSION

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MS behavior of BQB labeled PCs

138

The typical chemical labeling of PCs by BQB was shown in the Figure 2. The results show that

139

all the thiol groups from the PCs could react with BQB, and the derivatives had several parent

140

molecule ions with different charge states in the positive ESI-MS (Figure S1). This phenomenon

141

might be attributed to the fact that the proton release of the carboxy groups of derivatives during the

142

ionization process, and then result in the derivatives being contained one or several negative

143

charges. It is benificial in identifying metabolites by multi-peaks in one analysis. Meanwhile, we

144

found that the intensity of ions from derivatives with the most charge states were highest among the

145

multiple parent molecule ions.

146

All the parent molecule ions were subjected to ESI-MS/MS analysis to investigate the

147

fragmentation behavior of BQB-labeled PCs. Figure 3 depicts the fragment ion spectra of BQB

148

labeled PC2 ([M]2+), PC3 ([M]3+), PC4 ([M]4+), PC5 ([M]5+) and PC6 ([M]6+). The results show that

149

two common product ions at m/z 130.1 and 218.1 were observed for all the derivatives. The product

150

ion of m/z 130.1 was assigned to the fragment ion generated from the quinoline moiety of BQB, and

151

m/z 218.1 was assigned to the fragmentation of the C-S bond in derivatives. However, compared

152

with the BQB labeled PCs, the fragment ion spectra of unlabeled PCs were much complex (Figure

153

S2).

154

Enhancement of detection sensitivity upon chemical labeling

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The PCs can be ionized in ESI-MS with positive ion mode. The MRM transitions of 540.1→

156

336.0, 772.2→233.2, 1004.3→233.1, 1236.4→464.9, and 1468.4→772.0 for unlabeled PC2, PC3,

157

PC4, PC5, and PC6were extracted, respectively, due to the highest signal intensity in the LC-MS 8


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158

analysis (Figure 4A). However, the ionization efficiency is normally poor, and the ionization

159

efficiency dropped from PC2 to PC6. Previous studies demonstrated that introducion of eaisly

160

ionizable group into target componds by chemical labeling could obviously increase the detection

161

sensitivities of analytes during ESI-MS analysis.28-33 In this respect, chemical labeling of PCs by

162

BQB that has a permanent charged moiety of quaternary ammonium may also largely enhance the

163

ionization efficiencies of thiol compounds during ESI-MS analysis.

164

Here we used the PC standards (PC2, PC3, PC4, PC5 and PC6) to evaluate the enhancement of

165

detection sensitivity upon chemical labeling by BQB under MRM mode. The detailed MRM

166

conditions of BQB labeled and unlabeled PCs were shown in the Table 1 and Table S1, respectively.

167

Figure 4B shows the extracted ion chromatograms of BQB labeled PC standards under MRM

168

transitions. The transitions of [M]n+→218.1 for BQB labeled PCs were extracted due to the high

169

signal intensity and low matrix interferences (Figure 4B). Although the PCs concentrations labeled

170

with BQB were 100-folds lower than unlabeled PCs, MS responses of BQB labeled PCs were much

171

greater improved. We calculated the LODs of the unlabeled and BQB labeled PCs under their own

172

optimized MRM conditions and the results show that the signal intensities of BQB labeled PCs

173

improved by 14.3 to 750.0 folds compared to the unlabeled PCs (Table 2). As shown in Figure 4B,

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the higher PC derivatives exhibited a strong peak tailing. The phenomenon may be due to the

175

residue Si-OH of silica-based stationary phase, which may strongly interact with the positively

176

charged group(s) of derivatives. The higher PCs of derivatives have more numbers of the charged

177

groups that lead to the strong peak tailing. We also found that the addition of formic acid at higher

178

concentration in the mobile phase could inhibit the tailing, however, it also caused the MS

179

intensities decreased. Take the good peak shape and high intensities for consideration, we selected

180

the formic acid concentration in the mobile phase was at 0.1% (v/v). 9



181

Stability of BQB labeled PCs

182

The stability of the derivatives may greatly influence the reproducibility and sensitivity in

183

determination of PCs. To examine the stability of the derivatives, the derivatives were left for 4 oC

184

and determined every 24 hours. The results indicated that the BQB derivatives of PCs could be

185

stored at 4 oC for at least one week without decrease of signal intensities (Figure S3). The RSDs

186

were 2.2, 5.9, 3.3, 5.1, and 1.4 for the BQB labeled PC2, PC3, PC4, PC5, and PC6, respectively. It is

187

worth noting that the PCs-BQB derivatives should be placed in the vial of poly ethylene to avoid

188

the adsorption by the glass (Si-OH) vial.

