Comprehensive screening and identification of fatty acid esters of

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Comprehensive screening and identification of fatty acid esters of hydroxy fatty acids in plant tissues by chemical isotope labeling-assisted liquid chromatography-mass spectrometry Quan-Fei Zhu, Jing-Wen Yan, Tian-Yi Zhang, Hua-Ming Xiao, and Yu-Qi Feng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02839 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 28, 2018

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Analytical Chemistry

1

Comprehensive screening and identification of fatty acid esters of

2

hydroxy fatty acids in plant tissues by chemical isotope

3

labeling-assisted liquid chromatography-mass spectrometry

4 5

Quan-Fei Zhu,† Jing-Wen Yan,† Tian-Yi Zhang, Hua-Ming Xiao, Yu-Qi Feng *

6 7

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of

8

Education), Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China

9

† These authors contributed equally to this work.

10

*To whom correspondence should be addressed. Tel.: +86-27-68755595; fax:

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+86-27-68755595. E-mail address: [email protected].

12 13 14

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ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of lipid

17

mediators

with

promising

anti-diabetic

and

anti-inflammatory

18

Comprehensive screening and identification of FAHFAs in biological samples would

19

be beneficial to the discovery of new FAHFAs and enable greater understanding of

20

their biological functions. Here, we report the comprehensive screening of FAHFAs in

21

rice and Arabidopsis thaliana by chemical isotope labeling-assisted liquid

22

chromatography-mass spectrometry (CIL-LC-MS). Multiple reaction monitoring

23

(MRM) was used for screening of FAHFAs. With the proposed method, we detected

24

49 potential FAHFA families, including 262 regioisomers, in tissues of rice and

25

Arabidopsis thaliana, which greatly extends our knowledge of known FAHFAs. In

26

addition, we proposed a strategy to identify FAHFA regioisomers based on their

27

retention on a reversed-phase LC column. Using the proposed identification strategy,

28

we identified 71 regioisomers from 11 FAHFA families based on commercial

29

standards and characteristic chromatographic retention behaviors. The screening

30

technique could allow for the discovery of new FAHFAs in biological samples. The

31

new FAHFAs identified in this work will contribute to the in-depth study of the

32

functions of FAHFAs.

33

Keywords: FAHFA, chemical isotope labeling, mass spectrometry, chromatographic

34

retention behavior, plant

35 36 2


properties.

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37

Introduction

38

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous

39

lipids with important physiological functions1. It has been reported that FAHFAs can

40

improve glucose uptake from the blood, enhance insulin secretion and relieve

41

obesity-associated inflammation in mammals. Therefore, theses natural lipids are

42

expected to be used for diabetes therapy1,2. The molecular structure of FAHFA is a

43

combination of two fatty acyl chains through an ester bond. In recent years,

44

lipidomics has been used to identify a total of 18 FAHFA families consisting of 5 FAs

45

(PA, PO, OA, SA, and DHA) and 6 HFAs (HPA, HPO, HOA, HSA, HLA, and HDHA)

46

in different combinations (Figure S1)1,3. The full names of all the fatty acids can be

47

found in Table S1. Furthermore, each FAHFA family consists of different ester

48

regioisomers, which are distributed differently in specific tissues in vivo1. For

49

example, 13/12-, 11-, 10-, 9-, and 5-PAHSA are present in wild-type mice serum at

50

0.4-2.5 nmol/L. In white adipose tissue, 9-PAHSA is the most abundant isomer at 100

51

pmol/g; followed by 13/12-, 11-, 10-PAHSA (20%-30% of 9-PAHSA); and 8-, 7-,

52

5-PAHSA (2-3 pmol/g)1.

53

FAHFAs can be synthesized endogenously and obtained exogenously from

54

foods1. Yore et al. demonstrated that FAHFA can be synthesized in vivo with HFA as a

55

precursor1. In addition, several PAHSA regioisomers were detected in mouse feed,

56

apple and broccoli, suggesting that FAHFAs may exist naturally in other plants, and

57

that mammals could obtained FAHFA through dietary intake1. However, current

58

studies on FAHFAs mainly focus on mammals1,4-6, and little knowledge is available 3



59

on the existence of FAHFAs in plants. Previous studies have shown that FAs and

60

HFAs are abundant in plants7-9, and they may act as precursors for the synthesis of

61

FAHFAs in plants. In addition, the FAs and HFAs in plants tissues are different from

62

those in mammals. Therefore, screening of FAHFAs in plants could lead to the

63

discovery of some new FAHFA families or members.

64

Mass spectrometry (MS) is the most powerful analytical platform for

65

lipidomics10-12. In previous studies, full scan13,14, precursor ion scanning15 and neutral

66

loss scanning16 modes of MS were used for non-targeted lipidomics analysis to

67

discover and identify novel lipids. However, there are several difficulties in screening

68

and identifying FAHFAs in plants by traditional lipidomics methods: (1) the

69

ionization efficiency of FAHFAs under negative ion mode is poor in electrospray

70

ionization (ESI), and the aforementioned scanning modes lack sufficient detection

71

sensitivity for low-abundance FAHFAs (approximately 0.2–15 nmol/L in serum and

72

10–2500 pmol/g in mice tissues) in vivo1; (2) few FAHFA standards are commercially

73

available, and relevant information on FAHFAs is not recorded in databases; and (3) it

74

is very difficult to obtain MSn spectra of multiple regioisomers that elucidate the ester

75

bond location due to their low abundances17. Therefore, the discovery and

76

identification of new FAHFAs remain challenging.

