Identification of Protein Components in Human Acquired Enamel
Pellicle and Whole Saliva Using Novel Proteomics Approaches*
Yuan
Yao
§,
Eric A.
Berg§¶
,
Catherine E.
Costello¶
,
Robert F.
Troxler
, and
Frank G.
Oppenheim
**
From the
Department of Periodontology and Oral
Biology, Boston University School of Dental Medicine and the
¶ Mass Spectrometry Resource and
Department of
Biochemistry, Boston University School of Medicine,
Boston, Massachusetts 02118
Received for publication, June 25, 2002, and in revised form, October 21, 2002
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ABSTRACT |
Precursor proteins of the acquired enamel
pellicle derive from glandular and non-glandular secretions, which are
components of whole saliva. The purpose of this investigation was to
gain further insights into the characteristics of proteins in whole saliva and in vivo formed pellicle components. To maximize
separation and resolution using only micro-amounts of protein, a
two-dimensional gel electrophoresis system was employed. Protein
samples from parotid secretion, submandibular/sublingual secretion,
whole saliva, and pellicle were subjected to isoelectric focusing
followed by SDS-PAGE. Selected protein spots were excised, subjected to
"in-gel" trypsin digestion, and examined by mass spectrometry (MS).
The data generated, including peptide maps and tandem MS spectra, were
analyzed using protein data base searches. Components identified in
whole saliva include cystatins (SA-III, SA, and SN), statherin, albumin, amylase, and calgranulin A. Components identified in pellicle
included histatins, lysozyme, statherin, cytokeratins, and calgranulin
B. The results showed that whole saliva and pellicle have more complex
protein patterns than those of glandular secretions. There are some
similarities and also distinct differences between the patterns of
proteins present in whole saliva and pellicle. MS approaches allowed
identification of not only well characterized salivary proteins but
also novel proteins not previously identified in pellicle.
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INTRODUCTION |
Human teeth are exposed to whole saliva
(WS),1 consisting
mainly of secretions derived from three pairs of major salivary
glands, which comprise parotid, submandibular, and sublingual glands. Protein components that have been identified in all of the major glandular secretions are proline-rich proteins (acidic, basic, and
glycosylated families), amylase, statherin, histatins, lysozyme, lactoferrin, lactoperoxidase, and secretory IgA (1-10), whereas cystatins and mucins have been identified in submandibular/sublingual secretions (9, 11-13). However, detailed understanding of the protein
composition in WS is still limited because of the lack of knowledge
about proteins in other contributors to whole saliva such as secretions
from minor salivary glands and gingival crevicular fluid. In addition,
little is known about modifications that occur on proteins during or
after secretion into the oral cavity.
The acquired enamel pellicle (EP) is a protein film thought to result
from the selective adsorption of precursor proteins present in WS onto
tooth surfaces. Because of its intimate contact with enamel surfaces,
the EP plays an important role in maintaining tooth integrity by
controlling the mineral solution dynamics of enamel. At its interface
with the oral environment, the EP exerts selectivity on bacterial
attachment and is involved in the initial stages of plaque formation
(14). Because of the limiting amount of proteins that can be harvested
from EP formed in vivo, previous investigations have
utilized sensitive but indirect approaches such as enzymatic assays and
immunologic detection to identify EP components (15-19).
One of the ways for direct identification of EP components is mass
spectrometry (MS). MS has undergone considerable advances in the
sensitive and specific analysis of biological materials (20). The
development of matrix-assisted laser desorption/ionization (MALDI) MS
by Hillenkamp and colleagues (21, 22) greatly increased the ability to
analyze non-volatile biomolecules. Since then, improvements in
MALDI-time-of-flight (MALDI-TOF) mass spectrometers and sample handling
methodologies have allowed very high throughput, primarily as a result
of the speed of data acquisition and greater tolerance of contaminants
(e.g. salts and detergents) by MALDI when compared with
other MS methods (23). The introduction of quadrupole orthogonal
time-of-flight (QoTOF) MS has provided yet another level of
sophisticated analysis (24, 25). The coupling of quadrupoles to the TOF
analyzer initially generated electrospray ionization (ESI) data with
high sensitivity (<10 fmol) and mass accuracy (<20 ppm) and allowed
for tandem experiments that give much more complete and reliable data
to facilitate protein identification and characterization (26). The
more recent addition of a MALDI source to the QoTOF mass spectrometer
gave these instruments additional flexibility (27). These advances in
MS have been employed in a variety of biological investigations
including cataloguing bacterial proteomes (28, 29), identifying
differences in protein expression in disease versus normal
cells (30, 31), and characterizing post-translational modifications of
specific proteins (32, 33).
