Effect of Astringent Stimuli on Salivary Protein ... - ACS Publications

Subscriber access provided by University of Newcastle, Australia

Article

Effect of Astringent Stimuli on Salivary Protein Interactions Elucidated by Complementary Proteomics Approaches Judith Delius, Guillaume Médard, Bernhard Kuster, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00436 • Publication Date (Web): 22 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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

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

Page 1 of 36

Journal of Agricultural and Food Chemistry

1

Effect

of

Astringent

Stimuli

on

Salivary

Protein

2

Interactions Elucidated by Complementary Proteomics

3

Approaches

4

Judith Delius1, Guillaume Médard2, Bernhard Kuster2, and Thomas Hofmann1*

5

6

7

1

Chair of Food Chemistry and Molecular Sensory Science, Technical University of Munich, Lise-Meitner-Straße 34, 85354 Freising, Germany

8

2

Chair of Proteomics and Bioanalytics, Technical University of Munich, Emil-

9

Erlenmeyer-Forum 5, 85354 Freising, Germany

10

11

12

*

To whom correspondence should be addressed

13

PHONE

+49-8161/71-2902

14

FAX

+49-8161/71-2949

15

E-MAIL

[email protected]

16 17 18

1 ACS Paragon Plus Environment


Page 2 of 36

19

ABSTRACT

20

The interaction of astringent substances with salivary proteins, which results in

21

protein precipitation, is considered a key event in the molecular mechanism

22

underlying the oral sensation of puckering astringency. As the chemical nature of

23

orally active astringents is diverse and the knowledge on their interactions with

24

salivary proteins rather fragmentary, human whole saliva samples were incubated

25

with supra-threshold and iso-intensity solutions of the astringent polyphenol (-)-

26

epigallocatechin gallate, the multivalent metal salt iron(III) sulfate, the amino-

27

functionalized polysaccharide chitosan, and the basic protein lysozyme. After

28

separating the precipitated proteins, the proteins affected by the astringents were

29

identified and relatively quantified for the first time by complementary bottom-up

30

and top-down mass spectrometry-based proteomics approaches. Major salivary

31

target proteins, that may be involved in astringency perception, are reported here

32

for each astringent stimulus.

33

34

KEYWORDS: Astringency, polyphenol, polycation, chitosan, oral cavity, mass

35

spectrometry, nano-LC-MS, Intensity-Based Absolute Quantification, iBAQ.

36 37


Page 3 of 36


38

INTRODUCTION

39

Since the 1950s various molecular mechanisms underlying oral astringency have

40

been postulated,1 such as complex formation between alimentary polyphenols

41

and salivary proteins2,3 and polyphenol-induced G protein-coupled signaling in

42

trigeminal neurons.4 LC-MS-based top-down proteomic approaches, which aimed

43

at characterizing the target salivary proteins complexing with polyphenols,

44

revealed astringent procyanidins to interact primarily with acidic proline-rich

45

proteins (PRPs) and statherin, while basic and glycosylated PRPs were not or

46

only weakly affected.5–7 HPLC analyses of intact proteins with ESI-MS and UV

47

detection, respectively, were albeit confined to a restricted range of small proteins

48

and peptides, as high molecular weight salivary proteins had been precipitated

49

with trifluoroacetic acid during sample work-up.5,7–9

50

Besides polyphenols, other compounds have been reported to induce a

51

puckering astringent orosensation, such as polyvalent cations of metal salts,10–12

52

basic proteins exhibiting a high isoelectric point,13,14 as well as poly-(β-(1→4)-D-

53

glucosamine, also referred to as chitosan.15,16 Compared to some preliminary

54

work performed with polyphenols,5–7 the salivary target proteins forming

55

complexes with these alternative astringent compounds are unknown, primarily

56

due to the lack of any in-depth proteomic studies.

