Salivary Proteome Patterns Affecting Human Salt Taste Sensitivity

Oct 5, 2017 - In order to investigate the role of perireceptor events in inter-individual variability in salt taste sensitivity, 31 volunteers were mo...
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Salivary Proteome Patterns Affecting Human Salt Taste Sensitivity Theresa Stolle, Freya Grondinger, Andreas Dunkel, Chen Meng, Guillaume Médard, Bernhard Kuster, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03862 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 8, 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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Salivary Proteome Patterns Affecting Human

2

Salt Taste Sensitivity

3 4

Theresa Stolle†, Freya Grondinger†, Andreas Dunkel†, Chen

5

Meng‡, Guillaume Médard‡, Bernhard Kuster‡ and Thomas

6

Hofmann†,*

7 8



9

University of Munich, Lise-Meitner Str. 34, D-85354 Freising, Germany

10 11

Chair of Food Chemistry and Molecular Sensory Science, Technical



Chair of Proteomics and Bioanalytics, Technical University of Munich,

Emil-Erlenmeyer-Forum 5, D-85354 Freising, Germany

12 13 14 15 16

*

To whom correspondence should be addressed

17

PHONE

+49-8161-71-2902

18

FAX

+49-8161-71-2949

19

E-MAIL

[email protected]

20

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ABSTRACT

2 3

In order to investigate the role of perireceptor events in inter-individual

4

variability in salt taste sensitivity, 31 volunteers were monitored in their

5

detection functions for sodium chloride (NaCl) and classified into sensitive (0.6

6

- 1.7 mmol/L), medium sensitive (1.8 - 6.9 mmol/L), and non-sensitive

7

subjects (7.0 - 11.2 mmol/L). Chemosensory intervention of NaCl sensitive

8

(S+) and non-sensitive panellists (S-) with potassium chloride, ammonium

9

chloride, and sodium gluconate, respectively, showed the salt-taste sensitivity

10

to be specific for NaCl. As no significant differences were found between S+-

11

and S--subjects in salivary sodium and protein content, salivary proteome

12

differences and their stimulus-induced dynamic changes were analysed by

13

tryptic digestion, iTRAQ labelling, and LC-MS/MS. Differences in the salivary

14

proteome between S+- and S--subjects were found primarily in resting saliva

15

and were largely independent on the dynamic alterations observed upon salt

16

stimulation. Gene ontology (GO) enrichment analysis of key proteins, i. e.

17

immunoglobulin heavy constant y1, myeloblastin, cathepsin G and kallikrein,

18

revealed significantly increased serine-type endopeptidase activity for the S+-

19

group, while the S--group exhibited augmented cysteine-type endopeptidase

20

inhibitor activity by increased abundances in lipocalin-1, cystatin-D, -S, and -

21

SN,

22

transepithelial sodium transport by cleaving the y-subunit of the epithelial

23

sodium channel (ENaC) and protease inhibitors have been shown to reduce

24

ENaC-mediated sodium transport, the differentially modulated proteolytic

25

activity patterns observed in vivo for S+- and S--subjects show evidence of

26

playing a crucial role in affecting human NaCl sensitivity.

respectively.

As

proteases

have

been

suggested

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to

facilitate

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KEYWORDS: Salt taste, taste sensitivity, saliva, salivary proteome, sodium

2

chloride, gene ontology enrichment analysis

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INTRODUCTION

2 3

Dietary salt intake is essential for vertebrates and plays a key role in the

4

homeostatic regulation of water balance, pH, osmotic pressure, and nerve

5

conductance.1 However, excess sodium chloride has been correlated to

6

elevations in blood pressure and increased risk of cardiovascular diseases2.

7

To efficiently develop low-sodium food products without compromising on

8

salty taste, it is fundamental to understand the oral mechanisms involved in

9

salivary peri-receptor events and sodium-induced ion channel pharmacology.

10

Several mechanisms of salt taste transduction have been reported, one

11

of them comprises the epithelial sodium chloride channel (ENaC). The human

12

ENaC is amiloride sensitive3, consists of an α, β, y and δ subunit4 and is

13

primarily expressed in the apical ends of taste receptor cells4,5. Salt taste is

14

subsequently perceived when sodium passively enters the receptor cell along

15

a concentration gradient transducing neuronal signals in a cascade of

16

reactions.5 Consequently, the ENaC alone does not explain salt taste

17

behaviour completely6 and further pathways, e. g. vanilloid receptor TRPV17,

18

ion channel TRPML38 or claudin-based permeability barriers9, are suggested

19

to be involved in signal transduction. While salt taste perception of the sodium

20

salts of chloride and gluconate, respectively, were reported not to be entirely

21

inhibited by amiloride6, the broad-band antiseptic compound chlorhexidin has

22

been discovered to inhibit salt taste sensations induced by the chloride salts

23

of lithium, sodium, potassium, and ammonium.10 The underlying mechanism

24

of the anion- and cation-specific inhibition of salt taste is not yet understood

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on a molecular level and could contribute to the elucidation of the

2

mechanisms of human salt taste perception.11

3

Although saliva is considered to play an important role in modulating

4

the activation of taste receptors and ion channels on chemosensory taste

5

cells, the fundamental principles of how saliva affects taste sensitivity during

6

oral food processing are largely unknown. As taste molecules need to be

7

solubilised in saliva prior to taste receptor activation, the volume of saliva

8

produced determines the final concentration of a target tastant rather than the

9

food matrix.12,13 For example, the endogenous sodium levels in saliva have

10

been found to define the threshold of salt taste perception.14 Moreover, taste

11

stimuli have been shown to modulate saliva flow and composition,15

12

elevated salivary flow rates could further be correlated to a lower sodium

13

sensitivity due to limited reabsorption within salivary ducts.16 At the same

14

time, buffering capacities are increased and consecutively, sour taste

15

perception is reduced17,18. Alternatively, salivary constituents, such as

16

peptides and proteins, may fine-tune taste receptors and ion channels,

17

respectively, to alter taste sensitivity, or may interact with taste stimuli, thus

18

affecting their concentration available for taste receptor activation. Orally

19

expressed

20

triacylglycerides and, consecutively, to release free fatty acids which are

21

bound by lipocalin-1, which is secreted by the von Ebner glands, and

22

transported to fatty acid sensitive receptors to mediate fat taste perception.19-

23

23

24

precipitated upon complexation with astringent polyphenols, thus leading to a

25

change

lipases,

for

instance,

have

been

found

to

and

hydrolyse

Salivary proline-rich proteins and basic histatins have been shown to be

in

lubrication

perceived

as

a

mouth-drying

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and

puckering

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orosensation.24-27 In accordance with this finding, low salivary levels of

