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Eur J Oral Sci 2015; 123: 390–395 Ó 2015 Eur J Oral Sci
DOI: 10.1111/eos.12214 European Journal of
Printed in Singapore. All rights reserved
Oral Sciences

Gina A. Castiblanco1, Dorothea

Identification of proteins from human Rutishauser2, Leopold L. Ilag3,
Stefania Martignon1, Jaime E.
permanent erupted enamel Castellanos4, Wilson Mej
ıa1
1
UNICA – Caries Research Unit, Universidad
El Bosque, Bogota
, Colombia; 2Department of
Medical Biochemistry and Biophysics,
Castiblanco GA, Rutishauser D, Ilag LL, Martignon S, Castellanos JE, Mejıa W. Karolinska Institute, Stockholm; 3Department
Identification of proteins from human permanent erupted enamel. of Environmental Science and Analytical
Chemistry, Stockholm University, Stockholm,
Eur J Oral Sci 2015; 123: 390–395. © 2015 Eur J Oral Sci Sweden; 4Dental Faculty, Universidad
Nacional de Colombia, Bogota
, Colombia
Proteins from the extracellular matrix of enamel are highly specific and necessary
for proper enamel formation. Most proteins are removed from the matrix by
enamel proteases before complete mineralization is achieved; however, some residual
protein fragments persist in the mineralized matrix of erupted enamel. So far, only
amelogenin peptides obtained by traditional bottom-up proteomics have been recov-
ered and identified in human permanent erupted enamel. In this study, we hypothe-
size that other enamel-specific proteins are also found in human permanent enamel,
by analysing human erupted third molars. Pulverized enamel was used to extract
proteins, and the protein extract was subjected directly to liquid-chromatography
Gina A. Castiblanco, UNICA – Caries
coupled to tandem mass spectrometry (LC-MS/MS) without a previous trypsin-
Research Unit, Universidad El Bosque, Av.
digestion step. Amelogenin and non-amelogenin proteins (ameloblastin and enam- Cra 9 #131A-02, Casa XII, Bogota
, Distrito
elin) were succesfully identified. The sequences of the naturally occurring peptides Capital 110121, Colombia
of these proteins are reported, finding in particular that most of the peptides from
E-mail: gcastiblanco@unbosque.edu.co
the amelogenin X-isoform come from the tyrosine-rich amelogenin peptide (TRAP)
and that some were identified in all specimens. In conclusion, our LC-MS/MS
method without trypsin digestion increased the coverage of identification of the Key words: amelogenin; dental enamel
enamel proteome from a few amelogenin peptides to a higher number of peptides proteins; enamelin; mass spectrometry
from three enamel-specific proteins. Accepted for publication August 2015

Erupted dental enamel is a highly mineralized bioceramic
(95 wt%, 80 vol%) with little organic material (3 wt%,
15 vol%), most of which is protein. However, during
development, enamel is composed of only 30 wt% of
mineral, with the gradual removal of proteins ultimately
resulting in complete mineralization (1). The role that
enamel proteins play in enamel formation is so critical
that mutations on structural proteins can impair
mineralization.
During the secretory stage of amelogenesis, large
amounts of structural proteins are secreted by amelo-
blasts. The most abundant protein is amelogenin, which
has highly conserved domains among species, is
expressed by both X and Y chromosomes (2), and
dominates the profile of secretion (3). In vitro evidence
suggests that amelogenin promotes crystal organization
and modulates crystal morphology (4, 5). Less informa-
tion is available on non-amelogenin matrix proteins.
Ameloblastin, the second most-abundant protein in
enamel, is co-secreted with amelogenin (6). Evidence on
interactions between these two proteins is available (7),
although their functional significance is still unknown.
The third most-abundant protein secreted is enamelin,
which has been found to be co-localized with amelo-
genin (8). Such cooperation is believed to have a role in
controlling enamel crystal morphology (9). Another
minor matrix protein – amelotin – was recently
reported; however, its structure and functions are yet to
be explored (10). Matrix metalloproteinase-20 (MMP-
20) and kalikrein-4 (KLK-4) are proteinases involved
in the removal of the structural proteins; the former
during the secretion and early-maturation stages, and
the latter during the maturation stage of enamel devel-
opment (11). A third protease, chymotrypsin C (calde-
crin), was found to be associated with enamel
development but its role has not been reported (12).
In-situ hybridization and immunohistochemistry
assays have demonstrated that the expression of amelo-
genin and enamelin ceases in the early and late matura-
tion stages (13–15). The proteases hydrolyse most of
the protein material to allow the thickening of indivi-
dual crystallites within the enamel rods but some pep-
tides are not removed, remaining in completely
mineralized enamel. With the increased use of mass-
spectrometry (MS) techniques, in 2009, an amelogenin
peptide of enamel from mummy and contemporary
molars was identified using matrix-assisted laser desorp-
tion/ionization tandem time-of-flight (MALDI-TOF/
TOF) MS and synthetic peptides (16). Later in 2011,
another five amelogenin peptides from ancient and con-
temporary teeth were reported using MALDI-TOF/
TOF (17). Nonetheless, only a limited number of
reports are available on the identification of proteins in
mature enamel (from erupted or non-erupted teeth).