57

The objective of the present study was, therefore, to assess the interaction

58

of human salivary proteins with representatives of the four different chemical

59

classes of oral astringents, namely the polyphenol (-)-epigallocatechin gallate

60

(EGCG) from green tea, Fe2(SO4)3 used as a haemostatic metal salt in dentistry,17



Page 4 of 36

61

lysozyme from hen egg white and poly-(β-(1→4)-D-glucosamine (chitosan), which

62

is derived from the chitinous exoskeleton of crustaceans and is used in food

63

processing.18 To achieve this, pooled human saliva should be mixed with the

64

astringent molecules and the protein composition of the centrifuged sediments

65

analyzed by means of a bottom-up proteomic approach using tryptic in-gel

66

digestion, followed by nano-LC-Orbitrap analysis. As this bottom-up proteomic

67

approach may not allow for detecting post-translational protein modifications,19

68

and is known to be affected by non-trivial protein miscleavage upon enzymatic

69

digestion, some proteins might get quantitatively underestimated.20 Therefore, the

70

intact salivary proteins remaining soluble upon treatment with astringents should

71

be analyzed by means of a complementary top-down proteomic approach to gain

72

a more comprehensive insight into the salivary target proteins interacting with

73

chemically diverse astringent substances.

74

75

MATERIALS AND METHODS

76

Chemicals. The following compounds and reagents were commercially obtained:

77

EDTA-free

78

Germany); trypsin (Promega, Madison, USA); (-)-epigallocatechin gallate (EGCG),

79

DMSO,

80

bicarbonate) (Sigma-Aldrich, St. Louis, USA); chitosan (~500 kDa, >92.6%

81

deacetylated,

82

Coomassie Brilliant Blue G-250 assay reagent, bovine serum albumin (Thermo

83

Scientific, Rockford, USA); colloidal Coomassie blue (Carl Roth, Karlsruhe,

Complete®

protease

inhibitor

(Roche

Diagnostics,

Penzberg,

iron(III) sulfate, lysozyme UniProt: P00698, TEAB (triethylammonium

Heppe

Medical

Chitosan

GmbH,

Halle/Saale,

Germany);


Page 5 of 36


84

Germany); Precision Plus ProteinTM (Bio Rad, München, Germany); NuPAGE®

85

sample buffer (Invitrogen, Carlsbad, USA); dithiothreitol,

86

(Amresco, Solon, USA); acetic acid, formic acid (Merck, Darmstadt, Germany).

87

Solvents were of HPLC grade: acetonitrile, methanol (J. T. Baker, Deventer,

88

Netherlands); acetone (Alfa Aesar, Karlsruhe, Germany), ethanol (Merck,

89

Darmstadt, Germany).

iodoadacetamide

90

Human Saliva Collection. Unstimulated, whole saliva was freshly

91

collected in ice-cooled centrifuge tubes from seven healthy volunteers (two male,

92

five female, 23-30 years) at a fixed time in the morning of each sampling day to

93

minimize diurnal variations in salivary composition. Subjects were asked not to

94

eat or drink besides water at least one hour prior to donating saliva. Immediately

95

after collection, saliva was pooled and mixed with EDTA-free Complete® protease

96

inhibitor according to the manufacturer’s instructions. This sample was

97

centrifuged twice (5 min at 4800 g, then 30 min at 16000 g, each at 4 °C) to

98

remove cells and debris and to gain a clear saliva sample as the supernatant for

99

immediate use in further experiments (Figure 1).

100

In Vitro Protein Precipitation Assay. Saliva (0.8 mL) was mixed in a 4:1

101

ratio (v/v) with aqueous stimulus solutions containing the astringent substances in

102

concentrations

103

previously reported,12 that is (-)-epigallocatechin gallate (0.2 mL, 5.0 mmol/L),

104

iron(III)sulfate (0.2 mL, 5.0 mmol/L), lysozyme (0.2 mL, 0.5 mmol/L), and chitosan

105

(0.2 mL, 2 µmol/L, adjusted to a pH of 5.0 with aqueous 0.1 mmol/L HCl), followed

106

by incubation at room temperature for 15 min. In a control experiment, saliva was

107

mixed in a 4:1 ratio (v/v) with ultrapure water (0.2 mL; control) obtained from a

of

comparable

supra-threshold

astringency

responses

as



Page 6 of 36

108

Milli-Q Gradient A10 system (Millipore, Schwalbach, Germany), and was

109

incubated at room temperature for 15 min. Thereafter, the samples were

110

centrifuged for 15 min at 16000 g and the precipitates were separated from the

111

supernatants for further analysis. Biological replicates were prepared on three

112

independent days.