77

Our previous study demonstrated the introduction of an appropriate functional

78

group on the FAHFA structure could improve its detection sensitivity and

79

chromatographic separation18. Based on this, we established a new strategy using

80

chemical isotope labeling-assisted liquid chromatography (CIL-LC-MS) for 4


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81

comprehensive screening and identification of FAHFAs in rice and Arabidopsis

82

thaliana.

83

2-dimethylaminoethylamine (DMED) and d4-DMED, were used to selectively and

84

efficiently react with the carboxyl group of FAHFAs (Figure S2). The

85

DMED/d4-DMED-labeled FAHFAs can generate two characteristic neutral losses

86

using collision-induced dissociation (CID). We designed the potential FAHFA

87

precursor ions by screening the HFAs (precursors of FAHFAs) with carbon-chain

88

lengths from C6 to C24 and then combining with the essential FAs reported in plants.

89

According to the characteristic neutral loss of DMED/d4-DMED-labeled FAHFAs in

90

CID, the corresponding product ions can be inferred. In this respect, non-targeted

91

screening of potential FAHFAs in plants can be performed with high sensitivity and

92

selectivity in multiple-reaction monitoring mode (MRM). Subsequently, we

93

investigated the LC retention behavior of DMED-labeled FAHFAs on a

94

reversed-phase column and found that the retention of FAHFA regioisomers was

95

related to the ester bond position and the number of carbon atoms in the structure.

96

Using the spectral data of identified FAHFAs (retention factors, ester position and

97

carbon atom number), we constructed a three-dimensional (3-D) prediction model to

98

putatively identify new FAHFA regioisomers. We found 49 FAHFA families (262

99

regioisomers) in rice and Arabidopsis thaliana, and 11 FAHFA families (71

100

regioisomers) were accurately identified. The established method is a promising

101

analytical platform for the discovery and identification of new FAHFA compounds in

102

plant tissues.

In

this

strategy,

a

pair

of

isotope-labeling

5


reagents,


Page 6 of 32

103 104

Experimental section

105

Chemicals and reagents

106

All of the FAHFA standards, including 13-PAHSA, 12-PAHSA, 10-PAHSA,

107

9-PAHSA, 5-PAHSA, 13-OAHSA, 12-OAHSA, 10-OAHSA, 9-OAHSA, 5-OAHSA,

108

13-SAHSA, 12-SAHSA, 10-SAHSA, 9-SAHSA, 5-SAHSA, 13-POHSA, 12-POHSA,

109

10-POHSA, 9-POHSA, 5-POHSA, and 9-PAHPA were purchased from Cayman

110

Chemical.

111

10-HDAHSA and 7-PDAHSA were synthesized by XuKang Medical Science and

112

Technology Co., Ltd. (Xiangtan, Hunan, China). 1H–NMR spectrum and MS spectra

113

were shown in Figure S3. 2-dimethylaminoethlamine (DMED) was supplied by J&K

114

Chemical (Beijing, China). The isotope reagent, d4-DMED was synthesized according

115

to our previously described method19.

(Arbor,

Michigan,

USA).

9-MAHSA,

9-PDAHSA,

9-PAHMA,

116

Chromatographic grade acetonitrile (ACN), acetone, isopropanol (IPA),

117

chloroform, methanol (MeOH) and ethyl acetate (EtOAc) were purchased from Tedia

118

Co. Inc. (Fairfield, OH, USA). Formic acid, ammonium hydroxide (NH3·H2O),

119

2-chloro-1-methylpyridinium iodide (CMPI), and triethylamine (TEA) were of

120

analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai,

121

China). The water used throughout the study was purified on a Milli-Q apparatus

122

(Millipore, Bedford, MA). Strong anion exchange solid phase extraction cartridges

123

(SAX SPE-cartridges, 3 mL, 200 mg) were supplied by Weltech Co. (Wuhan, China).

124

Stock solutions of all FAHFAs were prepared in EtOAc at a concentration of 200 6


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125

µg/mL for each and stored at -20°C. CMPI, TEA, DMED, and d4-DMED were

126

prepared in HPLC-grade ACN at 20 µmol/mL.

127

Plant materials

128

Rice (Oryza sativa ssp. indica cv. Zhenshan 97B) and Arabidopsis thaliana

129

(Columbia ecotype) were obtained from State Key Laboratory of Rice Biology at the

130

China National Rice Research Institute and grown in an artificial environmental

131

chamber greenhouse at 30°C under 16 h light/8 h dark photoperiods. 10 days old rice

132

leaves and 7 weeks old Arabidopsis thaliana were harvested, weighed, immediately

133

frozen in liquid nitrogen, and stored at −80°C.