Previous approaches have so far detected amelogenin
peptides in mature enamel and there is consensus that
these are the only protein remains found at this point.
However, with the continuous development of MS
techniques, such as liquid chromatography coupled
with tandem mass spectrometry (LC-MS/MS), a
broader range of information can be obtained from
samples containing small amounts of protein, such as
dental enamel. As the expression of the major struc-
tural proteins (amelogenin, ameloblastin, and enamelin)
ceases during the maturation stage, we tested whether
amelogenin is the only protein found in human perma-
nent erupted enamel or if traces of other proteins are
also retained in completely mineralized enamel. There-
fore, the aim of this study was to increase the coverage
of previous MALDI-TOF/TOF identifications of pro-
teins remaining in human permanent erupted enamel,
in the largest set of human molar samples reported so
far, as analysed by LC/MS-MS.
Material and methods
Enamel samples
Ethical approval was obtained from the Ethical Board at
Universidad El Bosque (UB 267-2010). Third molars
were collected from patients attending the Dental
School’s Oral Surgery Clinic of Universidad El Bosque in
Bogot
a, Colombia. The extractions were performed for
orthodontic reasons and patients were informed about
the purposes of the research and signed a tooth donation
form.
Teeth were washed with distilled water, brushed with a
dental drill, and classified according to the International
Caries Assessment and Detection System (ICDAS) by two
trained examiners. For this study, molars with an ICDAS
score of 0 (sound) were selected. The final sample con-
sisted of eight sound molars that were stored dry at
20°C until processed.
Extraction of enamel proteins
The molar tooth crowns were cut in 150-lm-thick longitu-
dinal sections using an Isomet 1000 diamond-coated saw
(Buehler, Lake Bluff IL, USA). From each section, the
outer third of the enamel was marked under a stereomi-
croscope and cut with a diamond rotatory instrument
(Jota, R€uthi, SG, Switzerland). The outer-enamel pieces
were ground in liquid nitrogen using a mortar and pestle.
The powdered enamel recovered for each tooth was
pooled separately and stored at 70°C until required for
processing. The protein-extraction technique was a slightly
modified version of the method described by PORTO et al.
(18). On the day of extraction, the enamel powder was left
at room temperature and dissolved in 12% trichloroacetic
acid (TCA) [containing N-ethylmaleimide (NEM), phenyl-
methanesulphonyl fluoride (PMSF), and phenantroline, as
protease inhibitors, all at a final concentration of 2 mM],
at a ratio of 200 ll of acid per milligram of enamel. After
1 h, sodium deoxycholate (200 lg ml 1) was added as
detergent and the reaction took place for 2 d at 0°C. The
solution was centrifuged at 2500 g and 4°C for 45 min.
Following centrifugation, the protein pellet was washed
Proteins in human enamel 391
Fig. 1. Flow chart of the procedure followed for the extraction
and identification of enamel proteins. Note that trypsin diges-
tion and gel electrophoresis were not used and the extract was
separated by liquid chromatography (LC) before the identifica-
tion of proteins by tandem mass spectrometry (MS/MS). FA,
formic acid; NEM, N-ethylmaleimide; PMSF, phenylmethane-
sulphonyl fluoride; TCA, trichloroacetic acid.
with acetone ( 20°C, 200 ll) and centrifuged again at
13,000 g and 4°C for 10 min. The washing step was
repeated and the sample was dried at room temperature
for 1 h. The pellet was resuspended in 200 ll of a 6 M
urea solution containing the same protease inhibitors
described before, and lyophilized by sublimation. All
reagents were purchased from Sigma-Aldrich (St Louis,
MO, USA) (Fig. 1).
LC-MS/MS
Extracted enamel peptides were dissolved in 100 ll of 5%
formic acid and cleaned with C18 StageTips, according to
the manufacturer’s description (Thermo Fisher Scientific,
Bremen, Germany). Eluted peptides were dried and resus-
pended in 3% acetonitrile (ACN) and 0.2% formic acid.
The LC-MS/MS analyses were performed on an Easy-nLC
system (Thermo Fisher Scientific) coupled directly online
to a QExactive mass spectrometer (Thermo Fisher Scien-
tific).
Peptide separation was performed on a 10-cm-long
fused silica tip column (SilicaTips New Objective) packed
in-house with 3 lm C18-AQ ReproSil-Pur (Dr. Maisch,
Ammerbuch-Entringen, Germany). Chromatographic sepa-
ration was achieved using an ACN/water solvent system
containing 0.2% formic acid. The gradient was set up as
follows: 3 48% ACN in 50 min, 48 80% ACN in 3 min,
and 80% ACN for 7 min, all at a flow rate of
300 nl min 1. The MS acquisition method comprised one
survey scan ranging from a mass-to-charge ratio (m/z) of
300 to an m/z of 1650, with a resolution of R = 70,000 at
an m/z of 400, followed by 10 data-dependent MS/MS
scans from the top 10 precursor ions with a charge state
of ≥2.
392 Castiblanco et al.