113

Analysis

of

Proteins

in

the

Supernatant

in

Saliva/Stimulus-

114

Incubations. The amount of protein in the supernatant solution was determined

115

colorimetrically using the Bradford assay. To achieve this, an aliquot (40 µL) of

116

the supernatant was mixed with a volume (1 mL) of commercial Coomassie

117

Brilliant Blue G-250 assay reagent in triplicates and absorption was determined at

118

595 nm. Protein concentrations were calculated based on a calibration curve set

119

up with standard solutions of bovine serum albumin. To identify salivary proteins

120

remaining in solution after astringent induced precipitation, the supernatants were

121

diluted with ultrapure water (50/50, v/v), membrane filtered (0.45 µm,

122

polyethersulfone membrane, Pall, Crailsheim, Germany), and directly injected

123

onto a 2.1 x 150 mm, 300 Å, XBridge Protein BEH C4 column (Waters, Milford,

124

USA) in an UltiMate® 3000 HPLC system (Dionex, Sunnyvale, USA) connected to

125

a Bruker micrOTOFTM mass spectrometer (Bruker Daltonics, Bremen, Germany).

126

Chromatography was conducted at a flow rate of 0.2 mL/min with acetonitrile

127

(solvent A) and water (solvent B), both containing 0.1% formic acid, as the mobile

128

phase and the following gradient: 0 to 15% solvent A within 6 min, then to 40%

129

within 30 min, followed by an increase to 95% within 10 min. High resolution mass

130

chromatograms were recorded in positive ionization (ESI pos.) mode scanning the

131

mass range between m/z 500 and 3000. Mass spectra of individual peptide and 6 ACS Paragon Plus Environment

Page 7 of 36


132

protein signals in the chromatogram were deconvoluted using the software

133

DataAnalysis (version 3.4, Bruker Daltonics) and compared to literature data.21

134

Analysis of Proteins in the Precipitate in Saliva/Stimulus-Incubations.

135

The protein pellets, obtained in the in vitro precipitation assay described above,

136

were washed twice with water, followed by acetone. Thereafter, pellets were

137

separated from solvent traces in a stream of nitrogen. To reduce disulfide bonds

138

in proteins, the pellets were re-suspended in a mixture (50/50, v/v; 280 µL) of

139

aqueous dithiothreitol (100 mmol/L) and NuPAGE® sample buffer (Invitrogen,

140

Carlsbad, USA), thermally treated at 95 °C for 10 min while shaking. In the

141

following, aliquots (20 µL) of an aqueous solution of iodoadacetamide (550

142

mmol/L) were added for alkylation of cysteine residues, and samples were kept in

143

the dark for 30 min.

144

SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE). Aliquots (20 µL)

145

of the processed sample material were loaded on a 4-12% NuPAGE® sodium

146

dodecyl sulfate polyacrylamide gel (Invitrogen, Darmstadt, Germany) in an XCell

147

Sure LockTM electrophoresis cell and run at 200 V for 5 min to separate detergent

148

and buffer components. Proteins were fixed with 2% acetic acid in 40% methanol

149

while gentle shaking the gels for 1 h. Gels were stained for 5 min with an aqueous

150

solution of 20% colloidal Coomassie blue and 20% methanol, followed by washing

151

with a solution of 5% acetic acid in 25% aqueous methanol, and finally by

152

washing with 25% ethanol for partial destaining.

153

To separate into distinct, visible protein bands, 30 µL sample aliquots were

154

additionally loaded on a gel and run at 200 V for 45 min. Precision Plus ProteinTM

155

unstained standard was used as a molecular-weight size marker. Except for a 7 ACS Paragon Plus Environment


Page 8 of 36

156

more intense gel staining for 90 min, the same protocol was used as described for

157

the short gel.

