peptidomic analysis of human acquired enamel pellicle

Copyright © 2007 John Wiley & Sons, Ltd. Biomed. Chromatogr. 21: 1107–1117 (2007)

DOI: 10.1002/bmc

Peptidomic analysis of human acquired enamel pellicle 1107ORIGINAL RESEARCHORIGINAL RESEARCH

Copyright © 2007 John Wiley & Sons, Ltd.

BIOMEDICAL CHROMATOGRAPHY

Biomed. Chromatogr. 21: 1107–1117 (2007)

Published online 21 May 2007 in Wiley InterScience

(www.interscience.wiley.com) DOI: 10.1002/bmc.830

Peptidomic analysis of human acquired enamel pellicle

Rui Vitorino,1,2 Maria João Calheiros-Lobo,3 Jason Williams,4 António J. Ferrer-Correia,1 Kenneth B. Tomer,4José A. Duarte,2 Pedro M. Domingues1 and Francisco M. L. Amado1*1Department of Chemistry, University of Aveiro, Aveiro, Portugal2CIAFEL, Faculty of Sport, University of Porto, Porto, Portugal3High-School of Health-North, Medical School of Dentistry, Portugal4Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, Research Triangle Park,

NC 27709, USA

Received 12 December 2006; revised 6 February 2007; accepted 7 February 2007

ABSTRACT: Human acquired enamel pellicle is the result of a selective interaction of salivary proteins and peptides with the

tooth surface. In the present work, the characterization of the peptides as well as the type of interactions established with the

enamel surface was performed. Peptides from in vivo bovine enamel implants in the human oral cavity were sequentially

extracted using guanidine and trifluoroacetic acid solutions and the fractions obtained were analysed by LC-MS and LC-MS/MS.

Based on the LC-MS data, six phosphorylated peptides were identified in an intact form, strongly adsorbed to the enamel surface.

Data from the LC-MS/MS analyses allowed us to identified 30 fragment peptides non-covalently bonded to enamel [basic proline-

rich proteins, histatins (1 and 3) and acidic proline-rich protein classes]. The tandem mass spectrometry experiments showed the

existence of a pattern of amide bond cleavage for the different identified peptide classes suggesting a selective proteolytic activity.

For histatins, a predominance of cleavage at Arg, Lys and His residues was observed, while for basic proline-rich proteins,

cleavage at Arg and Pro residues prevailed. In the case of acidic proline-rich proteins, a clearly predominance of cleavage of the

Gln–Gly amide bond was evident. Copyright © 2007 John Wiley & Sons, Ltd.

KEYWORDS: acquired enamel pellicle; whole saliva; LC-MS/MS; salivary peptides; proteolytic activity

*Correspondence to: F. M. L. Amado, Department of Chemistry,

University of Aveiro, 3810-193, Aveiro, Portugal.

E-mail: [email protected]

Abbreviations used: PRP, proline-rich protein; TFA, trifluoroacetic

acid.

Contract/grant sponsor: Intramural Research Program of the NIH,

National Institute of Environmental Health Sciences.

Contract/grant sponsor: Fundação para a Ciência e Tecnologia; Con-

tract/grant number: SFRH/BPD/14968/2004 and POCTI/QUI/5890/

2004.

INTRODUCTION

The acquired enamel pellicle is a protein film originat-

ing in the continuous exposure of enamel to whole

saliva. The adsorption of proteins and peptides and

subsequent pellicle formation is considered a dynamic

process influenced by several factors inherent to the

individual, such as the circadian cycle, oral microflora,

proteolytic activity and the tooth’s physical and chemi-

cal properties, as well as the location of teeth in the

mouth (Lendenmann et al., 2000; Carlen et al., 1998;

Vacca Smith and Bowen, 2000; Rykke and Sonju, 1991;

Li et al., 2003). As the enamel pellicle constitutes an

interface between teeth and the oral environment, it is

expected that its origin and composition should be of

major importance for the function it plays within the

oral cavity. The acquired enamel pellicle has been pro-

posed to be involved in several oral functions, such as

lubrication to prevent abrasive damage, acting as a selec-

tive permeable barrier that regulates the mineralization/

demineralization processes, and control of the dental

plaque microbial flora composition (Lendenmann et al.,

2000; Carlen et al., 1998; Vacca Smith and Bowen,

2000; Rykke and Sonju, 1991; Li et al., 2003). Using dif-

ferent methodologies, several authors have studied the

acquired enamel pellicle composition, which allowed

identification of mucins MG1 and MG2, amylase, albu-

min, IgA, S-IgA, proline-rich proteins (PRPs), cystatins,

lysozyme, carbonic anhydrase, lactoferrin and histatins

as major pellicle components (Carlen et al., 1998; Vacca

Smith and Bowen, 2000; Rykke and Sonju, 1991; Li

et al., 2003, 2004; Schupbach et al., 2001; Yao et al.,

2001, 2003; Vitorino et al., 2004a; Leinonen et al., 1999).