The data were searched against the SwissProt protein peptides were present in more than one sample and
database using MASCOT software version 2.3 (Matrix their masses ranged from 800 to 1,500 Da.
Science, London, UK) with Homo sapiens specified in the The protein with the highest score, and thus the
taxonomy. A protein score of ≥24 was chosen as the majority of identified peptides, was amelogenin in both
threshold for protein identification, and for peptide identi- X and Y isoforms (Tables 1 and 2). After checking the
fication the threshold of ≥10 was chosen.
location of the peptides within the amelogenin
sequence, we found that the majority belonged to the
N-terminal region, the tyrosine-rich amelogenin peptide
Results (TRAP; AA1-45), except for LHHQIIPVL and
LHHQIIPVLS, which belong to the central region. The
Identification of proteins and peptides
most frequent peptides (present in all specimens) were
Our results show that it is possible to identify proteins found to be located between AA17-42.
from erupted human enamel using LC-MS/MS without
trypsin digestion of the protein extract. Database
searches using Mascot suggested that significance
Discussion
thresholds were ≥39 to 42. We identified seven proteins
from eight human erupted third molars (Table 1). The To date, the proteome of dental enamel has been studied
highest scoring peptide spectrum of each protein identi- using traditional bottom-up proteomics (16–18). In this
fied is shown as supporting information (Figures S1– study we used a different approach: we did not perform
S7). Four of the identified proteins were specific for two-dimensional (2D) gel electrophoresis or trypsin
human enamel (amelogenin X/Y, enamelin, and amelo- digestion because enamel proteins are highly specific (19)
blastin) and three were non-specific. Of the specific pro- and are more likely to be found as peptides hydrolysed
teins, amelogenin isoform X was the most frequent and by proteases. We demonstrated, for the first time, that
had the highest protein score within the set of samples by using LC-MS/MS, enamel peptides were successfully
analysed, followed by amelogenin isoform Y, amelo- separated and sequenced, subsequently allowing enamel
blastin, and enamelin. The non-specific proteins identi- proteins to be identified. This method saves time for
fied were serum albumin, antithrombin, and ubiquitin identification of enamel proteins if it is to be performed
(Table S2). routinely (i.e. it may prove useful for non-destructive
Table 2 shows the sequences of the peptides which enamel protein-extraction procedures, such as those pre-
allowed the identification of Amelogenin X and Y iso- viously reported for sex identification of ancient speci-
forms. The peptides that lead to the identification of mens) (17). Our method increases the efficiency of
ameloblastin and enamelin are shown in Table S1. As identification of enamel-specific proteins, reducing the
we did not perform any trypsin digestion, the peptides occurrence of laboratory contaminants, such as keratin,
are exhibiting their natural cleavage sites. Most of the reported in similar studies (16–18). This was accom-
plished by using the external third of enamel and by the
omission of trypsin digestion. However, it should be
Table 1 taken into account that this step excludes higher-molecu-
Proteins identified from human permanent erupted enamel with lar-weight peptides and the conclusions do not consider
a protein score of ≥24: the enamel-specific proteins are shown other non-specific proteins that have been previously
in bold reported in erupted enamel (e.g. type I collagen) (20).
Protein
Amelogenin is the most abundant protein in devel-
Prot_acc Protein name score* Frequency† oping enamel (19) and thus is most likely to be
found with the highest scores in mature enamel, as
AMELX_HUMAN Amelogenin, X 132 8 our results confirm in Table 1. The expression of
isoform Homo proteins such as enamelin and ameloblastin is
sapiens reported to cease at the early maturation stage (14,
AMELY_HUMAN Amelogenin, Y 64 4
isoform Homo
15), so smaller amounts of these peptides (and thus
sapiens lower scores) are expected. Therefore, we decided to
AMBN_HUMAN Ameloblastin Homo 52 4 allow for lower protein (≥24) and peptide (≥10)
sapiens scores than the significance thresholds suggested by
ENAM_HUMAN Enamelin Homo 49 6 Mascot (≥39–42). In general, as defined by Mascot,
sapiens peptide scores are the ‘ 10*Log(P), where P is the
ALBU_HUMAN Serum albumin 60 2
Homo sapiens
probability that the observed match is a random
ANT3_HUMAN Antithrombin-lll 36 2 event’ and protein scores are ‘the sum of the highest
Homo sapiens ions score for each distinct sequence’. Therefore, the
UBP54_HUMAN Inactive ubiquitin 24 1 higher the score, the better. In our set of samples, at
carboxyl-terminal least one specimen had protein scores above Mascot
hydrolase 54 Homo ion significance thresholds.
sapiens
Previous studies have used up to five molar samples
*Highest score from eight samples. for MS identifications (17, 18, 21, 22); in contrast, our
†
Frequency over eight samples. prot_acc, accesion number. study used eight molars and thus is the largest set of
 Proteins in human enamel 393