Recently, our group carried out the first direct identification of
proteins found in EP formed in vivo using a MS approach with
samples separated on one-dimensional SDS gels (34). We have now
addressed complications that arise from the presence of multiple
components in an apparent single electrophoretic band by achieving
better resolution of the protein mixture through the use of
two-dimensional electrophoresis (2-DE). Application of 2-DE has only
been reported in a few investigations on proteins in WS (35-37), and
none so far on proteins found in in vivo formed EP. In none
of these EP studies were proteins identified using MS techniques. In
the present study, we used 2-DE to resolve and compare proteins from
glandular secretions, WS, and EP and identified selected components
from WS and EP using mass spectrometry.
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EXPERIMENTAL PROCEDURES |
Human Subjects--
Healthy non-medicated male and female
volunteers, ranging in age from 20 to 60 years, were selected. The
subjects exhibited no overt signs of gingivitis or caries. Saliva and
pellicle collection protocols were approved by the Institutional Review
Board of Boston University Medical Center, and informed consent was
obtained from each subject.
Collections of Glandular Secretions and WS--
Parotid
secretion (PS) from both glands was collected by means of a
Carlson-Crittenden device (38). Submandibular/sublingual secretion
(SMSL) was collected using a custom-fitted device consisting of a
plastic core covered with Impregum F Impression material (3M ESPE,
Seefeld, Germany). The flow of glandular secretion was provoked by
gustatory stimulation using sugar-free lemon-flavored candies.
Collection of WS was carried out under masticatory stimulation using a
uniform bolus of ParafilmTM (Fisher Scientific, Pittsburgh,
PA). The samples were kept on ice during the collection procedure.
Immediately after the collection, WS samples were centrifuged at
14,000 × g for 20 min in a microcentrifuge at 4 °C
to remove undissolved materials. Samples of glandular secretions and WS
supernatant collected from two subjects were pooled and stored in
200-µl aliquots at
20 °C. Protein concentrations were determined
using a micro-BCA protein assay (Pierce).
Harvesting of Human EP--
The collection procedure for the
in vivo EP was carried out as described (34). Briefly, each
donor was subjected to a thorough dental prophylaxis employing a coarse
pumice containing no additives. EP was then allowed to form on the
enamel surfaces over a 2-h period. Teeth in each quadrant were isolated
with cotton rolls, rinsed twice with water, and dried with air. A
Durapore PVDF membrane (Millipore, Bedford, MA) soaked in 0.5 M sodium bicarbonate buffer, pH 9.0, was held with a pair
of cotton pliers and used to swab the coronal two thirds of the
labial/buccal surfaces spanning from the central incisor to the first
molar. One PVDF membrane was used for the collection of pellicle in
each quadrant. The resulting four membranes from one subject were
placed into a polypropylene microcentrifuge tube. To recover pellicle
proteins from PVDF membranes, 1 ml of distilled water was added to each
tube and extraction of pellicle was carried out by vortexing the sample
for 30 s followed by sonication (Branson Cleaning Equipment Co.,
Shelton, CT) for 5 min in an ice bath at 4 °C. Control experiments
using two-dimensional electrophoresis (2-DE) showed that sonication of
PS under these conditions provoked undetectable fragmentation of
proteins/peptides. To separate the extraction liquid from the membrane,
a small needle-size (16-gauge) hole was placed on the bottom of the
tube followed by centrifugation in a microcentrifuge and the pellicle
extract was collected into a separate tube. Pellicle samples were then desalted using sequential dilution-centrifugation steps in an Amicon
microcentrifuge device (Millipore) with a molecular mass cut-off of
3000 Da. Desalted pellicle samples were then analyzed using a micro-BCA
protein assay to determine protein concentration.