134

Sample preparation

135

The overall procedure for sample extraction is summarized in Figure S4. First,

136

100 mg of plant materials (fresh weight) were frozen in liquid nitrogen, ground into

137

powder with liquid nitrogen, and transferred into a 5 mL centrifuge tube. Then, Bligh

138

and Dyer lipid extraction was performed20,21. A mixture of 1 mL MeOH, 1 mL H2O

139

and 2 mL chloroform was added to the plant powder sample tube. The mixture was

140

homogenized via ultrasonication for 30 min. The mixture was centrifuged, and the

141

organic phase containing the extracted lipids was collected and dried under a N2

142

stream. Subsequently, the extract was re-dissolved with 1 mL ACN containing 0.1%

143

NH3·H2O (v/v), followed by strong anion-exchange solid phase extraction (SAX SPE)

144

enrichment (3 mL, 200 mg). The SAX SPE-cartridge was preconditioned with 3 mL

145

ACN. The extract were introduced to the cartridge, and the cartridge was washed with

146

3 mL acetone/H2O (1/9, v/v), followed by 3 mL acetone. Analytes were eluted with 3 7



147

mL formic acid/acetone (1/99, v/v), and the eluate was evaporated to dryness at 40°C

148

under nitrogen stream.

149

According to our previous work, the purified extract was equally divided into

150

two portions and labeled with DMED and d4-DMED, respectively22. Briefly, 200 µL

151

ACN, 15 µL CMPI (20 µmol/mL) and 30 µL TEA (20 µmol/mL) were added to the

152

dried samples and mixed using a vortex mixer. The mixture was then incubated at

153

40oC for 5 min. Then, 30 µL DMED (20 µmol/mL) or d4-DMED (20 µmol/mL) was

154

added, and the mixture was incubated at 40°C for 1 h. Finally, the labeled solution

155

was dried under nitrogen gas.

156

Mass spectrometry analysis

157

The screening process was performed on a UHPLC-ESI-MS/MS system

158

consisting of a Shimadzu MS-8045 mass spectrometer (Tokyo, Japan) and a

159

Shimadzu LC-30AD HPLC system (Tokyo, Japan). The LC system was equipped

160

with two 30AD pumps, a SIL-30AC auto sampler, a CTO-20A thermostat column

161

compartment, and a DGU-20A5R degasser. Data acquisition and processing were

162

performed using LabSolutions software (version 5.53 sp2, Shimadzu, Tokyo, Japan).

163

For the screening of HFAs, LC separations were performed on an Acquity UPLC

164

BEH C18 column (2.1 × 50 mm, 1.7 µm, Waters) with a flow rate of 0.4 mL/min at

165

40°C. Formic acid in water (0.1%, v/v, solvent A) and ACN (solvent B) were

166

employed as mobile phases for the analysis of DMED-labeled HFA compounds. A

167

gradient for column equilibration of 0-2 min 5% B, 2-48 min 5% to 95% B, 48-54

168

min 95% B and 54-55 min 95% to 5% B was used. MRM analysis was performed 8


Page 8 of 32

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169

using positive ion mode. The source and ion transfer parameters were as follows: DL

170

temperature of 250°C; heat block temperature of 400°C; nebulizing gas flow of 3

171

L/min;

172

[DMED-HFA]+→[DMED-HFA － 63]+ and [d4-DMED-HFA]+→[d4-DMED-HFA －

173

67]+ or DMED- and d4-DMED-labeled HFA, respectively, were used as MRM ion

174

pairs. All precursor ions ([DMED-HFA]+ and [d4-DMED-HFA]+) for the MRM-MS

175

detection were selected according to the potential HFA structures, with carbon chain

176

lengths from C6 to C24. The acquired product ions were m/z [DMED-HFA－63]+) for

177

“light”-tagged and m/z [d4-DMED-HFA－67]+ for the “heavy”-tagged samples.

and

drying

gas

flow

of

15

L/min.

The

transitions

of

178

For the screening of FAHFAs, separations were also performed on an Acquity

179

UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm, Waters). Formic acid in ACN/water

180

(0.1%, 6/4, v/v, solvent A) and formic acid in IPA/ACN (0.1%, 9/1, v/v, solvent B)

181

were used as the mobile phases for the analysis of DMED-labeled FAHFA compounds.

182

A gradient for column equilibration of 0-26 min 20% to 90% B, 26-40 min 90% B,

183

and 35-37 min 90% to 20% B was used. The mobile phase flow rate was 0.4 mL/min,

184

and the column oven temperature was 40°C. For MRM screening of FA1HFA2, the

185

MRM ion pairs were selected based on the fragmentation pattern of DMED- and

186

d4-DMED-labeled FAHFA, respectively. Precursor ions were set as [DMED-FAHFA]+

187

and [d4-DMED-FAHFA]+ according to the desired m/z range, while product ions were

188

set as m/z [DMED-HFA－63]+ and m/z [d4-DMED-HFA－67]+ according to the

189

characteristic fragmentation pattern. The MRM parameters are listed in Table S2.

190

Calculation of retention factor 9



Page 10 of 32

191

For structure identification, the chromatographic retention factors (k) of FAHFA

192

candidates were calculated. The column dead time, tM, was measured using thiourea.

193

The retention time of FAHFA compounds, tR, were obtained by CIL-LC-MS analysis.

194

The calculation of retention factor is shown in Eq.1. ݇=

195 196

௧౎ ି௧౉ ௧౉

,

(1)

where tR is the retention time of an FAHFA compound, and tM is dead time.

