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Omics Technologies Applied to Agriculture and Food

Rapid evaporative ionization mass spectrometry based lipidomics tracking of grass carp (Ctenopharyngodon idellus) during in vitro multiple-stage digestion Yanan Lin, Haixing Wang, Wei Rao, Yiwei Cui, Xina Yu, Zhiyuan Dai, and Qing Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01644 • Publication Date (Web): 28 May 2018 Downloaded from http://pubs.acs.org on May 28, 2018

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Journal of Agricultural and Food Chemistry

Rapid evaporative ionization mass spectrometry based lipidomics tracking of grass carp (Ctenopharyngodon idellus) during in vitro multiple-stage digestion

Yanan Lin1, Haixing Wang2, Wei Rao3, Yiwei Cui1, Xina Yu1, Zhiyuan Dai1,4, Qing Shen1,4,*

1 Institute of Seafood, Zhejiang Gongshang University, Hangzhou, 310012, China 2 Zhejiang Province Key Lab of Anesthesiology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, 325035, China 3 Waters Corporation, Shanghai, 201206, China 4 The Joint Key Laboratory of Aquatic Products Processing of Zhejiang Province, Hangzhou, 310012, China

Conflict of Interest: All authors declare that they have no conflict of interest.

* Corresponding Author Prof. Qing Shen Tel: +86 0571 88071024. Fax: +86 15968148458. E-mail: [email protected]; [email protected]

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ABSTRACT

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A rapid evaporative ionization mass spectrometry (REIMS) method was

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developed for lipidomics tracking of Ctenopharyngodon idellus during in

4

vitro multiple-stage digestion. The REIMS conditions were optimized to be

5

the temperature of heating probe 500 ◦C, sample amount 30 mg, and flow rate

6

of auxiliary solvent 100 µLmin–1. The results showed that the phospholipids

7

were detected to be dominated with variety and quantity in the crude and

8

multiple-stage digested samples. The enzymatic effect on the phospholipids is

9

varied depending on the phospholipid classes, and the hydrolysis rate of

10

phospholipids increased as the degree of unsaturation of the acyl chain

11

increased. The principal component analysis (PCA) indicated that the ion at

12

m/z 809.61, 811.63, and 857.52 were the most noticeable species digested

13

during the process. This method exhibited great potential in fast lipidomics

14

profiling for inspecting the characteristics of nutritional lipid absorption

15

digestion in human gastrointestin.

16

KEYWORDS:

17

multiple-stage digestion; rapid evaporative ionization mass spectrometry;

18

phospholipids.

lipidomics;

Ctenopharyngodon

19

2


idellus;

in

vitro

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INTRODUCTION

21

Phospholipids are a class of amphiprotic biomolecules with important

22

physiological and nutritional functions for human body, which include a

23

hydrophilic head and a hydrophobic tail comprising the hydrocarbon chains of

24

two fatty acids.1 The biological membrane is constituted of lipid bilayer, and

25

its permeability and fluidity attribute to the composition and distribution of

26

phospholipids.2,3 Recently, researchers found that phospholipids can play a

27

significant role in lipid absorption regulation, activating cells, enhancing the

28

immunity and regeneration, and reducing hepatic lipid levels and

29

inflammation by clinical and biological experiments.4 In addition, with the

30

natural biocompatible, biodegradable and amphiprotic characteristics,

31

phospholipids have been widely utilized as emulsifier, surfactantas, liposome

32

membrane, and drug coat in food, cosmetics and pharmaceutical industries.5-8

33

Therefore, tracking the phospholipids in vitro digestion can simulate and

34

estimate their behavior in the human gastrointestinal process, providing a

35

basis for designing phospholipid containing products.

36

In vitro digestion simulating human gastrointestinal environment is

37

generally considered to be a safe, rapid, inexpensive method without ethical

38

restriction for investigating the gastrointestinal behavior of food and

39

pharmaceutical, and establishing the physicochemical phenomena that

40

governs their behavior.9,10 Currently, researchers used a static method that

3



41

simulate the mouth, stomach, and small intestine phases, since it is postulated

42

that passage of a sample through the mouth and stomach will appreciably

43

alter its subsequent behavior in the small intestine.11,12 Meanwhile,

44

gastrointestinal tract model can accurately mimic the sophisticated conditions

45

in vivo, such as salt, bile acids, variable digestive enzyme and pH, etc.9

46

Therefore, multiple-stage digestion is an effective tool simulating the transit of

47

phospholipids and analyzing their behavior through the human digestive

48

tract.