2

proteins could be correlated to an increased astringency perception,28 while

3

subjects, who were able to rapidly restore their initial protein contents, were

4

observed to be less sensitive to astringency.29 Moreover, low levels of the

5

protease inhibitor cystatin-SN found in saliva collected from caffeine-sensitive

6

subjects led to the suggestion that proteolytic events in the oral cavity may

7

alter taste sensations.30

8

The wide-ranging influence of saliva on chemosensory perception

9

raised the question as to whether the salivary composition and dynamic

10

changes induced by chemosensory stimulation plays a key role in salt taste

11

perception and, in particular, in inter-individual variability in salt taste

12

sensitivity. The objectives of the present study were, therefore, to classify

13

subjects according to their salt taste sensitivity, quantitatively measure their

14

saliva flow induced by the salty stimuli sodium chloride, potassium chloride,

15

ammonium chloride, and sodium gluconate, and to investigate time-

16

dependent dynamic changes in the salivary proteome by means of tryptic in-

17

solution digestion of whole saliva samples prior to and after chemosensory

18

stimulation, followed by protein quantitation using isobaric tags for relative and

19

absolute quantitation (iTRAQ) and nano-LC-MS/MS.

20 21 22

MATERIALS AND METHODS

23 24

Chemicals. The following compounds were obtained commercially:

25

acetonitrile (LC-MS grade, Sigma Aldrich, Steinheim, Germany), acetone

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(HPLC grade, Merck, Darmstadt, Germany), sigmaFAST protease inhibitor

2

tablet (Sigma Aldrich, Steinheim, Germany), coomassie (Bradford) protein

3

assay kit (ThermoFisher Scientific, Rockford, USA), formic acid (MERCK,

4

Darmstadt,

5

triethylammonium bicarbonate buffer (Sigma Aldrich, Steinheim, Germany),

6

dithiothreitol

7

(Proteomics Grade, Amresco, Solon, USA) and modified trypsin (sequencing

8

grade, Promega, Mannheim, Germany). For iTRAQ labelling, an iTRAQ

9

reagent 8plex multiplex kit and iTRAQ reagent multiplex buffer kit were

Germany),

(Sigma

urea

Aldrich,

(Sigma

Aldrich,

Steinheim,

Steinheim,

Germany),

Germany),

iodoacetamide

10

obtained from AB Sciex, Framingham, USA.

For sensory analysis,

11

ammonium chloride, potassium chloride, sodium chloride as well as sodium

12

gluconate were obtained from Sigma Aldrich, Steinheim, Germany and diluted

13

in bottled water. Water for sample preparation and chromatographic

14

separation was purified with an integral 5 system (Millipore, Schwalbach,

15

Germany).

16

Study Subjects. 31 healthy volunteers (18 female, 13 male, age 22-30)

17

were recruited from the Chair of Food Chemistry and Molecular Sensory

18

Science, Technical University of Munich, without any exclusion parameters

19

besides being in good health, non-smoking, and not under medication. All

20

volunteers, giving informed written consent to the work, were asked to brush

21

their teeth and rinse their mouth with water (100 mL) after breakfast and, with

22

the exception of water, not to consume any food products and smoking

23

articles, respectively, for 1 h prior to the experiment.

24

Sensory classification according to sodium chloride (NaCl) sensitivity, the

25

collection of unstimulated saliva (control sample, tC) as well as the subsequent

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measurement of salivary protein and mineral content was conducted using the

2

entire group of 31 panellists. After running through a sensory training protocol

3

to check the reproducibility of their sensory responses, 12 panellists were

4

selected to further evaluate salt stimuli by means of temporal dominance of

5

sensations (TDS). Additionally, the four most NaCl-sensitive (S+) and four

6

most NaCl-insensitive panellists (S-) were asked to perform threshold

7

determination tests with additional salt stimuli, to participate in stimulated

8

salivary flow measurements and time-dependent proteome analyses using

9

nano-LC-MS/MS.

10

Sensory Analyses. General Conditions. Experiments were conducted in

11

a sensory room with individual computerised booths at 20° - 25° C. Sensory

12

testing was performed in the morning and in triplicate analysis to reassure

13

statistical quality.

14

Detection Threshold Determination. Threshold concentrations were

15

determined by using triangle tests according to ISO 4120:2004. Briefly,

16

subjects were presented a series of three randomised glass vials, one

17

containing a salt solution (10 mL) and two containing only bottled water

18

(control, 10 mL). The panellists were then asked to identify the sample that is

19

different from the others. Aqueous sodium chloride solutions were presented

20

in increasing concentrations from 3.0 to 8.0 mmol/L in 1.0 mmol steps, sodium

21

gluconate and ammonium chloride were evaluated at levels of 1.0, 2.5, 5.0,

22

7.5, 10.0, 15.0 and 20.0 mmol/L and potassium chloride in increasing

23

concentrations of 1.0, 2.5, 5.0, 7.5, 10.0, 15.0, 22.5 and 30.0 mmol/L.

24

Subsequently, the threshold of each panellist was calculated by the geometric

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mean of the last not correctly identified concentration and the first correctly

2

identified concentration (ASU: L 00.90-9, 1999).

3

Temporal Dominance of Sensations (TDS). In order to record the TDS

4

in compliance with the measurement of salivary flow upon stimulation,

5

panellists were challenged according to a defined procedure as follows. The

6

panellists were asked to rinse their mouth with bottled water (2 mL) for 60 s

7

and, then, to expectorate. Thereafter, the panellists were requested to take an

8

aliquot (2 mL) of an aqueous stimulus solution of sodium chloride, potassium

9

chloride, ammonium chloride, or sodium gluconate (100 mmol/L each),

10

respectively, into the oral cavity and to imitate chewing motions for 15 s and,

11

then, to expectorate. Using a computerised system (FIZZ Software, v2.20E,

12

Biosystems, Couternon, France), TDS was performed using the following

13

attributes which were pre-defined in consensus with the panellists: salty,

14

sweet, sour, bitter, umami, astringent, soapy, and liquorice-like. The panellists

15

were asked to score the dominant attribute once the stimulus solution was

16

taken into the oral cavity. Each time the perception changed, the new

17

dominant attribute was scored until this attribute was not any longer

18

perceivable. After expectoration, taste perceptions were evaluated for further

19

60 s to measure time-dependent changes. The dominance rate, significance

20

level and lowest significant proportion value was calculated according to a

21

literature protocol.31

22

Subject Classification According to Sodium Chloride Sensitivity. In

23

order to classify panellists according to their individual sodium chloride

24

sensitivity, subjects were sensorially screened in their full detection range. To