197 198

Results and discussion

199

Overview of the strategy for screening and identifying FAHFAs

200

In this study, we established a chemical isotope labeling-assisted LC-MS method

201

for non-targeted screening and identification of trace-level FAHFAs in plant tissues

202

(Figure 1). To this end, Bligh and Dyer lipid extraction coupled with SAX-SPE was

203

employed to selectively extract and purify FAHFAs in rice and Arabidopsis thaliana

204

samples (For detail, see Figure S4). Then, the extract was divided into two equal

205

portions and labeled by DMED/d4-DMED reagents, respectively (For detail, see

206

Figure S2). Finally, the light and heavy labeled samples were mixed equally and

207

analyzed by LC-MS.

208

To determine possible components of FAHFAs, we first investigated the

209

distribution of HFAs in rice and Arabidopsis thaliana tissues. The precursor ion

210

information of DMED/d4-DMED-labeled HFAs, with carbon chain lengths from C6

211

to C24, was obtained; and the corresponding product ions of DMED- and

212

d4-DMED-labeled HFAs were speculated, based on the characteristic neutral loss of 10


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213

63 and 67 Da in CID. Thus, LC-MRM-MS strategy was used for the screening of

214

HFAs in rice and Arabidopsis thaliana tissues. Subsequently, we obtained the possible

215

precursor ions of the DMED/d4-DMED-labeled FAHFAs by combining the spectral

216

data of detected HFAs with that of reported essential FA species in plants, and the

217

corresponding product ions were inferred using the particular CID fragmentation

218

behavior of DMED- and d4-DMED-labeled FAHFAs. Therefore, a list of MRM

219

transitions for DMED- and d4-DMED-labeled FAHFAs could be constructed, and

220

MRM-MS detection was used for non-targeted screening of potential FAHFAs in rice

221

and Arabidopsis thaliana. The obtained peak pairs were extracted and only peak pairs

222

with similar retention times and intensities were considered FAHFA candidates.

223

Finally, the FAHFA candidates were confirmed using pure standards and comparing

224

the retention time, or putatively identified according to their retention behavior on a

225

C18 column.

226 227

Discovery of FAHFAs in plants

228

Fragmentation of DMED/d4-DMED-labeled FAHFAs

229

The fragmentation pattern of known FAHFAs provides important insight into the

230

discovery and identification of novel FAHFAs. Hence, we investigated the

231

fragmentation behavior of FAHFAs labeled with DMED and d4-DMED by tandem

232

MS analysis. Three FAHFA standards (9-PAHSA, 10-SAHSA and 9-PAHPA) were

233

used as the analytes. As shown in Figures 2A and 2B, the DMED/d4-DMED-labeled

234

9-PAHSA can produced dominant product ions at m/z 308.3 via CID. The product 11



Page 12 of 32

235

ions were formed through a neutral loss (63/67 Da) and a loss of PA fragment,

236

respectively. DMED/d4-DMED-labeled 9-SAHSA and 9-PAHPA exhibited similar

237

fragmentation patterns to those of DMED/d4-DMED-labeled 9-PAHSA (Figure

238

2C-2F). Therefore, the DMED/d4-DMED-labeled FAHFA generated characteristic

239

product ions (m/z [DMED-HFA－63]+ and m/z [d4-DMED-HFA－67]+) via CID,

240

which were formed through a neutral loss (63/67 Da) and a loss of FA fragment,

241

respectively. Using this unique fragmentation pattern, the m/z of potential FAHFAs

242

can be theoretically predicted if the FAHFA composition is known.

243

Screening analysis of hydroxy-fatty acids in plants

244

HFAs are important precursors for the synthesis of FAHFAs in vivo1,2. In order to

245

select possible precursor ions of FAHFAs, we investigated the distribution of HFAs in

246

tissues of rice and Arabidopsis thaliana.

247

We

investigated

the

MS

fragmentation

behaviors

of

DMED-

and

248

d4-DMED-labeled HFAs (3-hydroxystearic acid and 12-hydroxystearic acid). The

249

DMED/d4-DMED-labeled 3-HSA and 12-HSA generated a characteristic neutral loss

250

of 63.0/67.0 Da using CID (Figure S5) and formed the most abundant product ions at

251

m/z 308.3. With this fragmentation pattern, the m/z of the product ions of

252

DMED/d4-DMED-labeled HFAs can be easily predicted as m/z [M+H]+ 63.0/67.0, by

253

subtracting

254

DMED/d4-DMED-labeled HFA were [DMED-HFA]+/[d4-DMED-HFA]+, according

255

to the predicted structures of HFAs with carbon chain lengths from C6 to C24. The

256

corresponding product ions, m/z [DMED-HFA－63]+ and [d4-DMED-HFA－67]+ for

the

neutral

loss.

Therefore,

the

12


precursor

ions

of

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257

DMED- and d4-DMED-labeled compounds, respectively, were selected for MRM-MS

258

analysis.