158

In-Gel

Protein

Digestion.

Tryptic

digestion

was

carried

out

in

159

polypropylene 96-well plates under keratin-free conditions. Wells were alternately

160

washed twice with 0.1% formic acid, followed by pure ethanol, and water.

161

Digestion wells were then filled with 5 mM TEAB/ethanol (50/50, v/v), and SDS

162

gel spots were cut with a scalpel and placed in the 96-well plate. After destaining

163

twice with 5 mM TEAB/ethanol (50/50, v/v) at 55 °C, samples were dehydrated

164

with 100% ethanol and then incubated with trypsin (10 ng/µL in 5 mM TEAB) at 4

165

°C. After 15 min, the enzymatic reaction was stopped with 5% formic acid.

166

Samples were washed with 5 mM TEAB, extracted with a mixture (60/40, v/v) of

167

acetonitrile and 0.1% aqueous formic acid and, after separating the acetonitrile in

168

vacuum, samples were frozen and lyophilized.

169

Liquid Chromatography / Mass Spectrometry. Liquid chromatography

170

tandem mass spectrometry was performed by coupling an Eksigent nanoLC-Ultra

171

1D+ (Eksigent, Dublin, CA) to an LTQ Orbitrap XL instrument (Thermo Scientific,

172

Bremen, Germany). The protein digests were injected onto a 2 cm × 100 µm, 5

173

µm, trap column filled with Reprosil-Pur C18-AQ material (Dr. Maisch,

174

Ammerbuch, Germany) that was flushed with 0.1% aqueous formic acid (solvent

175

A) at a flow rate of 5 µL/min. After 10 min peptides were transferred onto a 40 cm

176

× 75 µm analytical column self-packed with Reprosil-Gold C18, 3 µm resin (Dr.

177

Maisch, Ammerbuch, Germany) and operated at a flow rate of 300 nL/min using a

178

gradient with 0.1% formic acid in water as solvent A and 0.1% formic acid in

179

acetonitrile as solvent B, both solvents including 5% DMSO to boost nanoESI 8 ACS Paragon Plus Environment

Page 9 of 36


180

signal response.22 Increasing solvent B from 4 to 32% over 225 min, the effluent

181

was sprayed via PicoTip fused silica emitters (New Objective, Woburn, MA) at a

182

spray voltage of 2.2 kV and a heated capillary temperature of 200°C. The LTQ

183

Orbitrap XL instrument was operated in data-dependent mode, automatically

184

switching between MS and MS2. Full scan MS spectra (m/z 300 – 1300) were

185

acquired in the Orbitrap at a resolution of 30,000 (m/z 400) using an automatic

186

gain control (AGC) target value of 1e6 charges. Full MS spectra were on the fly

187

re-calibrated using a DMSO-related lock mass of m/z 901.922718.22,23 Tandem

188

mass spectra of up to ten precursors were acquired in the ion trap after collision

189

induced dissociation (CID; AGC target value 5×103, normalized collision energy

190

of 35%). Precursor ion isolation width was set to 2.5 Th, the maximum injection

191

time for MS/MS was 100 ms and dynamic exclusion was set to 20 s.

192

Protein Identification and Intensity-Based Absolute Quantitation

193

(iBAQ) of Proteins. Data analysis was performed using MaxQuant (version

194

1.4.0.5)24 with the integrated search engine Andromeda25. For peptide and protein

195

identification, raw files were searched against the UniProt human (009606)

196

reference

197

complemented with P00698 (Gallus gallus lysozyme). Carbamidomethylated

198

cysteine was selected as fixed modification and oxidation of methionine as well as

199

N-terminal protein acetylation as variable modification. Trypsin/P was specified as

200

the proteolytic enzyme, with up to two missed cleavage sites allowed. Precursor

201

tolerance was set to 6 ppm and fragment ion tolerance to 20 ppm. Peptide

202

identifications required a minimal length of seven amino acids. All data sets were

203

adjusted to 1% peptide-spectrum match (PSM) and 1% protein false discovery

database

(downloaded

on

22/07/2013)

annotated

with

Pfam



Page 10 of 36

204

rate (FDR). Intensity-based absolute quantification (iBAQ) values, which are

205

proportional to the molar protein quantities in the samples, were used for

206

quantification. iBAQ values were retrieved from the MaxQuant software as the

207

raw peptide intensities divided by the number of theoretical peptides. Feature

208

matching between raw files was enabled, using a match time window of 2 min.