25

prevent excessive fatigue, the panellists were pre-classified into a highly

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1

sensitive, sensitive, non-sensitive and highly non-sensitive group according to

2

previous sodium chloride threshold determination. Recognition threshold

3

determination was then carried out by using 3-alternative forced-choice tests

4

(ISO 13301:2002) and using individually adapted concentration ranges for

5

sodium chloride as follows: 0.25 - 1.5 mmol/L (in 0.25 mmol steps) for highly

6

sensitive panellists, 1.0 – 6.0 mmol/L (in 1.0 mmol steps) for sensitive

7

panellists, 4.0 – 9.0 mmol/L (in 1 mmol steps) for non-sensitive panellists, and

8

9.0 – 14.0 mmol/L (in 1 mmol steps) for highly non-sensitive panellists. Test

9

samples were three-digit coded and each dilution step presented with two

10

blanks (water). The panellists of the respective sensitivity group were asked to

11

identify the sample with the highest salt taste intensity in a forced-choice set-

12

up. The threshold of each panellist was further calculated by the geometric

13

mean of the last not correctly identified concentration and the first correctly

14

identified concentration (ASU: L 00.90-9, 1999). The final classification into

15

sensitive and non-sensitive panellists was based on each panellist’s ability to

16

correctly identify the sodium chloride sample in different dilutions and on

17

independent days. Thereafter, psychometric functions were calculated for

18

each panellist using logistic regression and a 95 % confidence interval.

19

Saliva Collection and Stimulated Saliva Flow Measurement.

20

Unstimulated and stimulated saliva samples were collected twice in the

21

morning (9:00 and 11:00 a.m.) on three independent days following a highly

22

standardized procedure.15 In brief, the panellists were asked to rinse their

23

mouth with bottled water (8 mL, 60 s) and, after expectorating, to take a pre-

24

stimulus control sample (water, 2 mL) into the mouth, to imitate chewing

25

motions for 30 s and, then, to expectorate into a pre-weighed 10 mL cup to

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collect the “control sample” (tC). After a second mouth rinsing and

2

expectoration step (8 mL water, 60 s), the panellists were requested to take

3

an aliquot (2 mL) of an aqueous stimulus solution of sodium chloride,

4

potassium chloride, ammonium chloride, or sodium gluconate (100 mmol/L)

5

into the mouth, to imitate chewing motions for 15 s and, then, to expectorate

6

into pre-weighed 10 mL cups to produce the “stimulus sample” (t0).

7

Thereafter, bottled water (2 mL) was taken into the mouth and, after imitating

8

chewing motions (30 s), panellists were asked to expectorate into pre-

9

weighed 10 ml cups to deliver a first “post-stimulus sample” (t30). Repeating

10

this last assay step afforded a second “post-stimulus sample” (t60). Each

11

saliva sample collected (tc, t0, t30, t60) was weighed and a protease inhibitor (3

12

µL of a solution of sigmaFAST protease inhibitor tablet in 100 mL water) as

13

well as an internal standard solution of lithium hydroxide (50 µL, 5.01 g/L

14

LiOH·H2O) was added. After vortexing (10 s), samples were sonicated (5

15

min), left for equilibration (10 min) and stored at - 80° C until use. Stimulus-

16

induced changes in saliva secretion were calculated by dividing the weight of

17

the stimulus sample (t0) as well as post-stimulus samples (t30, t60) by the

18

weight of the control sample (tC).

19

Preparation of Pooled Saliva Samples for Proteomics Analysis. An

20

aliquot (0.5 mL) of each sample collected as described above was pooled

21

according to sensitivity (sensitive vs. non-sensitive) and collection step (tC, t0,

22

t30, t60). Pooled samples were then vortexed (10 s), left for equilibration (10

23

min), sonicated (5 min), and kept at -80° C until further sample preparation.

24

Quantitation of Protein Content in Saliva Samples. The total protein

25

content of individual or pooled saliva samples was determined using the

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coomassie (Bradford) protein assay kit. Saliva samples were centrifuged (10

2

min, 4° C, 12,000 rpm) and an aliquot of the supernatant (5 µL) was placed in

3

a microplate well. The coomassie reagent was equilibrated to room

4

temperature and an aliquot (250 µL) was added to each sample. Afterwards,

5

samples were carefully mixed (30 s) and incubated with the exclusion of light

6

(10 min). The mixture was then spectrophotometrically analysed at λ=595 nm

7

using water as a reference. Standard calibration curves were equally obtained

8

by using bovine serum albumin (BSA) in saliva-relevant dilution series of 500,

9

250, 167, 125, 83, 42 and 25 µg/mL. Sample preparation and measurement

10

was performed by means of four technical replicates.

11

Quantitation of Minerals in Saliva Samples. Saliva samples were

12

centrifuged (10 min, 4° C, 12,000 rpm), an aliquot (250 µL) of the supernatant

13

was diluted with acetone (750 µL), centrifuged (10 min, 4° C, 13,200 rpm),

14

and the resulting protein pellet extracted with acetone/water (30/70, v/v; 100

15

µL). After centrifugation (10 min, 4° C, 13,200 rpm), the supernatants were

16

combined and separated from solvent under a stream of dry nitrogen (30 min,

17

35° C, 100 kPa) until dryness, the residues were solved in water (500 µL) and,

18

then, the cations were analysed by means of ion chromatography on a 250 × 4

19

mm Dionex IonPacTM AS19 RFICTM analytical column (Thermo Scientific,

20

Idstein, Germany) with a 30 min isocratic gradient (7 mmol/L methane sulfonic

21

acid) using a Dionex ICS-2000 ion chromatography system equipped with an

22

integrated DS 6 heated conductivity detection (Thermo Scientific, Idstein,

23

Germany) and a self-regenerating suppressor (Dionex CSRS Ultra II 2mm,

24

AutoSuppression Recycle Mode, 14 mA, underground conductivity 1.3 µS).

25

External calibration was carried out using the chloride salts of lithium, sodium,

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potassium, ammonium, magnesium, and calcium at concentrations ranging

2

from 0.1 to 3.6 µmol/L. Data were collected at a rate of 5 Hz and evaluated

3

using the Chromeleon Software (Version 7.1 Thermo Scientific).

4

Tryptic In-Solution Digestion and iTRAQ Labelling. Saliva samples

5

were centrifuged (10 min, 4° C, 12,000 rpm), the protein content determined

6

as described above and an aliquot (50 µg protein) of the supernatant was

7

taken for further sample preparation. Tryptic in-solution digestion was based

8

on the sample’s total protein content instead of volume and consequently, the

9

following preparation steps had to be individually adjusted. Solid urea was

10

added to reach a final concentration of 6 to 8 mol/L. Afterwards, 1 M TEAB

11

and 1 M DTT were added to final concentrations of 40 mM/L and 10 mM/L,

12

respectively. Samples were then incubated for 45 min at 56° C for subsequent

13

reduction. Alkylation of cysteine residues was carried out by adding 550

14

mmol/L iodo acetamide (1:22, v/v), followed by incubation for 60 min at room

15

temperature in the dark. Samples were then diluted using 50 mM TEAB (1:4,

16

v/v). Trypsin (20 µg) was diluted in 50 mM TEAB (100 µL) and an aliquot (2.5

17

µl) added to the samples to start tryptic digestion. After incubation (37° C, 4 h),

18

an aliquot (2.5 µL) of the trypsin solution was added again and samples

19

incubated overnight at 37° C. The enzymatic reaction was stopped by adding

20

concentrated formic acid (50 µL). Samples were then slowly applied onto the

21

top of SPE cartridges (SepPak tC18 1cc 50 mg) which were primed with an

22

aliquot (2 mL) of acetonitrile/water/formic acid (80/15/5, v/v/v) and equilibrated

23

with an aliquot (3 mL) of aqueous formic acid (water/formic acid, 95/5, v/v).