189

Optimization of the TCEP and BQB

190

We investigated the effects of the contents of TCEP and BQB on the efficiencies of reduction

191

of disulfide bonds and chemical labeling. Plant extract (without Cd stress) spiked with PCs (PC2-5,

192

0.1 µM; PC6, 0.5 µM) was used as the samples. To minimize the effect of instrumental variation, the

193

same amounts of BQB-d7-labeled PCs (PC2-5, 0.1 µM; PC6, 0.5 µM) were used as internal standards

194

and added to the samples prior to LC/MS analysis.

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PCs are sensitive to oxidation during plant sample preparation and storage. The oxidation can

196

occur in both intermolecular and intramolecular reactions via disulfide bond formation. El-Zohri et

197

al. found intermolecular-oxidized PC2 at m/z 1077 and PC3 at m/z 1543,16 and the

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intramolecular-oxidized PC2 at m/z 770 were also observed in previous study.3 Thus, in the current

199

study, the reducing agent was added to avoid the oxidation of PCs before chemical labeling. The

200

reducing agent of TCEP ranging from 0 to 50 nmol were investigated and the content of BQB was

201

fixed at 30 nmol. As shown in Figure S4A, the peak intensity ratio (analyte/IS) of PCs derivatives

202

did not obvisouly change with the increase of TCEP from 0 to 10 nmol. We reason that the PC

203

standards added in the plant extracts were all reduced PCs and were labeled with BQB immediately. 10


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However, the peak intensity ratio (analyte/IS) dropped with the increase of the TCEP amounts from

205

10 to 50 nmol, especially for PC6, indicating that the efficiencies of chemical labeling may be

206

suppressed by the excess content of TCEP. Consequently, 10 nmol of TCEP was used in the

207

following experiments.

208

Because plant sample mixture was much more complex than pure standards and BQB would

209

be consumed by the target compounds as well as other thiol-containing interferents in plant matrix,

210

the amounts of labeling reagent were further optimized in the range of 1-30 nmol. As shown in

211

Figure S4B, the peak intensity ratio (analyte/IS) of derivatives increased as the increase of BQB

212

content from 1-10 nmol and then reached a plateau when BQB contents exceeded 10 nmol. The

213

highest chemical labeling efficiencies of PC were achieved using 10 nmol of BQB. For more

214

reliable quantitation of PCs in plant tissue, we used 20 nmol of BQB in the following experiments.

215

Method validation

216

A series of experiments including linearity, LODs and LOQs, recoveries and precisions were

217

performed to validate the proposed method under optimized conditions. The resutls show that good

218

linearity was obtained with regression coefficient (R2) higher than 0.9931 (Table S2). The residual

219

plots show that a plot of the standardized residual versus the standardized predicted value revealed

220

no particular pattern (i.e. data were randomly distributed around 0), no more than 5% of cases had a

221

standardized residual absolute value above 2, and the Durbin–Watson statistic were 2.1, 2.2, 1.2, 2.3,

222

and 2.3 for the PC2, PC3, PC4, PC5, and PC6, respectively (between 1.5 and 2.5), indicating that the

223

residuals were uncorrelated (Figure S5).34 The LODs and LOQs for five PCs were in the range of

224

0.1-1.6 nM and 0.4-5.0 nM, respectively. Table 3 shows the comparison of the LODs obtained

225

between our method and other methods reported in literature.3,

226

obtained by the current method are much lower than those given in literatures, especially for the 11


11, 15, 17-19

Generally, the LODs


227

LODs of PC4, PC5 and PC6, which were about 7-17 times lower than the lowest values reported in

228

previous studies. 3, 15, 18

229

The recoveries of five PCs were in the range of 71.6-93.1%, and the intra- and inter-day RSDs

230

were less than 9.8% and 16.3%, respectively (Table S3), indicating that the accuracy and precision

231

of the proposed method were satisfactory for the determination of PCs in plant samples.

232

Quantitation of endogenous PCs in plant samples

233

In the present work, after 10-day growing, rice samples were treated with Cd at different

234

concentrations (0, 10, 100, 500 and 1000 µM) and collected after Cd stress for 3 hours, 3 days and 7

235

days. The real rice pictures under Cd stress with different concentrations and time was shown in the

236

Figure S6. As can be seen, the rice under all the Cd concentration show same appearance (green) at

237

the Cd treatment time of 3 h and Day 3. However, at the treatment time of Day 7, the rice leaves

238

turned yellow starting from the 100 µM Cd concentration, and the inhibitory effects in plant growth

239

were also observed at the Cd concentrations of 500 and 1000 µM.