259

Using this method, we determined the HFAs in tissues of rice and Arabidopsis

260

thaliana. The extracted ion chromatograms for DMED- and d4-DMED-labeled HFAs

261

are shown in Figure S6 in the Supporting Information. DMED- and d4-DMED-labeled

262

samples were combined 1:1 (v/v) and analyzed by LC-MS in MRM mode. The

263

extracted peak-pairs were selected as candidates of HFAs based on the following

264

criteria: they have a fixed mass difference of 4.0 Da (i.e., M d4-DMED-labeled－M

265

DMED-labeled ＝ 4.0 Da), approximately identical retention times and peak heights.

266

In total, 15 HFA families, including 86 regioisomers, were determined in the tissues

267

of rice and Arabidopsis thaliana (Table S3). These HFAs were used as a reference for

268

the precursor ions of potential FAHFAs in rice and Arabidopsis thaliana. Among the

269

discovered HFAs, 15 HFA families (86 regioisomers) were detected in rice, and 12

270

HFA families (49 regioisomers) were detected in Arabidopsis thaliana (Table S3).

271

Screening of FAHFAs in plants

272

Taking the correlation of DMED/d4-DMED-labeled FAHFA structures and

273

MS/MS fragmentation into account, MRM was used for non-targeted screening of

274

FAHFAs in rice and Arabidopsis thaliana tissues. The precursor ions of

275

DMED/d4-DMED-labeled

276

[d4-DMED-FAHFA]+, according to the possible combinations of detected HFAs and

277

reported essential FAs. The corresponding product ions were [DMED-HFA－63]+ and

278

[d4-DMED-HFA－67]+, according to the characteristic fragmentation pattern (Table

FAHFAs

were

13


[DMED-FAHFA]+

and


279

S2). Along this line, a list of potential FAHFAs was constructed, and a comprehensive

280

LC-MS-based non-targeted workflow was established to discover potential FAHFAs

281

in rice and Arabidopsis thaliana extracts.

282

An equal amount of rice or Arabidopsis thaliana sample was labeled with

283

DMED and d4-DMED, respectively. Then, the light and heavy labeled samples were

284

mixed and analyzed by LC-MS. A pair of DMED- and d4-DMED-labeled compounds

285

with similar retention times and intensities was assigned as a potential FAHFA. The

286

overlaid extracted ion chromatograms of FAHFAs are shown in Figure 3. For example,

287

the peak pairs of PAHPAs with similar intensities and retention times were the same

288

in the extracted ion chromatograms at m/z of 581.5→280.2 and m/z of 585.5→280.2

289

from the DMED- and d4-DMED-labeled samples, respectively (Figure 3), suggesting

290

these candidate compounds are potential PAHPA regioisomers.

291

A total of 49 FAHFA families (262 regioisomers) were identified, of which 48

292

FAHFA families (229 regioisomers) were detected in rice and 37 FAHFA families

293

(173 regioisomers) were detected in Arabidopsis thaliana. This indicates that

294

FAHFAs are abundant in tissues of rice and Arabidopsis thaliana. The distribution of

295

these discovered FAHFAs in rice and Arabidopsis thaliana are shown in Figure 4, and

296

the detail information is provided in Table S4. Among the detected 49 FAHFA

297

families, 41 families of FAHFAs were discovered in this study, and this finding

298

greatly extends our knowledge of FAHFAs. It should be noted that the FAHFAs in

299

rice (48 families, 229 regioisomers) are more abundant than those in Arabidopsis

300

thaliana (37 families, 173 regioisomers), which is consistent with the distribution of 14


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301

detected HFAs in rice and Arabidopsis thaliana.

302 303

Identification of FAHFAs in plants

304

Confirmation of FAHFAs using pure standards

305

The detected FAHFAs were first confirmed using commercially available

306

standards. A total of 21 FAHFA regioisomers, including 5-, 9-, 10-, 12-, 13-PAHSAs,

307

5-, 9-, 10-, 12-, 13-SAHSAs, 9-, 10-, 12-, 13-OAHSAs, 9-, 12-POHSAs, 9-PAHPAs,

308

9-MAHSA, 9-PDAHSA, 9-PAHMA, and 10-HDAHSA could be confirmed based on

309

the retention time and fragmentation patterns. The comparison of the extracted ion

310

chromatograms of the standard compounds and detected FAHFAs is shown in Figure

311

S7. However, few standard FAHFAs are available commercially, which restricts the

312

analysis of all FAHFAs detected in this study. Moreover, since FAHFAs are a newly

313

discovered class of lipid molecules, relevant information on FAHFAs is not recorded

314

in databases. Therefore, by analyzing the tandem MS spectra of DMED-labeled

315

FAHFAs, the composition of new FAHFAs (FA and HFA) can be confirmed, but the

316

determination of the ester position of the regioisomers in low abundance remains

317

challenging.

318

Putative annotation of FAHFAs using chromatographic retention

319

We investigated the retention behavior of different ester regioisomers of one

320

family on a C18 column and found out that the position of the ester group on the fatty

321

acyl chain was closely related to the retention of the regioisomers on the C18 column.

322

Indeed, as exemplified by the elution profile of PAHSAs and OAHSAs, the retention 15



323

time increased when the ester group was positioned closer to the carboxylic acid

324

moiety (Figure S8). As shown, the correlation between the retention factor (log10k)

325

and the ester position of different regioisomers in the PAHSA and OAHSA family is

326

significant, and the determination coefficient (R2) values are greater than 0.99 (Figure

327

S8). These results suggest that the ester position of each regioisomer from the same

328

FAHFA family can be determined by its retention behavior on the C18 column.