24

For SPE separation, the sample-loaded cartridges were washed with an

25

aliquot (5 mL) of aqueous formic acid (water/formic acid, 95/5, v/v) and the

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collected

upon

rinsing

with

an

aliquot

Page 14 of 47

1

effluent

(1.2

mL)

of

2

acetonitrile/water/formic acid (80/15/5, v/v/v) was dried under nitrogen (37° C,

3

4 h, 50 kPa).

4

For iTRAQ labelling, dried peptide extracts were reconstituted in 50 µL

5

dissolution buffer (iTRAQ Reagent - Multiplex Buffer Kit), vortexed for 30 s,

6

centrifuged (1 min, 5,000 rpm), and an aliquot (10 µL) added to the individual

7

iTRAQ reagent solutions (25 µL), which was prepared by centrifuging the

8

iTRAQ reagents (iTRAQ labels 113 – 121) for 1 min at 5,000 rpm at room

9

temperature, adding isopropanol (50 µL), followed by vortexing (30 s) and

10

centrifugation (1 min, 5,000 rpm). Sample mixtures were again vortexed (30 s)

11

and incubated (RT, 2 h, 500 rpm). An aliquot (5 µL) of each individually

12

labelled sample (iTRAQ 113 – 121) was combined, concentrated under

13

vacuum to dryness (- 80° C, 6 h), and excessive iTRAQ reagent degraded by

14

re-dissolving the sample in 5 % aqueous formic acid. The mixture was then

15

vortexed (30 s) and lyophilized (- 80° C, 12 h).

16

Before LC-MS analysis, sample clean-up was conducted using stage

17

tips (Empore C18 material) which were primed with aliquot (2 × 100 µL) of

18

acetonitrile/water/formic acid (80/15/5, v/v/v) and equilibrated with an aliquot

19

(150 µL) of aqueous formic acid (water/formic acid, 95/5, v/v). The labelled

20

sample mixtures were subsequently reconstituted in 5 % aqueous formic acid

21

(100 µL) and loaded onto the prepared stage tip (50 µL per stage tip). The

22

effluent was collected and loaded once again. The loaded sample was

23

washed with aliquot (150 µL) of aqueous formic acid (water/formic acid, 95/5,

24

v/v) and the effluent collected upon rinsing with an aliquot (100 µL) of

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acetonitrile/water/formic acid (80/15/5, v/v/v) was collected and lyophilized (-

2

80° C, 6 h).

3

Liquid Chromatography Electrospray Ionisation Mass

4

Spectrometry (LC-ESI-MS). Proteome analyses were performed using the

5

LC-MS/MS Eksigent nanoLC-Ultra 1D+ system (Eksigent, Dublin, CA)

6

coupled to an Orbitrap Velos mass spectrometer (Thermo Fisher Scientic,

7

Bremen, Germany). Dried samples were dissolved in 0.1 % aqueous formic

8

acid (20 µL), an aliquot (5 µL) was then loaded onto a 100 µm × 2 cm trap

9

column packed with 5 µm C18 resin (Reprosil-PUR AQ material, Dr. Maisch)

10

and flushed with 0.1% aqueous formic acid for 10 min at a flow rate of 5

11

µL/min. Peptides were then transferred onto a 75 µm × 40 cm analytical

12

column packed with 3 µm C18 resin (Reprosil-Gold C18 material, Dr. Maisch).

13

Samples were separated using a 110 min gradient from 4 to 32 % of a solvent

14

mixture A of acetonitrile containing 5% DMSO and 0.1 % formic acid in solvent

15

mixture B containing water with 5% DMSO and 0.1% formic acid at a flow rate

16

of 300 nL/min and directly transferred into the Orbitrap Velos mass

17

spectrometer operated in the positive electrospray ionisation mode.32 The

18

mass spectrometer was operated in data-dependent acquisition mode (DDA)

19

recording spectra by automatically switching between MS1 and MS2. Full

20

scan MS1 spectra were generated in the range of 360 to 1300 m/z at a

21

resolution of 40,000 using an automatic gain control (AGC) target value of 106

22

charges with a maximum injection time of 200 ms. Internal calibration was

23

carried out using a dimethyl sulfoxide cluster (m/z 401.922720). Further, the

24

top 10 peptide precursor peaks (isolation window 2 Th) were fragmented via

25

high-energy collisional dissociation (HCD) with a normalised collision energy

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of 36 % using an AGC target value of 4 · 104 with a maximum injection time of

2

200 ms. Fragment ions were recorded with a fixed first mass of 100 m/z at a

3

resolution of 7,500 and dynamic exclusion was set to 20 s.

4

Protein Identification and Label-Based Relative Quantitation

5

(LBQ). LC-MS/MS data were analysed using MaxQuant33 (version 1.5.2.8.)

6

and the integrated search engine Andromeda.34 For peptide and protein

7

identification raw files of biological replicates were searched against the

8

UniprotKB database with the taxonomy Homo sapiens (009606, version

9

2013_07_22) containing PFAM annotations for each entry and then, only

10

unique peptides taken for protein inference (Table S2). Carbamidomethylated

11

cysteine was selected as a fixed modification whereas oxidation of methionine

12

and N-terminal acetylation was set as a variable modification. Enzyme

13

specificity was set to trypsin/P with up to two missed cleavages allowed.

14

Peptide identifications further required a minimum of seven amino acids.

15

Quantitation was performed on iTRAQ reporter ions and mass tolerances set

16

to 6 ppm for precursors and 20 ppm for fragment ions. All data sets were

17

further adjusted to 1 % false discovery rate on the level of proteins and

18

peptide spectrum matches and filtered for contaminants. Assuming that the

19

total sum of protein intensities per iTRAQ channel is the same across all

20

channels (Tag 113 – 121), intensities were adjusted using total-sum

21

normalisation.