49

The rapid expanding research ﬁeld, lipidomics, is built on the advances in

50

multiple analytical technologies, such as liquid chromatography (LC), gas

51

chromatography (GC), mass spectrometry (MS), and nuclear magnetic

52

resonance (NMR) spectroscopy.13-15 One of the major analytical platforms in

53

current lipidomics practice is a multidimensional mass spectrometry

54

(MDMS)-based shotgun method.16,17 Hydrophilic interaction chromatography

55

(HILIC) or reverse phase LC coupled to MS/MS is also commonly used for the

56

separation and detection of phospholipids because of its resolution, sensitivity

57

and reproducibility.18 However, the analytical procedure of these techniques is

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complex and labor-costing. Recently developed REIMS is a new and efficient

59

technology which can realize real-time and accurate identification of tissues

60

and allow in vivo, in situ tissue analysis.19,20 REIMS lipidomic profile displays

61

many complex phospholipid molecular species originating from the cell

62

membrane and make the analysis of lipids with minimal sample preparation 4


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and analysis time in a few seconds.21,22 By far, REIMS has been successfully

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applied to identify and classify the biological tissues,23 detect and resect tumor

65

as the intelligent knife of medical surgery,21 characterize microorganisms,22

66

and etc. Therefore, the application of REIMS in the research field of food

67

science and technology has bright further, while related reports and

68

publications are very rare.

69

In this study, a REIMS method was developed and optimized for tracking

70

the phospholipids in Ctenopharyngodon idellus during in vitro multiple-stage

71

digestion. The results can contribute to revealing the characteristics of lipid

72

nutrition absorption and the principle of release of phospholipid anchored

73

compound in human gastrointestinal digestion.

74

MATERIALS AND METHODS

75

Materials and reagents.

76

U·g–1), gastric pepsin (2000 U·mg–1) from porcine gastric mucosa, gastric

77

lipase (3000 Ug–1), pancreatin (2500 Umg–1 based on lipase) from porcine

78

pancreas and bile salts from bovine bile were obtained from Sigma Aldrich (St.

79

Louis, MO, USA). Chloroform, methanol, propan-2-ol, and acetone were

80

chromatography grade from Merck (Darmstadt, Germany). The other

81

chemicals and reagents are standard analytical grade and purchased from

82

Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Salivary α-amylase from human saliva (1500

5



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Sample preparation and lipid extraction. The grass carp (C. Idellus)

84

was selected as a representative sample because it is an important economic

85

fish species in China containing medium content of fat. Fresh fish samples (C.

86

Idellus, weight ca. 3 kg) were purchased from Wal-Mart Stores, Inc.

87

(Hangzhou, China), placed in ice at a fish/ice ratio of 1:2 (w/w), and

88

transported to the Institute of Seafood, Zhejiang Gongshang University,

89

Hangzhou. Upon arrival, five fish samples without evident sign of parasite

90

were collected and steamed. The white muscle from both sides of the fish

91

sample was obtained by the mean of a mechanical deboner after removing the

92

internal organs, heads, and tails of fish. Then, the muscle sample was ground

93

to uniformity and stored at 4 ◦C. Five parallel samples from different fish

94

bodies were prepared, and four technical replicates were applied.

95

For extracting the crude lipid (including phospholipids, free fatty acids,

96

glycerides, etc.), about 10 g of sample was weighed and homogenized with 30

97

mL mixture of chloroform and methanol (2:1, v/v). Then, 10 mL of water was

98

added, and the mixture was oscillated and centrifuged at 4 ◦C at 10000 r·min–1

99

for 10 min. The organic phase (lower phase) was collected by pipette making

100

sure to avoid the solid sample and upper phase. The residue was re-extracted

101

with 20 mL of chloroform for another two times. Afterward, the collections of

102

organic phase were combined and condensed by a rotary evaporator at 50 ◦C.

103

After cooling down, 300 mL pre-cooled acetone (-20 ◦C) was poured into the

104

mixture with vigorous shaking. The phospholipids were obtained by collecting 6


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the precipitation and nitrogen drying. The total amount of phospholipids at

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different digestion stage (g/100 g) was calculated using the equation, Quantity

107

= Wp/Ws × 100, where Wp is the weight of phospholipids extracted from the

108

digestion juice at different stage (g), while Ws is the weight of the initial fish

109

sample (g).