329

We investigated the retention behavior of different families of FAHFAs with

330

identical ester position on a C18 column. We found that the retention time, when FA

331

or HFA is given, is a function of the length of the carbon chain. For example, the

332

retention time of 9-MAHSA, 9-PDAHSA, 9-PAHSA and 9-SAHSA (compounds with

333

the same precursor, 9-HSA) on the C18 column increased linearly with their FA

334

carbon number (Figure S9). The result demonstrates that the retention behavior of

335

FAHFAs obeys the well-known carbon number rule23,24 and, thus, would be helpful in

336

identifying FAHFAs.

337

Taking the relationship of retention time and ester position into account, we

338

constructed the prediction curves for the determination of the ester positions of

339

unknown regioisomers from PAHSA, SAHSA and OAHSA families. The log10k

340

values of the regioisomers were calculated according to Eq. 1, and regression curves

341

for different FAHFAs were constructed by plotting log10k versus the ester position

342

from known regioisomers (Figure 5). For example, good linearity between log10k and

343

ester position for PAHSA regioisomers (5-, 9-, 10-, 12- and 13-position, Figure 5A

344

black squares) was achieved. Using the linear regression equation, the ester positions 16


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345

of unknown PAHSA regioisomers could be predicted by their log10k values (Figure

346

5A, red circles). Therefore, the ester positions of unidentified PAHSA regioisomers

347

(3-, 4-, 6-, 7-, 8-, 11-, 14- and 15-position, red circles) could also be determined. In

348

this way, the regioisomers of SAHSA and OAHSA families were identified (Figure

349

5B and 5C, red circles).

350

The ester position rule is suitable for the identification of unknown regioisomers

351

within a given FAHFA family. However, identifying unknown regioisomers by this

352

rule is impossible without pure standards for calibration. To overcome this limitation,

353

we constructed prediction curves based on the carbon number rule. For example, the

354

relationship of log10k and FA carbon number is shown in Figure 6A. Then, prediction

355

curves were generated from the linear regression of identified FAHSAs with identical

356

ester positions. Each line represents a defined ester position, and the data points that

357

correlate with the lines represent the regioisomers at the corresponding ester position.

358

For example, the data points that correlate with the line of the 5-position can be

359

defined as 5-FAHSAs, and thus 5-MAHSA, 5-PDAHSA and 5-HDAHSA can be

360

identified (Figure 6A, red line). Along this line, the 9-, 10-, 12-, and 13- ester position

361

of unknown regioisomers from MAHSA, PDAHSA, HDAHSA and AAHSA families

362

were putatively identified in the same way (Figure 6A). Similarly, the retention of

363

PAHFA families on a C18 column also conforms to the carbon number rule. Therefore,

364

a new PAHFA regioisomer, 9-PAHAA, was identified (Figure 6B).

365

Construction of prediction model for identification

366

Using the carbon number rule, we identified FAHFA regioisomers that share the 17



367

same ester position with the standards. However, the difficulty in identifying those

368

isomers with ester positions different from the standards remained. Therefore, we

369

utilized the above-identified FAHSAs as standards to construct a three-dimensional

370

(3-D) prediction model for identifying the ester position of unknown regioisomers

371

from different families of FAHSA. The 3-D scatter plots were constructed with three

372

factors: log10k, ester position and FA carbon number of the identified FAHSAs (Figure

373

7, blue dots). Fortunately, the 3-D scatter plots were fitted with a binary linear

374

regression equation: y＝－0.0096x1 ＋0.026x2 ＋1.34, where y denotes log10k, x1

375

denotes the position of the ester, and x2 denotes the FA carbon number of FAHFAs.

376

The coefficient of determination (R2) of the fitting equation was 0.9912, and the

377

standard deviation was 0.0052, indicating that a good correlation (Figure 7, pink plane)

378

between the three factors exits. Therefore, the fitting equation (prediction model) can

379

be utilized to accurately identify the ester position of unknown regioisomers from

380

different families of FAHSA.

381

Accounting for the measured log10k value and FA carbon number in the fitting

382

equation, the ester position of unidentified FAHSAs was obtained. For example, the

383

ester position of an unknown MAHSA (log10k = 1.56; MA carbon number = 14) was

384

estimated to be 15.17, using the fitting equation. Thus the unknown MAHSA was

385

identified as 15-MAHSA. Using this method, 19 other regioisomers from MAHSA,

386

PDAHSA, HDAHSA and AAHSA families, were putatively identified as 4-, 8-, 11-,

387

14-, 15-MAHSA, 4-, 6-, 7-, 8-, 11-, 14-, 15-PDAHSA, 4-, 6-, 8-, 11-HDAHSA and 4-,

388

11-, 15-AAHSA (Figure 7, red dots). 18


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389

To verify the reliability of the prediction model, we synthesized 7-PDAHSA

390

(purity > 95%, see Figure S3-E), a new FAHFA identified according to above

391

established prediction model. By comparing the extracted ion chromatogram of

392

DMED-labeled PDAHSAs from rice with pure 7-PDAHSA, the structure was

393

confirmed (Figure S10). Additionally, the prediction model could be used to predict

394

regioisomers of different FAHFA families that share an identical HFA or FA. However,

395

due to the limited availability of standards, the current prediction model is only

396

suitable for the identification of saturated FAHSA regioisomers. To construct

397

prediction models for the identification of more FAHFAs, we plan to obtain more

398

FAHFA standards that could be chemically or biologically synthesized.