It is assumed that the formation of the acquired

enamel pellicle takes place in two stages, the first one

being characterized by an almost instantaneous adsorp-

tion of proteins to the enamel surface (Carlen et al.,

1998; Jensen et al., 1991; Lamkin et al., 1996). In this

phase, the initial adsorption of salivary components

occurs by electrostatic attraction forces developed


DOI: 10.1002/bmc

1108 R. Vitorino et al.ORIGINAL RESEARCH

between the charged groups of the macromolecules

and the phosphate and calcium ions of enamel. Indeed,

calcium ions present in the crystal construction of

enamel showed a stronger tendency to migrate to saliva

than did phosphate ions, contributing to the creation of

a double-layer: negative charges of phosphate ions on

the tooth surface and the opposite charged calcium ions

at the boarder phase (Yin et al., 2003; Gorbunoff and

Timasheff, 1984; Moreno et al., 1984). Therefore, basic

proteins are bound primarily by electrostatic interac-

tions between the amino groups and the phosphate ions

of the hydroxyapatite, whereas the acidic proteins are

bound specifically by complexation of their carboxyl

and/or phosphate groups to calcium sites. The predomi-

nant groups of salivary proteins which show selective

adsorption to enamel include small components such as

acidic PRPs, statherin and histatins, usually thought of

as pellicle precursors (Jensen et al., 1991; Moreno et al.,

1984; Bennick et al., 1983). A strong adsorption is

exhibited by the phosphorylated forms of those small

proteins. It has been described that these components

bind immediately when in contact with the enamel sur-

face and do not increase thereafter, while molecules

such as histatins bind slowly and progressively over

time (Vacca Smith and Bowen, 2000). Those peptides

are capable of phosphate or carboxyl-terminal anion

exchange reactions with the enamel surface as described

for statherin (Hay and Moreno, 1979; Schlesinger and

Hay, 1977; Stayton et al., 2003). In association with the

process of adsorption to hydroxyapatite, soluble pellicle

precursor proteins are transformed into a less soluble

or even insoluble protein film. This insolubility may

also arise from post-secretory processing of pellicle pro-

teins, which includes cross-linking and proteolysis (Yao

et al., 1999, 2000; Lamkin et al., 2001). In fact, in vitro

studies showed the existence of a proteolytic pattern

for adsorbed salivary proteins (Lamkin et al., 2001) and

that proteolysis can occur after protein adsorption to

hydroxyapatite, as shown by Bennick et al. (1983) and

Jensen et al. (1991). To date, several authors have stud-

ied salivary peptides in order to elucidate possible

cleavage pathways and post-translational modifications

that occur after its secretion from salivary glands. From

these studies, Castagnola and coworkers described a

possible cascade of fragmentation on histatins which

follows a trypsin-like pathway, and characterized a

complex of several isoforms from basic PRPs and acidic

PRPs (Castagnola et al., 2004; Messana et al., 2004;

Inzitari et al., 2005). In another study, Hardt et al.

(2005) characterized the peptidome of parotid saliva

by ultrafiltration, finding mainly peptides belonging to

acidic PRPs and histatin 1 classes.

Because of the importance of salivary peptides on

pellicle formation, the main goal of the present study is

the in vivo elucidation of peptide pellicle composition

and how salivary peptides interact with the enamel

surface. To achieve this, a combination of LC-MS and

LC-MS/MS was used for the separation and charac-

terization of human acquired enamel pellicle peptides

using a sequential extraction with guanidine (6 M) and

trifluoroacetic acid (TFA, 2%) from enamel implants.

MATERIAL AND METHODS

Chemicals. HPLC-grade acetonitrile (Riedel, Seelze, Germany),

TFA (Fluka, Buchs, Switzerland), guanidine (Sigma-Aldrich,

Madrid, Spain) and Milli-Q grade water were used.

In situ pellicle formation. For in situ pellicle formation,

two healthy, non-medicated male subjects (aged 24 and 28)

were selected according to visual oral examination performed

by an experienced dentist. The subjects showed no presence

of caries or signs of other oral pathologies. The subjects

refrained from eating and drinking for 3 h before and during

pellicle formation. Small rectangular pieces of enamel (about

10 mm long and 4 mm high) were prepared from labial

surfaces of bovine incisors (Hannig et al., 2005). The enamel

pieces were bonded on the labial surfaces of the upper and

lower first molars according to the accepted adhesion pro-

tocols for tooth restoration, as close as possible to the

gingival. The enamel implants were then thoroughly cleaned

with fluoride-free paste with a synthetic brush and copious

rinsed with an air–water spray to eliminate any possible con-

tamination with saliva previous to pellicle formation. After

2 h, the enamel pieces were removed from the mouth with

proper bracket debonding forceps used in orthodontics.

Peptide extraction. Sequential extractions were performed

as described previously for in vitro experimental conditions

(Vitorino et al., 2005), using solutions of guanidine (6 M)

and TFA (2%). Briefly, after enamel implant wash with

water, 300 µL of guanidine (6 M) were added to the enamel

pieces. Pellicle peptide extraction was performed by vigorous

vortexing for 120 s. Then the extract was centrifuged at

6000 rpm, 4°C, for 15 min, the resultant supernatant was

removed and the enamel pieces were further washed with

water under vigorous vortexing followed by a spin at

6000 rpm. After this, 300 µL of 2% TFA were added to

enamel pieces and after 2 h the suspension was centrifuged

and the supernatant recovered for further analysis. Adsorbed

salivary peptides were isolated by passing the supernatant

extracts through a 30 kDa cut-off membrane (Centricom 30,

Millipore, USA). All fractions were subsequently analysed by

LC-MS. Blanks were obtained with bovine enamel pieces

treated with the same sequential extraction procedure without

performing the incubation step.