110

In vitro multiple-stage digestion.

111

idellus during in vitro digestion, artificial simulated oral, gastric and intestinal

112

digestion was conducted following the harmonized protocol published by

113

Minekus et al.9 and Martínez-Las et al.24

To track the phospholipids of C.

114

At the oral digestion stage, salivary α-amylase was added to the simulated

115

salivary fluid (SSF) to a final concentration of 75 U·mL–1, and the mixture was

116

combing with the prepared sample by a ratio of 1:1 (w/v). The mixture was

117

properly homogenized in a 50 mL falcon tube and incubated with agitation for

118

2 min at 37 ◦C. For gastric phase simulation, pepsin and gastric lipase were

119

added into the simulated gastric fluid (SGF) to a final concentration of 2000

120

U·mL–1 and 120 U·mL–1 respectively, and mixed with the sample after oral

121

digestion at a ratio of 1:1 (v/v). The pH was adjusted to 3 with HCl (1 M), and

122

the mixture was incubated at 37 ◦C for 2 h with continuous shaking (50 rpm).

123

Pancreatin is a mixture of enzymes extracted from porcine pancreas

124

containing trypsin, pancreatic amylase and pancreatic lipase. Because lipid

125

digestion was the focus of this study, the addition of pancreatin was on the

7



126

basis of the lipase activity. Finally, the simulated intestinal fluid (SIF) with

127

2000 U·mL–1 of pancreatin and 10 mM of bile salts were added into the

128

gastric mixture at 1:1 (v/v) ratio, and the pH was adjusted to 7 with NaOH (1

129

M). Afterward, the mixture was incubated at 37 ◦C and shook at 50 rpm for 2 h.

130

At the end of each stage, the samples were taken to the boiling water bath

131

immediately for 6 min to inactivate the enzymes. The phospholipids were

132

extracted and weighed by the aforementioned methods. The experiment was

133

performed in triplicate.

134

The content of phospholipids of C.

Instruments and parameters.

135

idellus during in vitro multiple-stage digestion was investigated by REIMS

136

(Waters Co., Ltd., Shanghai, China) which mainly contained the electric

137

heating probe (WSD151, Weller, Germany) and a quadrupole time-of-flight

138

mass spectrometer (Xevo G2-XS, Waters Co., Ltd., Shanghai, China) coupled

139

with a commercial available REIMS interface. Mass spectrometer was

140

calibrated with sodium formate before analysis every day. For REIMS analysis,

141

the aerosol formed via the electric heating probe was aspirated into the MS via

142

a PTFE tube by a Venturi pump driven by 2 bar nitrogen and mounted on the

143

REIMS interface. In REIMS interface, the auxiliary solvent of propan-2-ol was

144

introduced into the interface via a stainless steel capillary (1/16ʹʹ O.D., 0.002ʹʹ

145

I.D.) with leucine enkephalin added for lock mass correction. The aerosol and

146

solvent were mixed and guided to a heated helix collision surface operated at

147

around 750 ◦C to improve signal intensity and avoid contamination. The 8


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148

REIMS spectra were obtained at the rate of 1 scan/second between m/z 50 –

149

1200 in negative mode and accumulated for each analysi. The MS data were

150

analyzed by the statistical software package Masslynxv4.1. Only the peaks with

151

signal-to-noise higher than 10 were selected and analyzed. For mass spectrum

152

processing, background subtraction was applied to reduce the interring noise,

153

automatic peak detection was used to center the spetrum, and then lock mass

154

(Leu-enkephalin, m/z 554.2615) was run to calibrate the spectrum for exact

155

mass.

156

Statistical analysis.

157

statistical analysis. The relative content of each peak was calculated using a

158

peak area normalization method. Statistical analysis and the calculation of

159

mean, standard deviation, and level of significance were performed by using

160

SPSS 16.0 software (SPSS Inc.,Chicago, IL, USA). The ions showing significant

161

difference were examined with one-way ANOVA (p < 0.05). 20 samples for

162

each digestion stage was applied to principal component analysis (PCA) for

163

the determination of the main sources of variability (phospholipid peaks and

164

relative contents) presented in the data sets to establish the relation between

165

crude and digested samples with phospholipids.

166

RESULTS AND DISCUSSION

167

Optimization of REIMS conditions.