Table 2
Naturally occurring peptides identified from amelogenin isoforms X and Y obtained from eight samples of human permanent erupted
enamel (peptide score ≥10)

Peptide No. of Mass

Prot_acc Protein name sequence Frequency amino acids (Da)

AMELX_HUMAN Amelogenin, X isoform Homo sapiens HPGYINF 4 7 846.4

LHHQIIPV 5 8 955.6
QSIRPPYP 4 8 957.5
YEVLTPLK 8 8 961.5
YQSIRPPY 5 8 1023.5
LHHQIIPVL 1 9 1069.6
PGHPGYINF 2 9 1001.5
YQSIRPPYP 8 9 1119.6
SYEVLTPLK 7 9 1048.6
LHHQIIPVLS 2 10 1155.7
YQSIRPPYPS 8 10 1206.6
YQSIRPPYPSY 8 11 1370.7
PHPGHPGYINF 1 11 1234.6
MPLPPHPGHPG 4 11 1151.6
YQSIRPPYPSYG 8 12 1427.7
MPLPPHPGHPGY 5 12 1314.6
LPPHPGHPGYINF 4 13 1445.7
YQSIRPPYPSYGY 3 13 1589.8
SIRPPYPSYGYEP 2 13 1524.7
RPPYPSYGYEPMG 2 13 1528.7
YQSIRPPYPSYGYE 2 14 1719.8
PLPPHPGHPGYINF 2 14 1542.8
SIRPPYPSYGYEPM 6 14 1671.8
IRPPYPSYGYEPMG 4 14 1641.8
MPLPPHPGHPGYIN 7 14 1542.7
YQSIRPPYPSYGYEP 7 15 1815.8
MPLPPHPGHPGYINF 6 15 1688.8
SIRPPYPSYGYEPMG 5 15 1728.8
QSIRPPYPSYGYEPMG 4 16 1857.8
YQSIRPPYPSYGYEPM 7 16 1962.9
LHHQIIPVLSQQHPPTH 1 17 1984.0
YQSIRPPYPSYGYEPMG 8 17 2019.9
YQSIRPPYPSYGYEPMGG 5 18 2076.9
LPPHPGHPGYINFSYEVLTPLK 1 22 2475.3
MPLPPHPGHPGYINFSYEVLTPLK 3 24 2719.4

AMELY_HUMAN Amelogenin, Y isoform, Homo sapiens HPGYINF 2 7 846.4

YQSMIRPPY 3 9 1170.5
YQSMIRPPYS 4 10 1257.6
YQSMIRPPYSS 3 11 1344.6
YQSMIRPPYSSY 2 12 1506.7
YQSMIRPPYSSYG 1 13 1564.7
LPPHPGHPGYINF 1 13 1445.7
MPLPPHPGHPGYIN 1 14 1542.7
MPLPPHPGHPGYINF 2 15 1689.8