Liquid chromatography and mass spectrometry analysis.LC-MS was performed using a Waters 2690 (Alliance, Waters

Corporation, Saint-Quentin, France) HPLC system at a flow

rate of 0.30 mL/min connected to a splitter (Acurate, LC

Packings, Amsterdam, Netherlands) to obtain a flow rate of

15 µL/min, compatible with the column used and the mass

spectrometer (QTOF2, Micromass, UK) electrospray source

operated in the positive mode at 3.0 kV. The HPLC column


DOI: 10.1002/bmc

Peptidomic analysis of human acquired enamel pellicle 1109ORIGINAL RESEARCH

was a Discovery C18

15 ! 0.32 mm, 5 µm particle size

(Supelco, Sigma-Aldrich, Madrid, Spain). The eluents were:

(A) water–0.05% TFA and (B) acetonitrile–0.05% TFA).

Peptides were separated using the following gradient pro-

gram: 0–5 min, 5% B; 5–60 min, linear increase to 44% B;

60–70 min, linear increase to 70% B. For each run 5 µg of

peptide extract from each subject were loaded onto the system.

LC-MS/MS analyses were performed using an Agilent

Nanoflow Proteomics Solution (Agilent Technologies, Cali-

fornia, USA). Twenty microlitres of each sample (5 µg of

peptide extract) were injected onto a C18

trapping column

(Zorbax 300SB-C18

, 5 µm particle size, 5 ! 0.3 mm, Agilent

Technologies) using an autosampler. The sample was washed

over the trapping column for 10 min with 95% buffer A

(water–0.1% formic acid), 5% buffer B (acetonitrile–0.1%

formic acid) at a flow rate of 10 µL/min. The flow was then

reversed over the trapping column, and the sample was eluted

onto a 150 ! 75 µm Zorbax 300SB capillary analytical C18

column with 3.5 µm particle size (Agilent Technologies) at a

flow rate of 0.3 µL/min. The analytical column was directly

plumbed into a nanoelectrospray ionization source (operated

at 1.9 kV), which was sampled into the quadrupole ion trap

mass spectrometer. A linear gradient of 5% buffer B to 65%

buffer B was run over a period of 65 min. The column was

then washed with a 10 min gradient from 65 to 95% buffer B,

followed by an 10 min hold at 95% buffer B. The column was

then re-equilibrated in 5% buffer B prior to future analyses.

Peptides were analysed by data-dependent acquisition of full-

scan mass spectra and MS/MS scans for the most abundant

ions. Spectra were collected in Standard-Enhanced MS mode,

with automated switching for MS/MS acquisition in Ultrascan

mode with automated selection of precursor ions.

Peptide identification

The data obtained from the LC/MS analyses were processed

using Masslynx software (Micromass, Manchester, UK). Spec-

tra corresponding to the detected peaks were deconvoluted

using MaxEnt 1 and 3 programs to yield monoisotopic masses.

Experimental masses were further compared with available

known theoretical peptide masses in the Swiss-Prot (http://

www.expasy.ch) and/or NCBI (http://www4.ncbi.nlm.nih.gov/

entrez/query.fcgi) databases. Identifications were considered

positive when differences between theoretical and experimen-

tal mass values were less than to 100 ppm.

The LC-MS/MS obtained data were processed by search-

ing the MS/MS spectra against the NCBI non-redundant

database (NCBInr, 3 January 2005) in Homo sapiens using

SpectrumMill software (Agilent Technologies, California,

USA) without enzyme specified or modifications. Mass toler-

ance was set to 0.15 Da for MS and MS/MS. The algorithm

used to create peak lists was Data Extractor, which is from

Agilent. The settings for this extractor are: all scans were

processed; scans were merged if they were within ±1.5 m/z

and ±240 s. MH+ values were allowed from 300 to 6000 and

sequence tag lengths from MS/MS were required to be >2.

The precursor charge assignment was set to ‘find’ with a

maximum charge of 6, a minimum signal/noise of 10 and

the ‘find 12C’ function enabled. Positive identifications

where accepted when the confidence level was greater than

95%.

RESULTS

LC-MS has been used for the analysis of salivary

peptides (Hardt et al., 2005; Vitorino et al., 2004b;

Messana et al., 2003; Castagnola et al., 2003). This

approach has allowed the identification of peptides

belonging to the basic PRPs, acidic PRPs, histatins and

statherins. As previously reported for in vivo and

in vitro experimental conditions (Vitorino et al., 2005),

we performed the analysis of the guanidine- and

TFA-extracted fractions of the acquired enamel pellicle

peptides (accounting for ca. 38.5 ± 3% of the total

adsorbed protein). In general, LC-MS experiments

show mainly peptides belonging to two classes: histatins

and proline-rich proteins. A large number of unidenti-

fied low-molecular-weight components were detected

on the guanidine extracts: It was also possible to detect,

although in trace amounts, the presence of the follow-

ing intact peptides: II-2, histatin 1, PRP3 and statherin

(Table 1). Some unidentified low-molecular-weight

components were also detected in the TFA extract, but

the same intact peptides found in the guanidine extract

(Fig. 1) predominate in this fraction. IB1 and PRP1

were also found with low relative abundance in this

fraction.