168

was performed by online mass spectrometric analysis of the aerosol produced

MS spectral data in centroid mode was exported for

The REIMS study of phospholipids

9



169

by electric heating evaporation.22 The temperature of sampling probe, sample

170

volume, and flow rate of auxiliary solvent are critical and significant factors

171

for the analysis of phospholipids. These factors were evaluated to optimize the

172

REIMS conditions. The intensities of the peaks at m/z 783.52, 809.61, 835.54,

173

857.52, and 883.54 were selected as inspection indicators.

174

The temperature of sampling probe exerted significant influence on the

175

intensity of phospholipid. In this study, the effect of temperature was

176

evaluated in the range from 350 to 550 ◦C (Figure 1A). The results indicated

177

that the sample can’t be sufficiently evaporated and ionized to produce aerosol

178

at low temperature. With the increase of temperature, the signal intensities of

179

the five selected peaks had a clear upward trend, and the maximum values

180

were reached at 500 ◦C. When the temperature exceeded 500 ◦C, the intensity

181

values decreased significantly, because high thermal energy can cause

182

excessive sample evaporation and ionization and produce aerosol which

183

contain many impurities. Therefore, the temperature of 500 ◦C was a superior

184

condition which can be adopted for subsequent optimization tests.

185

Sample volume is an important parameter for REIMS study, because a

186

small amount of sample makes insufficient aerosol during evaporation, while

187

excessive sample volume will dilute the thermal energy, leading to reduced

188

peaks of phospholipids. The effect of sample volume on the ionization of

189

phospholipids was depicted in Figure 1B, which indicated that there is a

10


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positive correlation between the signal intensity of phospholipid and the

191

sample volume. The intensities of the selected peaks increased in the range

192

from 15 to 30 mg of sample, and kept steady when the sample volume further

193

increased to 35 mg. Therefore, 30 mg digestive sample is enough for REIMS

194

analysis.

195

In addition, a steady flow of isopropyl was used as auxiliary solvent and

196

introduced directly into the atmospheric interface to enhance the signal

197

intensity of phospholipid and wash the ion transfer pipeline. The effect of the

198

loading flow rate of auxiliary solvent (10, 20, 50, 100 and 150 µLmin–1) on the

199

signal intensity and stability of phospholipids were investigated. As shown in

200

Figure 1C, the signals of phospholipid became more and more intensive when

201

the flow rate was increased gradually from 10 to 100 µLmin–1, and the highest

202

values were obtained at 100 µLmin–1. When the flow rate of auxiliary solvent

203

was continuously increased, a light drop of the signals was occurred, and the

204

signals of phospholipid became unstable and fluctuated dramatically.

205

Therefore, the flow rate of auxiliary solvent was set at 100 µLmin–1 for

206

promising a series of intensive and high resolution signals.

207

Total volume of phospholipids at different digestion stage.

208

prerequisite to compare the total quantity of phospholipids at different

209

digestion stages for tracking the changes of phospholipids. The digestion

210

efficiency and quantity of phospholipids in vitro oral, gastric and intestinal

11


It is a


211

digestion were evaluated and compared with that of non-digested sample and

212

the results were presented in Figure 2. The phospholipid digestion efficiency

213

of non-digested sample was set as the start point (0%). There is a slight

214

increase of digestion efficiency in the oral digestion stage (5.89%), and this

215

upward trend became significant during the gastric digestion stage (25.22%).

216

The highest digestion efficiency was obtained at the intestinal digestion stage

217

(61.75%). The absolute phospholipid content was also depicted in the Figure.

218

This value decreased in sequence during the oral, gastric and intestinal

219

digestion, and the trend of drop became more and more fierce along with the

220

digestion process. In particular, the absolute phospholipid content fell from

221

1.37 to 0.26 (%) in the intestinal digestion stage. This phenomenon could be

222

explained by the diversity and plenty of phospholipase in intestinal tract. After

223

the ingestion of food, phospholipids were mainly physically digested in the

224

mouth and stomach. The function of the oral cavity was mainly the

225

mechanical mastication and homogenization, and the stomach is subjected to

226

primary emulsification of lipid by gastric acid and reduce the surface tension

227

of lipids. In intestinal digestion, the SIF was composed of bile salts and

228

pancreatin from porcine pancreas containing lipase, phospholipase and

229

cholesterol esterase. Pancreatic lipase can catalyze the hydrolysis of fatty acids

230

of phospholipids and release fatty acids and acyl lysophospholipids. In

231

addition, bile salts and enzymes have a synergistic effect on the hydrolysis of

232

phospholipids.25

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The phospholipids in fish tissue can be

233

Identification of phospholipids.