22

Statistical Analysis of Protein Data. Label-based protein quantitation

23

(LBQ) data obtained were analysed using Microsoft Excel and the R statistical

24

programming environment. Protein intensities were log10 normalised, median-

25

centred and the batch effect removed where applicable. Principal component

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analysis (PCA) was then conducted on normalised LBQ intensities of three

2

biological replicates for each salt taste challenge. The differences between the

3

proteome pattern of NaCl-sensitive and non-sensitive panellists were further

4

analysed performing a two-sided t-test on logarithmised and median-centred

5

LBQ intensities with a Benjamini-Hochberg FDR cut-off of 5 %.35 Quantitative

6

differences were considered to be statistically significant for an adjusted log10

7

p-value < 2 and a fold-change of log10 fc > 0.2. Dynamics upon stimulation

8

were analysed by performing an analysis of variance (ANOVA) for each

9

collection step in the non-sensitive and sensitive group. Normal distribution

10

and homogeneity of the data set was further confirmed by the Shapiro-Wilk

11

test (p-value > 0.05) and the Levene’s test (p-value > 0.05). Consecutively,

12

significant dynamic changes (p-value < 0.2) were illustrated for each

13

sensitivity group in heatmaps using the ward.D2 cluster method. Data were

14

further row-scaled and displayed in relation to the samples’ mean (z-score).

15

Resulting key proteins were taken for gene ontology enrichment analysis

16

using the GOrilla tool36,

17

reviewed human proteome (Homo sapiens, UP000005640).38

37

(v21052016) and compared to the complete,

18 19 20

RESULTS AND DISCUSSION

21 22

Subject Classification According to NaCl-Mediated Salt Taste

23

Sensitivity. Aimed at determining the detection threshold concentration for

24

NaCl, a total of 31 healthy volunteers performed triangle tests on aqueous

25

NaCl solutions in increasing concentrations from 3.0 up to 10.0 mmol/L.

26

Although an average threshold concentration of 6.2 mmol/L was determined 17 Environment ACS Paragon Plus

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1

for the overall panel, individual thresholds were not normally distributed and

2

indicated the presence of groups exhibiting different oral salt sensitivity

3

(Figure S1).

4

In order to increase the precision of the detection threshold

5

determination by avoiding excessive fatigue by reducing the number of

6

samples to be tested, first, panellists were pre-classified according to their

7

previous performance and, then, challenged with individually adapted, smaller

8

concentration ranges of NaCl solutions using a three alternative forced choice

9

(3-AFC) set-up forcing the panellists to identify the sample which is higher or

10

lower in salt taste by using a skimming strategy39. Individual detection

11

threshold concentrations for sodium chloride ranged from 0.6 to 11.2 mmol/L

12

and were used to classify panellists into sensitive, medium sensitive, and non-

13

sensitive subjects in accordance with their individual threshold concentration

14

ranges of 0.6 - 1.7, 1.8 - 6.9, and 7.0 - 11.2 mmol/L, respectively (Table 1).

15

As detection thresholds may be affected by day-to-day variability of the

16

panellist’s performance,40 the individuals’ reproducibility to correctly identify

17

threshold level sodium chloride samples had to be taken into account in order

18

to identify suitable panellists for the following proteome studies. To achieve

19

this, psychometric functions were calculated for each panellist using logistic

20

regression models based on the panellist’s daily performance at each test

21

concentration of sodium chloride. Consistently sensitive and non-sensitive

22

panellists were identified on the basis of detection threshold and dose-

23

response curve characteristics (Figure 1). Panellists P12, P14, P16 and P31,

24

showing a low slope with high values of correct identification (1.0), were

25

classified ‘sensitive’ (S+), whereas subjects P2, P5, P9, and P10, exhibiting an

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even curve linearity with low values of correct identification (0.0), were

2

classified ‘non-sensitive’ (S-). These eight subjects were selected for further

3

studies.

4

To answer the question as to whether the subjects’ sensitivity for

5

sodium chloride is correlated to their sensitivity for other mineral salts, the

6

individuals’ detection thresholds of potassium chloride, ammonium chloride,

7

and sodium gluconate were determined by using a triangle test design and

8

logistic regression models were calculated for the NaCl sensitive (S+) and

9

non-sensitive groups (S-) (Figure S2). Although S+-panellists seemed to be

10

also somewhat more sensitive to other salts when compared to the S--

11

panellists, a clear classification was not possible due to the biological variance

12

between individuals (Table S1), thus indicating that both the anion as well as

13

the cation play a role in subjects’ salt taste perception.

14

Influence of Counter Ions on Salt Taste Perception. In order to

15

investigate how the counter-ion of salts affects both the sensory perception

16

and salivation, the temporal dominance of sensations (TDS) was measured in

17

conjunction with the collection of stimulated saliva. Therefore, panellists were

18

first challenged with sodium chloride (100 mmol/L), followed by isomolar

19

concentrations of potassium and ammonium chloride to study the influence of

20

the cation, as well as sodium gluconate (100 mmol/L) to visualize the impact

21

of the anion. While sodium chloride was primarily perceived as salty,

22

potassium chloride and ammonium chloride, in contrast, were mainly

23

perceived as bitter (Figure 2A). However, ammonium chloride additionally

24

triggered salt taste perception, although to a minor extent only. Since the

25

cation substitution resulted in a change in dominant taste quality, the cation

19 Environment ACS Paragon Plus

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1

might play a significant role in the perception of saltiness. The sodium

2

gluconate stimulus triggered dominantly salty and soapy taste qualities. The

3

decreased dominance of salt taste might be due to a reduced sodium influx

4

into taste cells when substituting chloride with the less permeable organic ion

5

gluconate9,41,42 or a decreased paracellular anionic conductance due to a

6

larger anion.9,41 Generally, an increasing dominance in astringency was

7

perceived after expectoration of aqueous salt solutions, which may be

8

explained by time-dependent changes in the proteome pattern and a resulting

9

alteration in saliva viscosity.

10

Influence of Protein and Mineral Content on Salt Taste Sensitivity.

11

As differences in salivary flow rate and salivary mineral levels have been

12

shown to affect taste perception,14,16 the volume, mineral and protein

13

concentration were determined in non-stimulated saliva (control sample tC)

14

collected from the 31 previously classified panellists. On average, the saliva of

15

sensitive panellists revealed marginally higher protein concentrations

16

demonstrating a mean value of 173 mg/L compared to non-sensitive panellists

17

exhibiting 155 mg/L (Figure S3). Non-sensitive panellists, in contrast,

18

exhibited higher salivary concentrations of sodium and potassium (2.9 and

19

11.2 mmol/L) when compared to sensitive panellists (2.1 and 9.9 mmol/L)

20

(Figure S3). However, due to a large inter-subject variability, these findings

21

were not significant and remained indicative only. Consequently, further

22

analyses were conducted to evaluate salt taste perception.