Table 1. Identified salivary peptides on acquired enamel pellicle by LC-MS on guanidine and TFA extracts. (a) Guanidine extract

and (b) TFA extract

Salivary Experimental Theoretical Acession Sequence

peptides MW MW pI number fraction

Histatin 1 4926.17 4926.23 9.1 123,134 a/b

Histatin 5 3035.53 3035.554 10.55 123,143 a/b

IB-1 9548.46 9588.51 10.1 131,006 b

II-2 7604.4 9.81 a/b

PRP1 15,514.2 15,514.1 4.63 131,008 b

PRP3 11,161.15 11,161 4.11 131,008 a/b

Statherin 5348.69 5378.45 6.64 134,947 b

a refers to guanidine extraction.

b refers to TFA extraction.


DOI: 10.1002/bmc


peptide fragments (18 out of 30) were found only in the

guanidine extract, 12 were found in both fractions and

two were observed only in the TFA extract. Generally,

the low-molecular-weight peptides found in the TFA

extracts were of low relative abundances, as shown for

the peptide of m/z 1819.0 (6-fold increase on guanidine

extracts compared with TFA extracts, obtained from

the ratio of the ion currents for the ion of m/z 1819.0 in

both fractions) or for the peptide of m/z 1565.5 (25-fold

increase on guanidine extracts when compared with

TFA extracts).

Several peptides were identified in the MS/MS

experiments as fragments of basic PRPs, with diverse

points of cleavage along the main chain (Fig. 4). The

observed peptide fragments arise via fragmentations of

the C-terminal and of the middle region of the primary

sequence in the case of IB8a and IB9 and of the

middle region of the N-terminus of IB1. Owing to the

sequence similarity among different b-PRPs, some of

the fragments detected can derive also from other

b-PRPs. Similarly to basic PRPs, the fragmentation of

Figure 2. Total ion chromatogram of the small peptides on guanidine and TFA extract obtained by LC-MS/MS. A similar

chromatogram profile is observed for both extracts.

Figure 1. Total ion chromatograms of acquired enamel pellicle peptides on guanidine and TFA

extract obtained by 1D-LC-MS.

The bulk of the unknown low-molecular-weight com-

ponents (<2200 Da), which were observed as doubly

and triply charged ions, were detected at a retention

time between 30 and 35 min in the LC-MS experi-

ments. To achieve a better separation and consequently

an increase the number of possible identified peptides,

we used a longer, linear gradient program in the LC-

MS/MS experiments. The LC-MS/MS chromatograms

for both the guanidine and TFA extracts are similar

(Fig. 2). All the peptides detected were sequenced by

MS/MS and identified by automated interpretation of

the peptide fragmentation pattern. The CID experi-

ments performed on doubly charged ions led to the

observation of peaks corresponding to y and b fragment

ions while the experiments performed on peptides with

more than 2 charges led to the detection mainly of y

fragments (Fig. 3). As a result, 30 peptides were identi-

fied and assigned with high confidence as fragments

belonging to basic PRPs, acidic PRPs and histatins

(Table 2). Comparing the guanidine and TFA extracts,

it is possible to observe that most of the identified


DOI: 10.1002/bmc


Tab

le 2.

Id

en

tifi

ed

fragm

en

t p

ep

tid

es o

n acq

uir

ed

en

am

el

pell

icle

b

y L

C-M

S/M

S o

n gu

an

idin

e an

d T

FA

extracts.

(a) G

uan

idin

e extract an

d (b

) T

FA

extract

Acessio

n

Sali

vary p

ep

tid

es

MW

pI

nu

mb

er

Sco

re

Pep

tid

e m

ass

Seq

uen

ce

Fractio

n

Basic

P

RP

fam

ily

Sali

vary co

ncavil

in-A

b

ind

ing p

ro

tein

(C

on

1)

37,2

77.4

12.1

71,9

11,4

90

171.0

51961.0

32

GP

PP

QG

GS

KS

RS

SR

SP

PG

KP

a

1565.4

59

SQ

GP

PP

PG

KP

QG

PP

PQ

a,b

1136.7

62

PQ

GP

PP

QG

GS

KS

a

1556.6

95

SA

RS

PP

GK

PQ

GP

PP

QG

a

1680.9

04

GP

PR

PP

QG

GR

PS

RP

PQ

a

Pep

tid

e fragm

en

ts w

ith

C

on

1 h

om

olo

gy

Basic

p

ro

lin

e-ric

h p

ep

tid

e P

-E

(IB

-9)

37,2

77.4

12.1

71,9

11,4

90

36.3

22720.3

91

SP

PG

KP

QG

PP

PQ

GG

NQ

PQ

GP

PP

PP

GK

PQ

a

Basic

p

ro

lin

e-ric

h p

ep

tid

e IB

-8a

6023.7

11.7

4131,0

09

19.2

81924.0

0P

PP

GK

PQ

GP

PP

QG

DK

SR

SP

a

Basic

p

ro

lin

e-ric

h p

ep

tid

e IB

-1

12,4

27.7

10.9

3105,4

17

34.2

11243.6

43

SP

PG

KP

QG

PP

PQ

Ga

Sali

vary p

ro

lin

e-ric

h p

ro

tein

p

recu

rso

r9530.5

9.7

131,0

06

51.0

61300. 6

64

SP

PG

KP

QG

PP

PQ

GG

a

(C

lon

es C

P3,

CP

4 an

d C

P5) [C

on

tain

s:

1767.9

52

SP

PG

KP

QG

PP

PQ

GG

NQ

PQ

a

Basic

p

ep

tid

e IB

-6;

Pep

tid

e P

-H

]1746.8

71

PQ

GP

PP

PG

KP

QG

PP

PQ

GD

a

PR

B1L

p

recu

rso

r p

ro

tein

38,5

46.0

11.2

22,1

44,9

10

36.9

42042.0

41

GP

PP

QG

DK

SR

SP

QS

PP

GK

PQ

a

Sali

vary p

ro

lin

e-ric

h p

ro

tein

p

recu

rso

r32,5

96.3

11.1

9130,9

95

29.1

7

(C

lon

es C

P3,

CP

4 an

d C

P5) [C

on

tain

s:

Basic

p

ep

tid

e IB

-6;

Pep

tid

e P

-H

]

Pro

lin

e-ric

h p

ro

tein

3 p

recu

rso

r8182.3

9.7

121,2

64,4

97

102.6

31315.7

16

GP

GR

IP

PP

PP

AP

Ya**

(P

ro

lin

e-ric

h p

ep

tid

e P

-B

)

[C

on

tain

s:

Pep

tid

e P

-A

; P

ep

tid

e D

1A

]

Acid

ic P

RP

fam

ily

Sali

vary acid

ic p

ro

lin

e-ric

h p

ho

sp

ho

pro

tein

17,0

16.5

4.6

3131,0

08

136.8

42083.9

14

GP

PQ

QG

GH

QQ

GP

PP

PP

PG

KP

Qa,b

1/2

p

recu

rso

r (P

RP

-1/P

RP

-3) (P

RP

-2/P

RP

-4)

1818.9

58

GP

PQ

QG

GH

PR

PP

RG

RP

Qa,b

(P

IF

-F

/PIF

-S

) (P

ro

tein

A

/pro

tein

C

)1731.8

67

GP

PQ

QG

GH

PP

PP

QG

RP

Qa,b

[C

on

tain

s:

Pep

tid

e P

-C

]1471.7

15

GR

PQ

GP

PQ

QG

GH

QQ

a**

1380.7

24

GP

PQ

QG

GH

PR

PP

Rb

1866.9

21

GP

PP

QG

GR

PQ

GP

PQ

GQ

SP

Q(-)

a,b

**

His

tati

n fam

ily

His

tatin

1

6962.9

9.1

123,1

34

122.6

72618.1

13

HH

SH

RE

FP

FY

GD

YS

NY

LY

DN

(-)

a,b

1443.5

71

YG

DY

GS

NY

LY

DN

(-)

b**

1963.8

03

EF

PF

YG

DY

GS

NY

LY

DN

(-)

a,b

2119.9

04

RE

FP

PF

YG

DY

GS

NY

LY

DN

(-)

a,b

His

tatin

3

6145.1

10.3

1123,1

43

54.5

71718.8

73

RK

FH

EK

HH

SH

RG

Ya

1491.7

67

DS

HA

KR

HH

GY

KR

a,b

*

1766.9

31

DS

HA

KR

HH

GY

KR

KF

a*

1356.7

76

KR

HH

GY

KR

KF

a

3035.5

22

DS

HA

KR

HH

GY

KR

KF

HE

KH

HS

HR

GY

a,b

1118.5

96

RK

FH

EK

HH

a,b

1287.6

09

HE

KH

HS

HR

GY

a

a refers to

gu

an

idin

e extractio

n.

b refers to

T

FA

extractio

n.

Fragm

en

t p

ep

tid

es in

b

old

an

d assig

ned

asteris

ks w

ere p

revio

usly

id

en

tifi

ed

in

w

ho

le sali

va b

y C

astagn

ola

et

al.

(2005) w

hil

e a d

ou

ble

asteris

k refers to

p

ep

tid

es id

en

tifi

ed

b

y H

ard

t et

al.

(2005) o

n p

aro

tid

sali

va.


DOI: 10.1002/bmc


Figure 3. MS/MS fragmentation pattern performed on the doubly charged ion [m/z 722.35) identified as a peptide fragment of

histatin 1 (A) and on the triply charged ion [m/z 695.31) identified as a peptide fragment of acidic-PRP1 (panel B). Both y and b

fragmentations are present.

Table 3. Position of the aminoacid residues involved on peptide bond cleavages

R K D P N Q S G E H F Y A

IB1 97 72, 92 91 73, 75, 76 50 51

IB9 37, 61 56 55 38, 41, 15 16

42, 62

IB8a 37 55 38 15, 56 16

Con1 78, 139, 114, 238 132 17, 58, 59, 32, 93, 254, 270, 18, 79, 140, 31, 92, 153,

201, 260 77, 115, 269 133, 154 384 202, 239, 259 215, 214,

255, 385

PRP1 106 76, 93, 77, 94, 111,

110, 120, 121, 132

131

Histatin 1 22 17 23 18, 21 26 27

Histatin 5 12 5, 11 20 15, 19 14 4

histatin 5 occurs at the C-terminal, middle and N-

terminal regions. In the case of acidic PRPs and of

histatin 1, the observed peptide fragments were due to

fragmentation in the N-terminal sequence (Fig. 5).