399

Of the detected 49 FAHFA families (262 regioisomers), 11 FAHFA families (71

400

regioisomers, Table S5) were using pure standards for 8 families (21 regioisomers,

401

Table S5) and chromatographic retention behaviors for 7 families (50 regioisomers,

402

Table S5). Moreover, an additional 38 FAHFA families (191 regioisomers, Table S5)

403

were found, but their ester positions could not be annotated in the current study.

404

To verify the applicability of the prediction model, we screened and identified

405

FAHSA regioisomers in wheat seeds. As shown in Figure S11, a total of 12

406

regioisomers from the PAHSA family were detected in wheat seeds, and the x1 values

407

of these PAHSA regioisomers with different ester positions, were calculated using the

408

binary linear regression equation (y＝－0.0096x1＋0.026x2＋1.34). Thus, the ester

409

positions of the screened PAHSA regioisomers in wheat seeds were determined. The

410

results demonstrate that the established 3-D prediction model can be used to directly 19



411

identify regioisomers from FAHSA families in a variety of biological samples.

412 413

Conclusion

414

In this study, we report a comprehensive screening of FAHFAs using

415

CIL-LC-MS in MRM mode. This approach dramatically improved the detection

416

sensitivity and selectivity of FAHFAs by selecting paired-peaks with defined mass

417

differences for MRM scanning mode. In addition, we proposed a novel strategy for

418

identifying the ester position of FAHFA regioisomers based on the characteristic

419

chromatographic retention rules on a C18 column. This strategy overcomes the

420

difficulties in identifying regioisomers when commercial standards are not available

421

or when the compounds are not reported in databases. Using this method, we detected

422

49 FAHFA families, including 262 regioisomers in rice and Arabidopsis thaliana,

423

among which 71 regioisomers from 11 FAHFA families were further confirmed based

424

on commercial standards and chromatographic retention behaviors. This method

425

provides a promising tool for the discovery and identification of new FAHFAs in

426

biological samples.

427 428

ASSOCIATED CONTENT

429

Supporting information

430 431

Supporting Information Available: Table S1 – S5; Figure S1 – S10. This material is available free of charge via the Internet at http://pubs.acs.org.

432 20


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433 434

Notes The authors declare no competing financial interest.

435 436

Acknowledgements

437

The work is supported by the National Key R&D Program of China

438

(2017YFC0906800), the National Natural Science Foundation of China (21475098,

439

21635006, 31670373).

440 441

21



442

Reference

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(1) Yore, M. M.; Syed, I.; Moraes-Vieira, P. M.; Zhang, T. J.; Herman, M. A.; Homan, E. A.; Patel, R. T.;

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(3) Kuda, O.; Brezinova, M.; Rombaldova, M.; Slavikova, B.; Posta, M.; Beier, P.; Janovska, P.;

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Lee, J.; Chen, S. L.; Peroni, O. D.; Dhaneshwar, A. S.; Hammarstedt, A.; Smith, U.; McGraw, T. E.; Saghatelian, A.; Kahn, B. B. Cell 2014, 159, 318-332. (2) Muoio, D. M.; Newgard, C. B. Nature 2014, 516, 49-50. Veleba, J.; Kopecky, J. Jr.; Kudova, E.; Pelikanova, T.; Kopecky, J. Diabetes 2016, 65, 2580-2590. (4) Brezinova, M.; Kuda, O.; Hansikova, J.; Rombaldova, M.; Balas, L.; Bardova, K.; Durand, T.; Rossmeisl, M.; Cerna, M.; Stranak, Z.; Kopecky, J. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 2018, 1863, 126-131. (5) Nelson, A. T.; Kolar, M. J.; Chu, Q.; Syed, I.; Kahn, B. B.; Saghatelian, A.; Siegel, D. Journal of the American Chemical Society 2017, 139, 4943-4947. (6) Kolar, M. J.; Nelson, A. T.; Chang, T.; Ertunc, M. E.; Christy, M. P.; Ohlsson, L.; Harrod, M.; Kahn, B. B.; Siegel, D.; Saghatelian, A. Anal Chem 2018, 90, 5358-5365. (7) Lu, C.; Fulda, M.; Wallis, J. G.; Browse, J. The Plant Journal 2006, 45, 847-856. (8) Farmer, E. E.; Weber, H.; Vollenweider, S. Planta 1998, 206, 167-174. (9) Lu, C.; Fulda, M.; Wallis, J. G.; Browse, J. Plant Journal 2006, 45, 847-856. (10) Han, X.; Gross, R. W. Expert Review of Proteomics 2005, 2, 253-264. (11) Rainville, P. D.; Stumpf, C. L.; Shockcor, J. P.; Plumb, R. S.; Nicholson, J. K. Journal of Proteome Research 2007, 6, 552-558. (12) Chen, D.; Yan, X.; Xu, J.; Su, X.; Li, L. Metabolomics 2013, 9, 949-959. (13) Bird, S. S.; Marur, V. R.; Sniatynski, M. J.; Greenberg, H. K.; Kristal, B. S. Analytical Chemistry 2011, 83, 6648-6657. (14) Huan, T.; Li, L. Analytical Chemistry 2015, 87, 7011-7016. (15) Ejsing, C. S.; Duchoslav, E.; Sampaio, J.; Simons, K.; Bonner, R.; Thiele, C.; Ekroos, K.; Shevchenko, A. Analytical Chemistry 2006, 78, 6202-6214. (16) Houjou, T.; Yamatani, K.; Nakanishi, H.; Imagawa, M.; Shimizu, T.; Taguchi, R. Rapid Communications in Mass Spectrometry 2004, 18, 3123-3130. (17) Ma, Y.; Kind, T.; Vaniya, A.; Gennity, I.; Fahrmann, J. F.; Fiehn, O. J. Cheminformatics 2015, 7, 53. DOI: 10.1186/s13321-015-0104-4