209

Theoretical isoelectric points (pI) of the identified proteins were computed with the

210

pI/Mw tool on the ExPASy server (http://web.expasy.org/compute_pi).26

211

Statistical Analysis. Before analysis raw data were filtered in respect to

212

proteins displaying valid iBAQ values for all replicates of at least one experimental

213

condition. iBAQ raw data were median centered, and missing values were

214

imputed based on a Gaussian distribution using the Perseus algorithm to allow for

215

statistical analysis. Statistical significance was assessed by means of a one-way

216

ANOVAs. P-values were adjusted for multiple comparisons by the Benjamini-

217

Hochberg method.27 Quantitative differences in protein abundance within the

218

precipitates between the experimental conditions or sampling days were

219

considered to be statistically significant for adjusted p 0.05) for proteins marked with °.

595

Figure 6.

Selected base peak ion chromatograms of supernatant human

596

saliva

after

incubation

with

astringents

and

subsequent

597

centrifugation. Saliva was mixed (4/1, v/v) with (a) H2O (0.2 mL,

598

control) and the astringents (b) EGCG (0.2 mL, 5.0 mmol/L), (c)

599

Fe2(SO4)3 (0.2 mL, 5.0 mmol/L), (d) chitosan (0.2 mL, of 2 µmol/L),

600

respectively, and was then centrifuged. Chromatograms are plotted

601

as an overlay of the astringent treated sample (white) with the

602

control sample (grey) to expose the signals of the salivary proteins

603

affected by the respective astringent as a grey trace. For peak

604

assignment see Table 1.

605

606

607

608


Page 29 of 36

609 610 611 612


Figure 1 (Delius et al.)

613 614 615 616

617

618

619

620

621

622

623



Page 30 of 36

624

625 626 627 628 629 630


631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 30 ACS Paragon Plus Environment

Page 31 of 36

652 653 654 655 656 657 658 659



660 661 662 663 664 665 666 667 668 669 670 671 672 31 ACS Paragon Plus Environment


Page 32 of 36

673 674


679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 16

100%

75%

TCN1 TF MUC5B PRB3 SERPINA1 HV303 CST2 HV305 KLK1 GAPDH AZGP1 KV204 IGHA2 MUC7 LCN1 IGJ CSTB LTF PRH1 IGHM CST3 S100A9 LEG1 DEFA3 S100A8 CST5 IGHG1 PIGR BPIFA2 LYZ ALB IGLC2 CST1 CA6 IGKC IGHA1 CST4 AMY1A ZG16B PIP

18

TCN1 TF MUC5B PRB3 SERPINA1 HV303 CST2 HV305 KLK1 GAPDH AZGP1 KV204 IGHA2 MUC7 LCN1 IGJ CSTB LTF PRH1 IGHM CST3 S100A9 LEG1 DEFA3 S100A8 CST5 IGHG1 PIGR BPIFA2 LYZ ALB IGLC2 CST1 CA6 IGKC IGHA1 CST4 AMY1A ZG16B PIP

Σ IBAQ intensity (x 108)

Page 33 of 36 Journal of Agricultural and Food Chemistry

675 676 677 678


20

Chitosan

EGCG

Lysozyme

Fe2(SO4)3 Fe2(SO4)3

14

12

10

8

6

4

2

50%

25%

0%

33

ACS Paragon Plus Environment


701 702 703 704

Page 34 of 36


705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 34 ACS Paragon Plus Environment

Page 35 of 36

724 725 726 727



728 729 730

731



TOC graphic 176x115mm (150 x 150 DPI)

ACS Paragon Plus Environment

Page 36 of 36

Effect of Astringent Stimuli on Salivary Protein ... - ACS Publications

Recommend Documents