MS/MS analysis of histatins has shown a predomi-

nance (seven out of 10) of fragmentation in Arg, His

and Lys residues, as can be observed in Table 3

(labelled in bold on Fig. 5). Fragmentation at Ser20

(histatin 5) and Tyr27 (histatin 1) are also observed but

less extensively (two in 10 amino acid residues). For

basic PRPs, the cleavages sites observed involve

mainly Arg, Lys and Pro residues. Gln and Gly cleav-

ages are also observed in high abundance on salivary

concavalin-A binding protein (Con1). In the case of

acidic PRPs, one cleavage at Arg106 was observed

(which results on PRP3 formation), but the most abun-

dant amide cleavages were detected between Gln and

Gly residues (labelled in bold on Fig. 5), resulting in a

fragment consensus containing at least two proline

residues following Gly (GlnProPro).


DOI: 10.1002/bmc


Figure 4. Amino acid sequences of basic salivary proline-rich peptides IB1, IB8a and

IB9 (top panel) and histatin 3 (bottom panel). The peptide fragments observed in the

LC-MS/MS experiment are aligned below the protein sequence. All peptide bond cleav-

age sites are indicated in bold and underlined. Phosphorylated sites are indicated in

italic bold and underlined. < represents a pyroglutamic residue. Diverse points of cleav-

age along the main chain of the peptides were observed for basic-PRP and Con1.

Figure 5. Amino acid sequences of salivary proline-rich phosphoprotein 1/2 (top panel),

histatin 1 and histatin 5 (bottom panel). The peptide fragments observed in the LC-MS/

MS experiment are aligned below the protein sequence. All peptide bond cleavage sites

are indicated in bold and underlined. Phosphorylated sites are indicated in italic bold

and underlined. < represents a pyroglutamic residue. Predominant cleavages near the C-

terminal from the primary sequence of acidic-PRP and histatin 1 were observed.


DOI: 10.1002/bmc


DISCUSSION

In the present study, an experimental approach based

on the recovery of bovine teeth enamel implants after

2 h in the oral cavity of two human subjects was used

for in vivo pellicle collection. Bovine enamel presents

a high structural compatibility with human dental

hard tissues (Hannig et al., 2004) and was used recently

by Hannig et al. (2004, 2005), who have quantified

lyzosyme and amylase on in vivo enamel pellicle on

similar experimental conditions to those described in

this work. Moreover, to test possible contributions from

bovine enamel implants, blank LC-MS experiments

were performed using the same experimental proce-

dure, which showed the absence of contaminating

peptides (data not shown), confirming this strategy as a

valuable tool for in vivo experiments.

In order to dissociate pellicle protein and simultane-

ously characterize the bonding nature established between

salivary peptides and enamel surface, a sequential ex-

traction was performed based on guanidine cahotropic

properties (Del Vecchio et al., 2002) and on hydr-

oxyapatite solubilization by TFA, to remove the

remaining adsorbed proteins (Vitorino et al., 2005).

In most of the early studies, both in vivo and in vitro,

the characterization of acquired enamel pellicle was

performed based on bulk extractions procedures. As

shown by Moreno et al. (1984) and Gorbunoff and

Timasheff (1984), the nature of the interactions of

saliva components with enamel is crucial to the under-

standing of the mechanisms of its formation and its

function in tooth protection and bacterial adhesion.

The results found in this in vivo experiment clearly

show different compositions between the TFA and the

guanidine extracts and lead to the hypothesis that there

is a simple structural explanation for the different

strengths of interaction corresponding to the substitu-

tion of negative enamel ions and its complexation with

positive charges integrating the solid surface structure

(Vitorino et al., 2005).

In recent studies of whole saliva, Inzirati et al. (2005)

proteolysed with trypsin the isolated salivary peptides

obtained by HPLC pool collection. Several peptides

from basic and acidic PRP classes were then identified

by MS and MS/MS. This procedure allowed identifica-

tion of 18 different acidic PRPs isoforms (including

phosphorylated forms) and 15 different basic PRP

isoforms. All these peptides have molecular weights

over 3500 Da. Using HPLC separation and MS/MS,

Castagnola et al. (2004) analysed the low molecular

weight peptides (<3500 Da) whole saliva, and Hardt

et al. (2005) analysed parotid saliva samples. A cascade

of fragments resulting mainly from histatin 3, some

fragments resulting from PRPs (Hardt et al., 2005) and

the intact form of histatin 1 were observed (Csatagnola

et al., 2004). Only a few reports concerning pellicle

peptide analysis have been described. Lamkin et al.

(2001) reported the sequencing of several peptide

fragments in in vitro experiments belonging mainly

to histatins, statherin and cystatins classes. Yao et al.

(2001) reported the analysis of pellicle peptides by LC-

MS, finding components with a molecular weight in

the 1500–5400 Da range, attributed to statherin variant

V1 and of cystatin fragments. Following this, and taking

into consideration the importance of peptides as pellicle

precursors, we adopted a strategy based on the applica-

tion of LC-MS and LC-MS/MS for the analysis of pel-

licle extracts. This strategy allowed the separation and

identification of small peptides (by MS/MS pattern

search) as well as large peptides (through the com-

parison of the theoretical molecular weight with the

experimentally observed molecular weight). Our ex-

periments showed that guanidine extracts present

higher levels of fragmented peptides, which most

likely interact non-covalently with the enamel surface,

while mainly ‘intact’ peptides strongly interacting with

enamel surface are detected in TFA extracts.