234

evaporated and ionized in both positive-ion and negative-ion modes. However,

235

the signals of phospholipids in positive-ion mode were suppressed by

236

impurities, such as glycerides, because of the protonating competition during

237

the rapid evaporative ionization. The phospholipids in negative-ion mode are

238

obviously more abundant and informative, due to the substantial negative

239

charge of the headgroup.26 Therefore, negative-ion mode was used in the

240

subsequent experiments, and the extract of phospholipids from non-digested

241

C. idellus was used as a control group. The mass spectrum was recorded in

242

real-time as the electric heating probe touched the sample. The identities of

243

phospholipids were interpreted by investigating the characteristics of ions in

244

the database LIPID MAPS and comparing them with those in related

245

literature, and the results of phospholipids analysis at different digestion

246

stages were summarized in Table 1. Some ions with intensive signals were

247

confirmed by MS/MS analysis. For example, the ion of 885.5 could be

248

tentative identified as [PI(38:4)-H]- by exact mass value, which could be

249

confirmed by the characteristic fragments of m/z 283.1 (18:0) and m/z 303.1

250

(20:4), indicating the detailed structure [PI 18:0/20:4-H]-. The phospholipids

251

identified in non-digested sample, oral, gastric, and intestinal digested sample

252

majorly dominated in the m/z range of 700 to 950. A total of 35 phospholipid

253

molecular species were tentatively identified, which could be divided into

254

three phospholipid classes, including PC, PE, and PI. However, PS, PA and PG

13



255

were not observed in this experiment, and it could be attributed to the low

256

abundance of PS and PA in fish tissue and the rare existence of PG in animal

257

muscle tissue.27 As shown in the spectrum, the m/z of the most phospholipid

258

peaks came out as an odd number (such as 809.61), indicating that the

259

nitrogen-containing moiety of PC or PE has lost during ionization. It is usually

260

a good rule to speculate the molecular formula of phospholipids. For example,

261

the m/z 739.57 could be tentatively explained as [PC O-38:2-N(CH3)3]– or [PE

262

O-38:2-NH3]–, and the m/z of PC O-38:2 and PE O-38:2 became identical

263

after losing the nitrogen-containing moiety. This phenomenon is not

264

frequently observed in modern mass spectrometers which are usually

265

equipped with soft ionization techniques, such as electrospray ionization,

266

atmospheric pressure chemical ionization, and matrix assisted laser

267

desorption ionization. The intensive energy of electric heating probe make the

268

rapid evaporative ionization a kind of hard ionization, leading to the loss of

269

nitrogen-containing moiety possible. It is worth to mention that some peaks

270

were composed of more than one phospholipid species. As described, the

271

overlap of [PI 34:4 - H]–, [PE 44:6 - NH3]– and [PC 44:6 - N(CH3)3]– peaks

272

produced the peak at m/z 829.53. This phenomenon could be attributed to the

273

similarity of structure between PC with PE and the equivalent molecular mass

274

after losing some groups. Some phospholipid molecular species has positive

275

nutritional properties, such as m/z 795.51 (PC 42:9 & PE 42:9), m/z 929.51

276

(PI 42:10), etc., whose sn-1 and sn-2 moieties involved eicosapentaenoic acid

14


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or docosahexaenoic acid regarded as important protective agents for health

278

promotion and disease prevention.28

279

Influence of digestion on the quantity of phospholipids.