(18) Zhu, Q.-F.; Yan, J.-W.; Gao, Y.; Zhang, J.-W.; Yuan, B.-F.; Feng, Y.-Q. Journal of Chromatography B 2017, 1061, 34-40. (19) Hao, Y. H.; Zhang, Z.; Wang, L.; Liu, C.; Lei, A. W.; Yuan, B. F.; Feng, Y. Q. Talanta 2015, 144, 341-348. (20) Bligh, E. G.; Dyer, W. J. Canadian Journal of Biochemistry & Physiology 1959, 37, 911-917. (21) Zhang, T. J.; Chen, S. L.; Syed, I.; Stahlman, M.; Kolar, M. J.; Homan, E. A.; Chu, Q.; Smith, U.; Boren, J.; Kahn, B. B.; Saghatelian, A. Nat. Protoc. 2016, 11, 747-763. (22) Zhu, Q. F.; Zhang, Z.; Liu, P.; Zheng, S. J.; Peng, K.; Deng, Q. Y.; Zheng, F.; Yuan, B. F.; Feng, Y. Q. Journal of Chromatography A 2016, 1460, 100-109. (23) Meulebroek, L. V.; Paepe, E. D.; Vercruysse, V.; Pomian, B.; Bos, S.; Lapauw, B.; Vanhaecke, L. Analytical Chemistry 2017, 89, 12502-12510. (24) Yu, D.; Zhou, L.; Xuan, Q.; Wang, L.; Zhao, X.; Lu, X.; Xu, G. Anal Chem 2018, 90, 5712-5718.

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485

Figure legends

486

Figure 1. Overview of the procedure for the screening and identification of FAHFAs

487

in rice and Arabidopsis thaliana by CIL-LC-MS.

488 489

Figure 2. Fragmentation of DMED/d4-DMED-labeled products by MS analysis. (A)

490

DMED-labeled 9-PAHSA; (B) d4-DMED-labeled 9-PAHSA; (C) DMED-labeled

491

9-SAHSA; (D) d4-DMED-labeled 9-SAHSA; (E) DMED-labeled 9-PAHPA; (F)

492

d4-DMED-labeled 9-PAHPA. Highlighted in red are the dominant product ions;

493

highlighted in blue are the theoretical m/z.

494 495

Figure 3. Extracted ion chromatograms of FAHFAs labeled with DMED and

496

d4-DMED and analyzed by LC-MS using MRM mode.

497 498

Figure 4. Fingerprint chromatograms of discovered FAHFAs in plant tissues. In total,

499

(A) 48 FAHFA families (229 regioisomers, plots) were found in rice and (B) 37

500

FAHFA families (173 regioisomers, plots) were found in Arabidopsis thaliana.

501 502

Figure 5. Identification of PAHSA, SAHSA and OAHSA regioisomers by the ester

503

position rule. Regression curve of the measured log10k (k, retention factor) versus the

504

ester position of (A) PAHSAs, (B) SAHSAs and (C) OAHSAs by LC-MRM-MS

505

analysis. Black squares represent commercial standards, and red circles represent

506

predicted structures.

507 23



508

Figure 6. Identification of FAHFA regioisomers by the carbon number rule. (A)

509

Prediction of FAHSA species via the carbon chain length of FA. Black circles for

510

FAHSA standards, gray circles for unknown FAHSAs, red line for 5-position, blue

511

line for 9-position, green line for 10-position, orange line for 12-position and purple

512

line for 13-position. (B) Prediction of PAHFA species via the carbon chain length of

513

HFA. Black circles for 9-PAHMA, 9-PAHPA and 9-PAHSA standards, gray circles for

514

remainder PAHMAs and PAHAAs, red line for 9-position.

515 516

Figure 7. The prediction model, based on the log10k, ester position and carbon

517

number of FA, for saturated FAHSAs. Blue dots represent the confirmed compounds;

518

Red dots represent the predicted compounds.

519

24


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520

Figure 1

521 522

25



523

Figure 2

524 525

26


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526

Figure 3

527 528

27



529

Figure 4

530

28


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531


Figure 5

532

29



533

Figure 6

534 535

30


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536 537 538

Figure 7

539 540

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541

TOC

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Comprehensive screening and identification of fatty acid esters of

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