samples reported so far for the identification of enamel
proteins from erupted sound human molars using MS.
In this study, we sequenced a much broader set of
amelogenin peptides than previously reported, and six
were present in all samples (Table 2). This is the first
report on sequences of amelogenin peptides exhibiting
naturally occurring cleavage sites, revealing preferred
sites of the proteases reported so far for developing
enamel (MMP-20, KLK-4 and, more recently, chy-
motrypsin C) (12). Our finding also supports the con-
clusion that TRAP accumulates during the maturation
step and is retained in mature enamel.
In addition to amelogenin, we demonstrated that
some enamelin and ameloblastin peptides are also left
after the maturation process. The role of these protein
remnants – that in mature enamel are placed between
rod and inter-rod enamel – is the regulation of its
mechanical properties (23). Dental enamel is more flex-
ible and softer than pure hydroxyapatite as a result of
its organic content (24, 25), and this allows it to
behave as a coating that retains the tooth’s shape,
resisting fracture and wear during load-bearing func-
tion. Few organic components are found in the enamel
surface, as they are mainly present close to the dentin–
394 Castiblanco et al.
enamel junction (DEJ) (26). Nano indentation studies
(26, 27) suggest that the extensive removal of proteins
on the enamel surface (followed by restricted removal
of proteins towards the DEJ during enamel matura-
tion) is not a random event (28): the overall role of
this selective protein retention is the creation of a func-
tional gradient in hardness and stiffness in enamel (27).
Such a gradient may allow for a smoother transition
of mechanical stresses across the DEJ and into the
softer dentin (28), thereby improving tooth resistance.
Other minor roles of retained peptides could be the
definition of cleavage planes to deflect cracks, the
allowance of limited differential movement between
adjacent rods (29), and the maintenance of enamel
hierarchical structure by gluing hydroxyapatite crystals
together (30). The peptides from amelogenin, enamelin,
and ameloblastin, found in this study, could contribute
to this overall mechanical effect in mature dental
enamel.
It is important to stress that the amelogenin X-iso-
form peptides found in all specimens belong to the
same region of the protein sequence (AA 17–42),
which belongs to the TRAP and is highly conserved
among species. These peptides are not random cleav-
age products, as their N- and C-termini have been
previously reported as cutting sites for MMP-20 and
KLK-4 (31, 32). The secondary and tertiary structures
of these frequent peptides could form the so-called in
biomaterial science ‘sacrificial bonds’, molecular bonds
capable of sacrificing themselves to absorb mechanical
stress (30). Knowing the primary structure of the
remaining enamel peptides as they occur in erupted
enamel could be useful in the search for potential
domains able to serve as sacrificial bonds in erupted
enamel, which have already been proven to exist in
bone (33). Such investigations could lead to the under-
standing of enamel biomechanical behaviour and the
development of biomaterials with improved mechanical
properties.
The recovery of amelogenin peptides and the identifi-