23

Influence of Mineral Salts on Salivation. In order to investigate the

24

influence of mineral salts on saliva flow, NaCl sensitive and non-sensitive

25

panellists were asked to rinse their mouth with a pre-stimulus control sample

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(water), to imitate chewing motions for 30 s and, then, to expectorate into a

2

pre-weighed cup to collect the “control sample” (tC). Thereafter, sodium

3

chloride solutions (10, 100, 500 mmol/L, each six repetitions) were taken into

4

the oral cavity and, after imitating chewing motions for 15 s, expectorated into

5

a pre-weight cup to produce the “stimulus sample” (t0). Thereupon, the

6

panellists were requested to take an aliquot of water into the mouth, perform

7

chewing motions for 30 s and expectorate in a pre-weight cup to deliver a first

8

“post-stimulus sample” (t30). In order to measure time-dependent changes, the

9

latter step was repeated to produce a second “post-stimulus sample” (t60).

10

Statistical evaluation by means of the Tukey HSD-method and ANOVA

11

revealed stimulus concentrations of 100 and 500 mmol/L of sodium chloride to

12

trigger a significant change in saliva flow (p < 0.01) upon immediate

13

stimulation when compared to a blank experiment with water as the stimulus

14

(Figure S4).

15

Thereafter, the experiment was repeated by using potassium chloride,

16

ammonium chloride, and sodium gluconate (100 mmol/L), respectively, as

17

taste stimuli. Ammonium chloride demonstrated the greatest effects on

18

salivation triggering flow increases of 13.0 % upon stimulation (t0; p = 0.001)

19

and 7.8 % in the first post-stimulus sample (t30; p = 0.001) within both

20

sensitive and non-sensitive panellists (Figure 2B). In comparison, sodium

21

chloride solely induced a significantly elevated saliva flow immediately upon

22

stimulation at t0 (p-value = 0.006) with an increase of 6.4 % observed for the

23

sensitive group and 1.9 % in case of the non-sensitive group. In contrast,

24

chemosensory intervention with potassium chloride and sodium gluconate,

25

respectively, did not show any significant impact on salivary flow. In summary,

21 Environment ACS Paragon Plus

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1

neither counter-ions, nor the perceived taste quality were correlated to saliva

2

flow alteration and salivary flow rates seemed to play a minor role in sodium

3

chloride sensitivity.

4

Individual Variation and Dynamics of the Salivary Proteome. In

5

order to investigate a potential relationship between salt taste sensitivity and

6

the saliva proteome pattern and/or dynamic changes induced upon salt

7

stimulation, the corresponding saliva samples (tC, t0, t30, t60) collected in the

8

NaCl-intervention trial detailed above from salt taste sensitive (S+) and non-

9

sensitive subjects (S-) were pooled and, then, the salivary proteins identified

10

and relatively quantified by means of iTRAQ labelling, tryptic digestion and

11

nano-LC-MS/MS, followed by principal component analysis (PCA) to discover

12

differentially expressed proteome patterns. As displayed in Figure 3, the PCA

13

score plot demonstrates two main components affecting salivary proteome

14

alteration upon sodium chloride stimulation. The first component strongly

15

separated dynamic proteome alterations throughout the NaCl-intervention

16

(52.4 %) with time-dependent changes bouncing back to the initial proteome

17

pattern (tc) 60 s after stimulation (t60). In comparison, the second component

18

(28.3 %) separated the NaCl sensitive (S+) and non-sensitive group (S-). Since

19

this separation occurred to an equal extent in all saliva collection steps (tC, t0,

20

t30, t60), sodium chloride sensitivity may be influenced by a subject’s initial

21

proteome pattern rather than its stimulus-induced proteome alteration. This is

22

further substantiated by the observation that oral stimulation with potassium

23

chloride, ammonium chloride, or sodium gluconate (Figure S5) revealed the

24

same trends as found for sodium chloride (Figure 3). Moreover, the day-to-

25

day reproducibility and the low biological variability of the separation of the

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proteome data recorded for the resting saliva collected from NaCl sensitive

2

(S+) and non-sensitive subjects (S-) even in the intervention trials with

3

potassium chloride, ammonium chloride, and sodium gluconate, respectively,

4

further strengthened the hypothesis that sodium chloride sensitivity may

5

depend on the proteome pattern in resting saliva.

6

To gain a first insight into the key proteins causing the PCA separation

7

of the resting saliva collected from NaCl sensitive (S+) and non-sensitive

8

subjects (S-), t-tests with a 5 % FDR cut-off were applied and the significance

9

then plotted versus the fold change of the proteins detected. Extracting the

10

differentially expressed key proteins in non-stimulated saliva (tC) revealed the

11

non-sensitive subject group (S-) to exhibit significantly increased abundance in

12

both lipocalin-1 (LCN1), reported to play a role in fat taste perception, and

13

DMBT1 (malignant brain tumors protein 1) which is proposed to function as a

14

binding protein in saliva for the regulation of taste sensation (Figure 4).20,21 In

15

comparison, the salt-sensitive group (S+) demonstrated significantly increased

16

abundance in immunoglobulines IGHG1 and IGHG2, reported to play a key

17

function in oral health,38 as well as in cytoplasmic 2 actin (ACTG1) and

18

coronin-1A (CORO1A), respectively.

19

This analytical procedure was repeated for each saliva collection step

20

(tC, t0, t30, t60) as well as stimulus (Table 2). Across all data sets, lipocalin-1

21

(LCN1), cystatin-SN (CST1), cystatin-S (CST4) and cystatin-D (CST5) were

22

found to be top marker proteins for the non-sensitive (S-) group. Lipocalin-1 is

23

secreted by von Ebner glands which are located in the cleft of taste buds.44

24

Although lipocalin-1, through its ability to bind and transport hydrophobic

25

molecules,45 has been proposed to play a role in the sensory perception of fat

23 Environment ACS Paragon Plus

Journal of Agricultural and Food Chemistry

1

taste,20,21,38 it has not been described as a candidate protein affecting sodium

2

chloride sensitivity. Among the family of 14 human cystatins, cystatin-A,

3

cystatin-B, cystatin-C, cystatin-D, cystatin-S, cystatin-SA, and cystatin-SN

4

have been identified previously in saliva,38 cystatin-SN could be linked to

5

caffeine sensitivity30, 47 and the perception of astringency.29, 48

6

When compared to the data obtained for the S--group, the sensitive

7

group (S+) showed significantly increased abundance for immunoglobulin

8

heavy constant y1 (IGHG1), immunoglobulin heavy constant y2 (IGHG2)

9

chain C region, myeloblastin (PRTN3), cathepsin G (CSTG), and haptoglobin

10

(HP) across the various salty tastant challenges. While the up-mentioned

11

cystatins are released mainly from the major submandibular glands, the

12

immunoglobulins are known to be secreted by major and minor salivary

13

glands.49 In consequence, observed differences in proteome pattern between

14

S- and S+-subjects may be induced by the differential activation patterns of

15

salivary glands in both subject groups. Taking all these data into account, the

16

differences in sodium chloride sensitivity of subjects may be concluded to be

17

due to differences in the resting saliva proteome and is largely independent

18

from dynamic proteome alterations induced by chemosensory stimulation.