With the exception of histatin 5, all the peptides

found in the TFA extract correspond to the phos-

phorylated species. These phosphorylated peptides,

statherin acidic PRPs, histatin 1, IB1 and II-2 have

been extensively studied with respect to their ability to

adsorb to enamel surface participating on its pellicle

formation (Lendenmann et al., 2000; Carlen et al., 1998;

Vacca Smith and Bowen, 2000; Jensen et al., 1991; Yin

et al., 2006; Al-Hashimi and Levine, 1989). The high

affinity of these species for enamel is most probably

due to the negative N-terminal. For example, in the se-

quence of acidic PRPs, the first 11 N-terminal residues

of the acidic PRPs contain eight negative charges

including a phosphorylated serine residue at position 8

and in the case of statherin the first five N-terminal

residues are three negative acidic amino acid residues

and two phosphorylated serines, Ser2 and Ser3. A simi-

lar trend with negative charges and phosphorylated

serines is observable for the other species. The negative

N-terminal is extremely important for interactions with

hydroxyapatite, as reported by Boackle et al. (1999),

who found that, after cleavage of the N-terminal of the

acidic PRPs by human leukocyte elastase, the affinity

decreased by more than 70%. Studies investigating the

conformation of statherin on the hydroxyapatite surface

showed a helical conformation after its adsorption to

the surface of hydroxyapatite. The statherin N-terminal

pentapetide recognizes the hydroxyapatite surface and

establishes a strong interaction between the surface and

the phosphoserines and carboxylate side-chains groups.

Dynamics studies suggest a weak interaction for the

remainder of the helix with hydroxyapatite (Schlesinger

and Hay, 1977; Stayton et al., 2003). Despite the

absence of reports concerning the other phosphorylated

peptides, a similar mechanism could be suggested for


DOI: 10.1002/bmc


hydroxyapatite interaction by carboxylate groups and

phosphoserines with hydroxyapatite (Yin et al., 2006).

The histatin 5 sequence shows an aspartic residue on

the N-terminus (Asp1) (Fig. 5). The charge on the Asp

side chain is similar to that of phosphorylated residues

present on the N-terminal region and supports a similar

mechanism for the strong interaction of histatins with

the hydroxyapatite surface.

In this in vivo study we have found several fragments

arising from fragmentation of basic PRPs, acidic PRPs

and histatins non-covalently bound to the enamel sur-

face. In their study of histatin 3 fragmentation in saliva,

Castagnola et al. (2004) reported a cascade of frag-

ments from this peptide. In our work several histatin

fragments were observed; however, these belonged

only to histatin 5. Taking into account that the precur-

sor of histatin 5 is histatin 3, the absence of pellicle

fragments not shared between histatin 3 and 5 suggests

that the small fragments arise only from histatin 5 frag-

mentation. Underlining this suggestion, the observed

fragments cover the entire sequence of histatin 5. In the

case of basic PRPs, peptide fragments identified by

LC-MS/MS could be formed through fragmentation of

several protein precursors (at least four possibilities can

be assigned; Fig. 4) because this is a highly homologous

family (Table 2). These peptides, as observed for

histatin 5, arise from diverse cleavage points along their

primary sequence. For histatin 1, only C-terminal

fragmentations of the primary sequence were detected.

In this study, when compared with PRP3 relative

abundances, low relative abundances of PRP1 were

found strongly adsorbed (LC-MS of TFA extracts;

Fig. 1). This is in agreement with the work of Bennick

et al. (1983), who observed by crossed immuno-

electrophoresis the degradation of PRP1 to PRP3 and

smaller peptides after adsorption to enamel.

Comparing the identified peptide fragments with

those early observed on whole (Castagnola et al., 2004;

Messana et al., 2004; Inzitari et al., 2005) and parotid

saliva (Hardt et al., 2005), only six of the 30 fragments

identified have been previously observed. Our results

indicate that only a few classes of proteins and amino

acid residues are involved in proteolysis. Based on the

fragments identified, specific cleavage sites could be

assigned for the different classes of peptides. For

example, 80% (eight cleavage sites of the total of 10

observed for histatins) of the amide bond cleavages in

histatins occur at the Arg, His and Lys residues, while

for basic PRPs, the predominant cleavage sites occur

at the Lys, and Pro residues (four cleavage sites of

the total of six observed for this class). Some 85% of

the cleavage sites of acidic PRPs are located between

Gln and Gly residues. Altogether, this illustrates

the specificity of the proteolytic enzymes which pro-

mote the degradation of peptides on enamel pellicle.

Castagnola et al. (2004), in their characterization of

peptides in whole saliva, also observed a dominant

cleavage at Arg and Lys in histatin 3 and suggested that

a trypsin-like digestion pathway generated the histatin

3 cascade fragmentation. Proteolytic activity has been

reported in whole saliva in the last few years in associa-

tion with oral pathologies such as Sjögren’s syndrome,

and periodontitis (Dickinson, 2002; Pozo et al., 2005).

Among the enzymes implicated are metalloproteins

(MMP1 and 9; Chaussain-Miller et al., 2006), cathepsins

(B, H and L; Dickinson, 2002) and kallikrein. LC-MS/

MS of trypsin digests of whole saliva showed the pres-

ence of proteolytic enzymes including kallikrein (1 and

11), cathepsins (D and L) and carboxipeptidase H

(Wilmarth et al., 2004; Hu et al., 2005). The fragments

observed in our work could be attributed not only to

one enzyme, but to a combination of different enzymes.

