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Feb 22, 2017 - ABSTRACT: The interaction of astringent substances with salivary proteins, which results in protein precipitation, is considered a key ...
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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

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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.

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Journal of Agricultural and Food Chemistry

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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

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ABSTRACT

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

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

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

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

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salivary proteins rather fragmentary, human whole saliva samples were incubated

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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

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

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

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

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

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for each astringent stimulus.

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

35

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

36 37

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INTRODUCTION

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Since the 1950s various molecular mechanisms underlying oral astringency have

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been postulated,1 such as complex formation between alimentary polyphenols

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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

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at characterizing the target salivary proteins complexing with polyphenols,

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revealed astringent procyanidins to interact primarily with acidic proline-rich

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

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

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

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

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

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Besides polyphenols, other compounds have been reported to induce a

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

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

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

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

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complexes with these alternative astringent compounds are unknown, primarily

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

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

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of human salivary proteins with representatives of the four different chemical

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

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

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

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

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

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astringent molecules and the protein composition of the centrifuged sediments

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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

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

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

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

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intact salivary proteins remaining soluble upon treatment with astringents should

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

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a more comprehensive insight into the salivary target proteins interacting with

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chemically diverse astringent substances.

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MATERIALS AND METHODS

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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%

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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);

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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

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

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

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

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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

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

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immediate use in further experiments (Figure 1).

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

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

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concentrations

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

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

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

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

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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

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

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

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

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supernatants for further analysis. Biological replicates were prepared on three

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independent days.

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Analysis

of

Proteins

in

the

Supernatant

in

Saliva/Stimulus-

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

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

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

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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

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remaining in solution after astringent induced precipitation, the supernatants were

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

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

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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).

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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%

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

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

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

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protein signals in the chromatogram were deconvoluted using the software

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

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

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

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

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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

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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

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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

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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

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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

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

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the short gel.

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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

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

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

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

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

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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

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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

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

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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

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

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Protein Identification and Intensity-Based Absolute Quantitation

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

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

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

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reference

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complemented with P00698 (Gallus gallus lysozyme). Carbamidomethylated

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

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

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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

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

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

database

(downloaded

on

22/07/2013)

annotated

with

Pfam

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

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

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

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

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

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

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

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

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

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

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

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

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

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Hochberg method.27 Quantitative differences in protein abundance within the

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precipitates between the experimental conditions or sampling days were

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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

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Journal of Agricultural and Food Chemistry

Figure 1 (Delius et al.)

613 614 615 616

617

618

619

620

621

622

623

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625 626 627 628 629 630

Figure 2 (Delius et al.)

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

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Figure 3 (Delius et al.)

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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)

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675 676 677 678

Figure 4 (Delius et al.)

20

Chitosan

EGCG

Lysozyme

Fe2(SO4)3 Fe2(SO4)3

14

12

10

8

6

4

2

50%

25%

0%

33

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Figure 5 (Delius et al.)

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Figure 6 (Delius et al.)

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