240

The concentrations of the PCs in those rice tissues were determined by the developed method.

241

Figure 5 shows the typical extracted ion chromatogram of five PCs and their respective internal

242

standards from the plant treated with 100 µM Cd (Day 3). As can be seen, the BQB labeled PCs and

243

the IS (BQB-d7 labeled PCs) were coeluted on the chromatography, and the chromatographic

244

isotope effect of BQB and BQB-d7 labeled standards was about 0.1 min.

245

The quantitation results (Figure 6, Table S4 in Supporting Information) show that only PC2 and

246

PC3 were observed in the control samples (0 µM Cd), indicating that PC2 and PC3 can be

247

synthesized in plant even without heavy metal stress. For the rice under Cd stress, compared with

248

the control samples, the contents of PC2 and PC3 under Cd stress at the concentrations of 10-1000

249

µM dramatically increased. 12


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PC4 and PC5 were also detected in Cd stressed rice. It is worth noting that PC5 was firstly

251

detected in the rice tissues in our study. PC4 and PC5 were induced by the Cd concentration greater

252

than 10 µM, and PC4 could also be found in rice under 10 µM Cd at the treatment time of Day 3

253

and Day 7. The results indicate that PC4 was more easily synthesized than PC5 in rice. The

254

concentrations of PC2, PC3 and PC4 in rice at 100 µM Cd stress (Day 7) were consistent with

255

previous work for the measurements of PCs in rice stems with 50 µM Cd stress for 7 days.3

256

Generally, the contents of PCs in rice increased with the increase of the concentration and treatment

257

time of Cd, indicating that the PCs have the role of detoxification for plants under Cd stress.1

258

Comparison of the developed method with previous methods

259

Compared with previously published methods, our method has two major advantages. Firstly,

260

the detection sensitivity was significantly improved. As can be seen in the Table 3, the LODs of PC4,

261

PC5 and PC6 obtained by the current method were 7, 8 and 17 times lower than the lowest values

262

reported in the previous studies, respectively, and the LODs of PC2 and PC3 were also comparable

263

with those lowest values. Due to the high detection sensitivity, PCs can be easily quantitated using

264

only 5 mg rice tissues and PC5 was firstly detected in the rice tissues. Secondly, the accuracy for the

265

identification and quantitation of PCs were also enhanced. The PC standards labeled with the

266

BQB-d7 were used as the isotope internal standards, which can aviod matrix and ion suppression

267

effects in MS analysis (Figure 6). Taken together, the method can significantly improve the

268

detection sensitivity and the accuracy for the identification and quantitation of PCs in plants.

269

In conclusion, we developed a stable isotope labeling method combined with LC/MS analysis

270

for the sensitive detection of PCs in rice. The plant samples and PC standards were labeled with

271

BQB and BQB-d7, respectively, then mixed and analyzed by LC/MS. The BQB labeling improve

272

the detection sensitivities of PCs by 14–750 folds due to the introduction of a permanent charged 13



273

moiety of quaternary ammonium, and the BQB-d7 labeled standards were used as the internal

274

standards for quantitation to minimize severe matrix effect. Using the developed method, four

275

endogenous PCs (PC2, PC3, PC4 and PC5) were sucessfully identified and quantitated in only 5 mg

276

rice samples under Cd stress, and PC5 was firstly reported existence in rice tissues by this method.

277

The contents of PCs dramatically increased as the Cd concentrations and treatment time increased,

278

suggesting PCs have the roles of detoxification in plants under Cd stress. This highly sensitive and

279

reliable method can also be extended for the identification and quantitation of PCs in other samples.

280 281

ACKNOWLEDGMENTS

282

The authors thank the financial support from the National Basic Research Program of China

283

(973 program) (2012CB720601, 2013CB910702), the National Natural Science Foundation of

284

China (21475098, 91217309), and the Natural Scinence Foundation of Hubei Province, China

285

(2014CFA002).

286 287

Supporting Information Available: MRM transitions for PCs data (Table S1), data for calibration,

288

LOD and LOQ (Table S2), data for recoveries and precisions (Table S3), PCs in rice (Table S4), MS

289

spectra of BQB labeled PCs (Figure S1), MS/MS spectra of the unlabeled PCs (Figure S2), stability

290

of BQB (Figure S3), optimization of TCEP and BQB contents (Figure S4), residual plots (Figure

291

S5), pictures of rice under Cd stress (Figure S6). This material is available free of charge via the

292

Internet at http://pubs.acs.org.

293 294

AUTHOR INFORMATION

295

Corresponding Authors 14


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*Tel.: +86-27-68755595. Fax: +86-27-68755595. E-mail: [email protected].