19

To investigate dynamics upon salty tastant challenge, an analysis of

20

variance was performed for both the sensitive and non-sensitive group and

21

significant changes (p-values < 0.2) graphically heatmapped for the non-

22

sensitive group upon sodium chloride stimulation (Figure 5). The hierarchical

23

clustering revealed three main clusters accounting for initial salivary proteome

24

pattern, proteins affected upon stimulation and time-dependent changes,

25

respectively. The largest effects were shown upon stimulation and resulted in

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significantly increased abundances of lysozyme C (LYZ), antileukoproteinase

2

(SLPI), ankyrin repeat domain containing protein SOWAHA (SOWAHA) and

3

lactotransferrin (LTF), respectively. In comparison, lubricating mucin-5B

4

(MUC5B) as well as cystatin-SN (CST1) and cystatin-S (CST4) were found as

5

key proteins affected in a time-dependent manner and solely upregulated after

6

stimulation in samples t30 and t60. These findings were identical in both the

7

NaCl sensitive (S+) and non-sensitive group (S-) (Figure S5) and thus,

8

suggested that sodium chloride sensitivity is affected by the proteome pattern

9

in resting saliva only. The same trends were observed upon oral stimulation

10

with potassium chloride, ammonium chloride, or sodium gluconate (Figure

11

S5). Lysozyme C (LYZ) and antileukoproteinase (SLPI) were found to be key

12

proteins affected upon stimulation (Table S3, Figure S6). Mucin-5B (MUC5B),

13

in contrast, was significantly upregulated in post-stimulus samples t30 and t60

14

and consequently, key protein for time-dependent changes upon oral

15

stimulation. Being responsible for viscoelastic properties of whole saliva,38

16

mucins are secreted by sublingual and submandibular glands only.50 In doing

17

so, the contribution of specific salivary glands upon time-dependent changes

18

may suggest that dynamics upon stimulation alter proteome pattern by

19

differential activation of salivary glands.

20

Sodium Chloride Sensitivity and Underlying Biological Functions

21

of Saliva Proteins. In order to correlate key proteins in the context of sodium

22

chloride sensitivity to underlying biological functions, a gene ontology

23

enrichment analysis was conducted using the GOrilla tool.36,

24

this, key marker proteins for each sensitivity group were compared to the

25

complete, reviewed human proteome (Homo sapiens, UP000005640) as

25 Environment ACS Paragon Plus

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To achieve

Journal of Agricultural and Food Chemistry

Page 26 of 47

1

visualised in Figure 6. Intriguingly, marker proteins indicative for the sensitive

2

and non-sensitive group, respectively, demonstrated a highly significant

3

enrichment in contrasting biological functions. The non-sensitive group (S-)

4

demonstrated an augmented endopeptidase inhibitor activity (p-value =

5

8.74·10-9) resulting from an elevated secretion of lipocalin-1, cystatin-SN,

6

cystatin-S, cystatin-SA, and cystatin-D, respectively. In comparison, the

7

sensitive

8

endopeptidase activity (p-value = 8.52·10-8) which is linked to an increased

9

abundance in immunoglobulin y1, y2 and y3 chain C region as well as

10

lactotransferrin, cathepsin, and myeloblastin, respectively. It is interesting to

11

notice that serine proteases have been suggested to cleave the y-subunit of

12

the epithelial sodium channel (ENaC) to facilitate the transepithelial sodium

13

transport,51 while serine protease inhibitors have been shown to reduce

14

ENaC-mediated sodium transport.52 In consequence, the proteolytic activity

15

pattern of saliva, which has now been shown for the first time to be

16

differentially modulated between NaCl-sensitive and non-sensitive subjects,

17

may be concluded to play a crucial function in the modulation of sodium

18

chloride sensitivity. Therefore, enhanced salt sensitivity may be explained by

19

a facilitated transepithelial sodium transport through endoprotease-catalysed

20

cleavage

21

endoprotease activity may be considered to degrade some salivary proteins to

22

release salt-taste enhancing peptides such as arginyl-peptides which have

23

been shown to exhibit human salt taste enhancement53,

24

activation.5 Future studies are needed to clarify which of both mechanisms

25

plays the key role in endoprotease-enhanced salt taste sensitivity.

group

of

(S+)

exhibited

ENaC’s

a

y-subunit.51

significantly

Alternatively,

26 Environment ACS Paragon Plus

enriched

serine-type

increased

54

salivary

and ENaC

Page 27 of 47

Journal of Agricultural and Food Chemistry

1 2

Supporting Information Available

3

Tables S1 and S2 and Figures S1 to S6 are available free of charge via the

4

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

5 6

Acknowledgment

7

We are grateful to Givaudan Flavors Corporation for supporting this research

8

and thank Gesa Haseleu, Elisabetta Lubian, and Thorsten König for the many

9

inspiring and fruitful discussions.

10

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Table 1. Individual Detection Threshold Concentrations for Sodium Chloride (NaCl) and Classification According to NaCl Sensitivity Sensitive Subject

Medium sensitive

TC

Subject a

Non-sensitive

TC a

Subject

TC

No.

(mmol/L)

No.

(mmoL/L)

No.

(mmol/L)a

P12

0.6

P23

3.0

P28

7.0

P31

0.9

P29

3.5

P7

7.3

P16

0.9

P25

3.6

P8

7.8

P14

1.2

P30

3.7

P5

8.1

P13

1.4

P20

3.8

P4

9.1

P15

1.4

P27

4.4

P2

9.5

P17

1.7

P19

4.4

P10

10.8

P18

1.7

P11

4.5

P9

11.2

P26

1.7

P22

4.8

P1

5.0

P24

5.0

P21

5.0

a

Individual detection threshold concentration of sodium chloride (water) determined by means of a 3-AFC test.