The high content on lysine and arginine residues on

histatins makes these proteins extremely susceptible to

tryptic-like digestion. Most of the resulting fragments

found from the cleavage at the Arg and Lys residues

could be attributed to a trypsin-like pathway. Cleavage

of the Lys-His bond at Lys-17 of histatin 1 promotes

the release of the C-terminal peptide with a molecular

weight of 2618.11. After this, the cleavage at Arg–Xaa

between residues 22 and 23 of the primary sequence of

histatin 1 releases the peptide with a molecular weight

of 1963.8, or the cleavage between the residues Arg37

and Pro38 of the primary sequence of IB9 results in the

peptide with a molecular weight of 1746.87. Minor

cleavages at the Ser and Phe residues in the primary

sequence of histatins can also occur. Wong et al. (1983)

have reported that kallikreins are the responsible

enzymes associated with the generation of acidic PRP

C (now known as PRP3) before and after its secretion

from glandular tissues by a cleavage at the Arg106 resi-

due of acidic PRP A (now known as PRP1). Enzymes

which are able to cleave amide bond at His residues

(the other predominant cleavage on histatins) are also

required. It was previously reported by Xu et al. (1993)

that there existed a chymotryptic pathway which ad-

dressed the peptide-bond cleavage at the His residues.

Enzymes such as kallikreins or cathepsin members,

which work similarly to pepsin A via the chymotrypitic

pathway, may be involved in cleavages of histatin 3

leading to the observation of peptides with molecular

weights 1287.61, 1356.78 and 1766.93. The predominant

cleavages of acidic PRPs detected in LC-MS/MS ex-

periments occur between the Gln–Gly residues, which

is in agreement with the previous report by Jonsson

et al. (2001). This suggests that the presence of a cyclic

structure of the Pro residue which follows Gly could be

important for the stabilization of the reactive conforma-

tion. Furthermore, it should be emphasized that there

are no known enzymes able to cleave acidic PRPs at

Gln–Gly bonds, indicating the presence/activities of an

as yet unidentified enzyme.


DOI: 10.1002/bmc


In conclusion, we have used a strategy based on

LC-MS/MS to characterize the low-molecular-weight

peptides of in vivo enamel pellicle. Based on these

data, a pattern of proteolytic pathways for salivary

peptides on enamel pellicle was observed with pre-

dominant cleavages at Arg, Lys, Ser and Phe residues

for the majority of the peptides (except acidic PRPs,

where the predominant cleavages are at the Gln–Gly

bond). These data suggest the involvement of a com-

bination of proteolytic enzymes which selectively

degrades the salivary proteins and contributes to modu-

lation of the acquired enamel pellicle.

Comparing all detected peptides in both extracts

(guanidine and TFA) with the peptides identified in

whole saliva described in previous works (some with

similar experimental conditions: Castagnola et al., 2004;

Messana et al., 2004; Vitorino et al., 2004a, 2004b;

Hardt et al., 2005), it is possible to observe a small

number of species adsorbed to the enamel. This sug-

gests the existence of a selective adsorption process

and the predominance of the adsorption of intact

forms. The presence of carboxyl groups or phosphate

groups in some adsorbed peptides should contribute to

a strong interaction with the enamel surface. This may

also be a protection factor against proteolytic activity

involving the formation of covalent/non-covalent com-

plexes with other proteins. Additionally, this study

shows that the bulk of peptide fragments were derived

from proteins other than the most abundant known

salivary components also present on enamel pellicle

(albumin, amylase, mucins) (Carlen et al., 1998;

Li et al., 2003, 2004; Hannig et al., 2005). These results

are in agreement with Hardt et al. (2005) for the

low-molecular-weight species isolated from parotid

saliva by passage through a 10 kDa membrane fol-

lowed by their characterization using tandem mass

spectrometry.

Although based on only two subjects, this study con-

tributes to a better understanding of the composition of

acquired enamel pellicle because previous studies

(Rykke and Sonju, 1991; Li et al., 2003; Yao et al., 2001;

Al-Hashimi and Levine, 1989) have shown that in vivo

pellicle composition exhibits considerable inter-subject

consistency, and that the overall protein composition

of the pellicles displayed characteristics typical of the

saliva prevailing in the area of the mouth where the

pellicles were formed (Vacca Smith and Bowen, 2000).

Based on the results reported here, we hypothesize that

the general mechanism for pellicle formation involves

a strong interaction of peptides containing carboxyl

and/or phosphate groups and enamel surface and elec-

trostatic interactions of other protein species forming

several layers, as suggested already by other authors

(Gorbunoff and Timasheff, 1984; Moreno et al., 1984;

Hay and Moreno, 1979). Further research concerning

inter-subject variability and the importance of tooth

location for pellicle composition could be the key to the

understanding of diseases involving tooth destruction.

Acknowledgements

This research was supported in part by the Intramural

Research Program of the NIH, National Institute of

Environmental Health Sciences. The authors would like

to express their appreciation for the financial support

provided by the Fundação para a Ciência e Tecnologia

(FCT, grant nos SFRH/BPD/14968/2004 and POCTI/

QUI/5890/2004).

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