297

Notes

298

The authors declare no competing financial interest.

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15



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402 403 404

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405

FIGURE LEGENDS

406

Figure 1. The schematic diagram of the principle of stable isotope labeling coupled with LC/MS

407

analysis for PCs determination.

408 409

Figure 2. Chemical labeling reaction for PCs using BQB.

410 411

Figure 3. The MS/MS spectra of the BQB labeled PCs. A, PC2-BQB; B, PC3-BQB; C, PC4-BQB; D,

412

PC5-BQB; E, PC6-BQB.

413 414

Figure 4. Extracted-ion chromatograms between unlabeled and BQB labeled PC standards. (A)

415

Unlabeled PC2-5, 10 µM; PC6, 50 µM, 10 µL; (B) BQB labeled PC2-5, 0.1 µM; PC6, 0.5 µM, 10 µL.

416 417

Figure 5. The typical extracted ion chromatogram of PCs from the plant treated with 100 µM Cd

418

(Day 3) and their corresponding internal standards.

419 420

Figure 6. The contents change of PCs in rice with Cd stress at different concentrations and time.

19



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Table 1. The MRM transitions and optimal parameters of mass spectrometry for BQB labeled PCs. PCs-BQBf

Charge

Precursor ion

PC2-BQB

[M]2+

453.9

[M-H]+

906.7

[M]3+

441.3

[M-H]2+

661.3

[M]4+

435.1

[M-H]3+

579.6

[M-2H]2+

869.1

[M]5+

431.3

[M-H]4+

539.0

[M-2H]3+

718.4

[M]6+

428.6

[M-2H]4+

643.0

[M-3H]3+

856.9

PC2-BQB-d7

[M]2+

460.8

PC3-BQB-d7

[M]3+

448.4

PC4-BQB-d7

[M]4+

442.2

PC5-BQB-d7

[M]5+

438.4

PC6-BQB-d7

[M]6+

436.0

PC3-BQB

PC4-BQB

PC5-BQB

PC6-BQB

Product ion 130.1 218.1 777.4 749.3 130.1 218.1 130.1 596.8 130.1 218.1 130.1 536.9 804.3 740.0 130.1 218.1 218.1 499.9 130.1 675.2 130.1 218.1 130.1 578.0 814.0 137.1 225.1 137.1 225.1 137.1 225.1 137.1 225.1 137.1 225.1

DP / V

EP / V

50

7

90

10

45

6

55

7

41

7

41

8

100

7

45

6

38

6

50

7

40

7

37

7

90

5

50

7

45

6

41

7

45

6

40

7

20


CEP / V

CE / V

CXP / V

15

40

3

15

35

3

37

40

7

37

50

7

20

35

3

20

37

3

25

55

3

60

30

8

19

35

3

17

35

3

26

50

3

26

30

6

36

32

7

36

38

8

20

35

3

20

35

3

25

45

3

25

35

7

31

65

4

31

40

8

20

35

3

20

35

3

20

50

3

20

35

8

36

32

7

15

40

3

15

35

3

20

35

3

20

37

3

19

35

3

17

35

3

20

35

3

20

35

3

20

35

3

20

35

3

Page 21 of 29


Table 2. Comparison of LODs of unlabeled and BQB labeled PC standards under their own optimized MS conditions. LODs (nmol) PCs

Improved folds Unlabeled

BQB labeled

PC2

6.0×10-5

2.4×10-6

25.0

PC3

1.0×10-4

7.0×10-6

14.3

PC4

3.5×10-4

6.2×10-6

56.5

PC5

2.2×10-3

6.8×10-6

323.5

PC6

2.4×10-2

3.2×10-5

750.0

21



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Table 3. Comparison of limits of detection (LODs) with previous studies. LODs (fmol) Separation

Detection

References PC2

PC3

PC4

PC5

PC6

RP-HPLC

ESI-MS/MS

2.4

7.0

6.2

6.8

32.0

Present method

HILIC

ESI-MS/MS

20.0

60.0

75.0

375.0

530.0

3

RP-HPLC

FLD

140.0

140.0

140.0

RP-HPLC

ED

78.0

6.0

86.0

CE

MS

3.0

39.0

42.0

RP-HPLC

ESI-MS/MS

30.6

100.8

19

RP-HPLC

ESI-MS/MS

8800.0

2400.0

17

22


11

59.0

15

18

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Figure 1.

23



Figure 2.

24


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Figure 3.

25



Figure 4.

26


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Figure 5.

27



Figure 6.

28


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TOC graphic:

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Determination of Phytochelatins in Rice by Stable Isotope Labeling

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