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Table 2. Key Proteins Differentially Modulated between Sensitive and Non-sensitive Panellists before and upon Chemosensory Stimulation Gene names

Molecular function/biological process‡

log10 foldchange

log10 pvalue

-0.3 -0.6

3.1 3.4

0.3

3.4

0.3

2.8

0.3 0.3

3.3 3.2

0.2

4.3

0.3

3.3

0.3 0.2

4.0 2.2

0.2 0.2

2.2 2.7

-0.2

2.6

-0.3 -0.2 -0.2

2.4 2.4 3.8

-0.7

4.9

-0.4

3.1

-0.2 -0.3

2.9 2.3

-0.3 0.2 0.4

2.1 4.1 3.7

Sodium chloride DMBT1* LCN1*

ACTG1

CORO1A DEFA3 IGHG1 IGHG2 IGHG3 LTF S100A8 SPR1A TF

Protein binding Sensory perception of taste, endopeptidase inhibitor activity, small molecule binding, transporter activity Identical protein binding, cell junction assembly, response to calcium ion Protein C-terminus binding, calcium ion transport Antibiotic, antifungal, antiviral Antigen binding, immune response, endopeptidase activity Antigen binding, immune response, endopeptidase activity Antigen binding, immune response, endopeptidase activity Ca2+/Zn2+ binding, immune response Iron ion binding, iron homeostasis Potassium chloride

BPIFB2* CST1* CST4* DSC2* LCN1*

LYZ* ORM1* PIP*

TYMP* AMY2B HP

Lipid binding, post-translational protein modification Endopeptidase inhibitor activity Endopeptidase inhibitor activity Calcium ion binding, cell adhesion Sensory perception of taste, endopeptidase inhibitor activity, small molecule binding, transporter activity Catalytic activity, antimicrobial, protein binding Inflammatory response Endopeptidase activity, detection of chemical stimulus involved in bitter taste Transferase activity Digestion Hemoglobin binding, antioxidant activity 36 Environment ACS Paragon Plus

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HSPB1 PRTN3

Protein binding, response to virus Endopeptidase activity, peptidase activity

0.2 0.2

3.5 2.7

-0.3

2.1

-0.3

2.4

-0.7

3.0

-0.3

2.3

Ammonium chloride SERPINA1*

CST1*

Endopeptidase inhibitor activity, protease binding Lipid binding, post-translational protein modification Bicarbonate transport, Detection of chemical stimulus involved in perception of bitter taste Endopeptidase inhibitor activity

CST4*

Endopeptidase inhibitor activity

-0.3

2.1

DMBT1*

Protein binding

-0.2

2.0

FCGBP*

May be involved in the maintenance of the mucosal structure as a gel-like component of the mucosa Sensory perception of taste, endopeptidase inhibitor activity, small molecule binding, transporter activity Catalytic activity, antimicrobial, protein binding Endopeptidase activity, detection of chemical stimulus involved in bitter taste Hemoglobin binding, antioxidant activity Endopeptidase activity, peptidase activity Transketolase activity

-0.4

4.7

-0.8

3.9

-0.4

2.9

-0.3

2.0

0.3

2.3

0.3

3.7

0.3

3.3

BPIFB2* CA6.1*

LCN1*

LYZ* PIP*

HP** PRTN3** TKT**

Sodium gluconate BPIFA2*

Lipopolysaccharide binding, antimicrobial response, defense response to bacterium

-0.2

2.2

BPIFB1*

Lipid binding, antimicrobial response, innate immune response in mucosa

-0.4

2.3

BPIFB2*

Lipid binding, post-translational protein modification

-0.2

2.8

FCGBP*

May be involved in the maintenance of the mucosal structure as a gel-like component

-0.4

4.1

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LCN1*

PIP* SPARCL1* ZG16B* A2M** CRNN** CTSG** HP** IGHG1** KV204** MPO**

PRTN3** PPIA**

of the mucosa Sensory perception of taste, endopeptidase inhibitor activity, small molecule binding, transporter activity Endopeptidase activity, detection of chemical stimulus Calcium ion binding, signal transduction Carbohydrate binding, retina homeostasis Protein binding Calcium ion binding Endopeptidase activity, peptidase activity Hemoglobin binding, antioxidant activity Antigen binding, immune response, endopeptidase activity Response to food, response to mechanical stimulus, response to lipopolysaccharide Endopeptidase activity, peptidase activity Peptidyl-prolyl cis-trans isomerase activity

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-0.5

4.5

-0.2

2.7

-0.3

2.5

-0.2

2.0

0.3 0.2 0.4

2.5 2.1 2.4

0.3

4.2

0.2

2.1

0.2 0.2

2.3 2.3

0.3

3.2

0.2

2.0 ‡

* Key protein in the non-sensitive group. ** Key protein in the sensitive group. Source: UniProt protein database

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FIGURE LEGENDS

Figure 1.

Psychometric functions displaying the performance of sensitive and non-sensitive panellists to detect sodium chloride at different concentrations.

Figure 2.

(A) Graphical TDS representation for sodium chloride, potassium chloride, ammonium chloride, and sodium gluconate, respectively. (B) Time course of salivary flow increase induced by an aqueous stimulus solution (2 mL) containing sodium chloride, potassium chloride, ammonium chloride, or sodium gluconate (100 mmol/L each), respectively. The increase or decrease in saliva secretion upon stimulation was calculated by the weight difference of stimulus (t0) or post-stimulus samples (t30, t60) and the pre-stimulus sample (tc). The flow rates are shown for both individual panellists (thin line) as well as the median of each sensitivity group (thick line).

Figure 3.

Principal component analysis (PCA) of saliva samples upon sodium chloride stimulation. Biological replicates are comprised by convex polygons and colour-coded according to sensitivity and saliva collection step (tc, t0, t30, t60).

Figure 4.

Key proteins differentially modulated between the sensitive and non-sensitive group in unstimulated saliva samples. The volcano plot illustrates the proteome pattern before sodium chloride stimulation by plotting significance versus fold change (5 % FDR). Key proteins with a threshold of log10 p-value > 2 and log10 fold change > 0.2 are further labelled by their gene names.

Figure 5.

Dynamic changes of the salivary proteome upon sodium chloride stimulation. The hierarchical clustering at the top reveals initial proteome pattern, proteins affected upon stimulation and timedependent changes. The z-score illustrates the abundance of significantly in- or decreased proteins with a 5 % FDR cut-off and is row-scaled.

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Figure 6.

Gene ontology enrichment analysis of differentially expressed proteome pattern with regard to sodium chloride sensitivity. The significance of enriched biological functions within each sensitivity group is colour-coded. The hierarchical structure further illustrates the relation between enriched GO terms.

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Stolle, Grondinger, Dunkel, Meng, Médard, Kuester and Hofmann (Figure 1)

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Stolle, Grondinger, Dunkel, Meng, Médard, Kuester and Hofmann (Figure 2)

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Stolle, Grondinger, Dunkel, Meng, Médard, Kuester and Hofmann (Figure 3)

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Stolle, Grondinger, Dunkel, Meng, Médard, Kuester and Hofmann (Figure 4)

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Stolle, Grondinger, Dunkel, Meng, Médard, Kuester and Hofmann (Figure 5)

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Stolle, Grondinger, Dunkel, Meng, Médard, Kuester and Hofmann (Figure 6)

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TOC Graphic 147x81mm (150 x 150 DPI)

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