cation of enamelin and ameloblastin can also be of
great utility when comparing the proteomic profile of
sound enamel with enamel that has developmental
defects [e.g. dental fluorosis and molar/incisor
hypomineralization (MIH)]. As quantitative label-free
approaches are currently available to be performed
using data extracted from LC-MS/MS (34), our method
opens the possibility of performing quantitative studies
in this sort of samples.
Regarding the non-enamel-specific proteins
identified, we found ubiquitin, antithrombin, and
albumin in two out of eight samples. Serum albumin
and human antithrombin have been found in
defective enamel, such as that affected by MIH (21,
22); however, the sample used in this study was clini-
cally sound and these proteins were found in only
20% of the sample. The finding of such contaminants
may be attributed to adsorption of proteins from
body fluids (e.g. serum, saliva, and gingival crevicular
fluid) on the hydroxyapatite matrix of erupted
molars.
In conclusion, our method, which omits trypsin
digestion, is useful for the identification of enamel-
specific proteins. Besides the well-known presence of
amelogenin in erupted human enamel, we report the
occurrence of enamelin and ameloblastin peptides
exhibiting their naturally occurring sequences. This
finding supports and stimulates discussion on the role
of protein remnants in regulating the mechanical prop-
erties of enamel. Furthermore, this novel information
on the naturally occurring sequences of common amel-
ogenin X-isoform peptides can be used to study a
potentially highly conserved domain retained in the
mineral matrix for mechanical purposes. Finally, our
method can be reproduced for a more detailed study of
the enamel proteome at the peptide level and may be
useful for comparing sound and defective enamel.
Acknowledgements – This investigation was supported by COL-
CIENCIAS (442-2012), the scholarship ‘Young Researchers and
Innovators’ from COLCIENCIAS, granted to Gina Castiblanco
for 2 years, internal grants from Universidad El Bosque (PCI
2010-93/PCI-2011-242), and physical and human resources from
the Department of Environmental Science and Analytical Chem-
istry of Stockholm University and the Department of Medical
Biochemistry and Biophysics of Karolinska Institute. We thank
Dr Leopold Ilag and the Department of Environmental Science
and Analytical Chemistry of Stockholm University for hosting
the first author for 3 months during the development of this
research.
Conflicts of interest – The authors declare no conflicts of interest.
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Supporting Information
Additional Supporting Information may be found in the online
version of this article:
Table S1. Identified naturally occurring peptides from non-amelo-
genin proteins.
Table S2. Identified naturally occurring peptides from non-enamel
specific proteins.
Fig. S1. Spectrum of the highest scoring peptide of AMELX_HU-
MAN.
Fig. S2. Spectrum of the highest scoring peptide of AMELY_HU-
MAN.
Fig. S3. Spectrum of the highest scoring peptide of AMBN_HU-
MAN.
Fig. S4. Spectrum of the highest scoring peptide of ENAM_HU-
MAN.
Fig. S5. Spectrum of the highest scoring peptide of ALBU_HU-
MAN.
Fig. S6. Spectrum of the highest scoring peptide of ANT3_HU-
MAN.
Fig. S7. Spectrum of the highest scoring peptide of UBP54_HU-
MAN.