280

spectra of phospholipids extracted from non-digested crude sample and oral,

281

gastric and intestinal digested samples were depicted in Figure 3. It was

282

obvious that in vitro simulated digestion had a great influence on

283

phospholipids, not only on the mass spectrometric profile, but also on the

284

specific intensity of phospholipids. Before digestion, the peaks of phospholipid

285

in crude sample had the best signal-to-noise (S/N) ratio and signal diversity

286

involving 35 phospholipids species. The ion at m/z 809.61 was the most

287

abundant, which could be tentatively interpreted as the overlap of [PC 42:2 -

288

N(CH3)3H]– and [PE 42:2 - NH3]–, followed by the ions at m/z 857.52 ([PI

289

36:4 - H]–) and m/z 811.63 ([PC 42:1 - N(CH3)3H]– & [PE 42:1 - NH3]–). After

290

oral digestion, although there is a reduction of the signal intensities from

291

1.35e7 to 2.57e6, the diversity of phospholipid molecular species didn’t

292

changed much, because of the absence of lipase in the oral environment. The

293

lingual lipase secreted by lingual serous glands in mouth wasn’t considered

294

here, because it works on medium- and long-chain triglycerides only but not

295

phospholipids. Along with the progress of multistage digestion, the

296

background noise of the mass spectra became more and more serious, and the

297

signal intensity of phospholipid decreased to 9.72e5 and 4.22e5 for gastric and

298

intestinal digestion stages, respectively. The specific dynamic changes of 15


The REIMS


299

intensity were listed in detail in Table 1. After comprehensive analysis, it can

300

be seen that the absolute intensity (peak area, cps) were reduced for all the

301

phospholipids during the digestion process. The enzymatic effect on the

302

phospholipids is varied generally depending on the phospholipid classes. The

303

PI decreased more significantly than the PC and PE. For example, the most

304

intensive peak, m/z 857.52 ([PI 36:4 - H]–), decreased dramatically from

305

9.19% to 3.71% after gastrointestinal digestion, and some PI peaks were

306

digested completely, such as the m/z 879.50 ([PI 38:7 - H]–), 903.50 ([PI 40:9

307

- H]–), 931.53 ([PI 42:9 - H]–), etc. On the contrary, the relative percentage of

308

PC and PE increased gradually, attributing to the marked drop of PI. For

309

example, the relative percentage of m/z 811.63 at different stages were 6.03%

310

(oral phase), 10.21% (gastric phase), and 13.57% (intestinal phase). In

311

addition, it was observed that the phospholipids with high degree of

312

unsaturation are more easily digested. The polyunsaturated phospholipid

313

molecular species, 903.50 ([PI 40:9 - H]–), 931.53 ([PI 42:9 - H]–), and etc,

314

were digested completely. It has been reported that the hydrolysis rate of

315

phospholipids increased as the degree of unsaturated of the acyl chain

316

increased.25 The hydrolyzing selectivity of porcine pancreatic lipase is also

317

related to location of double bonds in the fatty acid, which appears to be

318

depending on the distance of the first double bond from the ester linkage

319

being hydrolyzed.29

16


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It is well known that PCA is an effective method

320

PCA statistical analysis.

321

used for reducing the number of dimensions present in the whole data matrix

322

with only a minimum loss of original information. To determine the main

323

variability and establish the relation between phospholipids and digestion,

324

PCA was applied to analyze the data produced by REIMS. It normalized the

325

relative amounts of the phospholipids in crude and digested samples in a

326

reduced dimension plot for revealing the difference between each digestion

327

stage. As shown in Figure 4A, there are generally three major clusters

328

observed in the score plot. The lipidomic profile data from crude and orally

329

digested samples were mixed into a single cluster. It can be explained by the

330

limited digestive effect in the oral digestion stage due to the absent of

331

phospholipase. The other two samples of gastric and intestinal digestion

332

stages were distinctly separated by using only the first two PCA components.

333

The separation effect of the second principal component (PC2) was not as

334

good as that of PC1. In detail, the first principal component (PC1) separated

335

the three clusters by explaining 65.8 cumulative percent (cum %) of the

336

variance in the REIMS data set, and PC2 explained 11.7 cum %. The first two

337

PCA components with 77.5 cum % of the total variance could be considered to

338

be significant enough to demonstrate the variation of the digestion stages

339

among these samples. Figure 4B revealed the loadings for the variables in the

340

first two PCA components clearly, in which the most influential phospholipid

341

molecular species could found out. It showed that most of the phospholipid

17



342

molecular species were clustered at the central zero line, whereas the rest were

343

dispersed far away from the center. The species with higher loading values

344

and maximum variance in the data were mostly influenced by the digestion

345

treatment. For instance, the ion at m/z 797.54 ([PG 38:4]–), m/z 809.61 ([PE

346

42:2 - NH3]– & [PC 42:2 - N(CH3)3]–), 811.63 ([PE 42:1 - NH3]– & [PC 42:1 -

347

N(CH3)3]–), 857.52 ([PI 36:4 - H]–), etc. were the most noticeable species, and

348

their content varied dramatically along with the digestion process. All of these

349

ions showing significant difference were examined with one-way ANOVA (p

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