Two-dimensional Gel Electrophoresis--
2-DE (28, 39, 40) was
carried out by isoelectric focusing (IEF) using pre-made immobilized pH
gradient (IPG) strips on the Protean IEF cell (Bio-Rad) followed by
SDS-PAGE using the Protean-II device (Bio-Rad). Approximately 50 µl
of PS, SMSL, WS, or pooled EP samples containing 100 µg of proteins
was mixed with 300 µl of IEF rehydration buffer in a focusing tray
upon which a 17-cm-long, pre-made IPG strip was added. Rehydration was
carried out in the tray under a constant voltage of 50 V for 12 h.
The voltage was then gradually increased to 10,000 V, and samples were
focused for an additional 6 h. To prepare the IPG strip for the
second dimension, the strip was first equilibrated in a buffer
containing 50 mM Tris-HCl, pH 8.8, 30% glycerol, 2% SDS,
6 M urea with 1% dithiothreitol (Sigma) for 10 min
at room temperature, followed by a second equilibration for 10 min
using the same buffer except that dithiothreitol was replaced by 4% iodoacetamide (Sigma). Subsequently, the IPG strip was applied horizontally on top of a 10% SDS-polyacrylamide gel (20 × 20 cm), and proteins/peptides were separated vertically for 16 h at a constant voltage of 105 V. The resulting two-dimensional gel was stained either with silver (Owl Separation System, Portsmouth, NH) or
with Sypro-Ruby (Molecular Probes, Eugene, OR). Gels with the
Sypro-Ruby staining were visualized under ultraviolet light using a
Gel-Doc 1000 Imager (Bio-Rad).
Mass Spectrometry--
Protein spots were excised from
two-dimensional gels using a sterile, cut pipette tip. Proteins
contained in the gel were digested with sequencing-grade trypsin
(Promega, Madison, WI) as previously described (41). Tryptic peptides
were extracted from gel pieces with 1% trifluoroacetic acid in 50%
acetonitrile and dried using a Speed-VacTM (Thermo Savant,
Holbrook, NY). The resulting samples were resuspended in 0.1%
trifluoroacetic acid and desalted using
ZiptipsC-18TM (Millipore) as per the
instructions from the manufacturer. Samples were then dried and
resuspended in 50% methanol with 1% formic acid and were analyzed
using both the Finnigan MAT Vision 2000 MALDI-TOF reflectron mass
spectrometer (Thermo Finnigan, San Jose, CA) equipped with an
ultraviolet laser (nitrogen, 337 nm) and the Applied
Biosystems/MDS-Sciex QStar Pulsari quadrupole/orthogonal acceleration TOF mass spectrometer (QoTOF) with nanospray and MALDI (UV
laser; nitrogen, 337 nm) sources (Applied Biosystems Inc., Framingham,
MA). The MALDI-TOF MS was used initially to both screen samples to
ensure adequate digestion and peptide recovery and to analyze samples
at higher mass ranges. The QoTOF was then used to obtain data with high
mass accuracy as well as to obtain tandem MS data. The MALDI matrix was
2,5-dihydroxybenzoic acid (DHB), and typically 50-200 laser shots were
summed for each spectrum. The laser power used was between 50 and 60%
when obtaining the Vision spectra and 30-33 µJ when obtaining the
QoTOF spectra. When obtaining QoTOF nanospray data, 1-µm nanospray
tips, pulled with a Sutter model P-87 micropipette puller, were used
with an ion source voltage of 1000-1300 V. For tandem data, nitrogen
was used as the collision gas and a range of operator-controlled
collision voltages (12-50 V for electrospray; 35-90 V for MALDI) were
employed. Spectra were analyzed systematically by manually
reconstructing mass data with tabulation of peaks having an area of
1.0% or greater relative to the largest peak. Peaks corresponding to
trypsin autolysis peptides were not included. Mass lists were used to
screen against tryptic fragment libraries including Mascot
(Matrix Sciences Ltd.; www.matrixscience.com), PROWL (Rockefeller
University and New York Universities; www.prowl.rockefeller.com),
Protein Prospector (University of California at San Francisco;
www.prospector.ucsf.edu), PepSea (Protana; 195.41.108.38) and
PeptideSearch (EMBL; www.mann.embl-heidelberg.de) to identify salivary
and pellicle components. Instruments were externally calibrated, and
identification of fragment mass matches used an error of 0.1% for data
obtained using the Vision 2000 MALDI-TOF MS and 50 ppm for data
obtained using the QoTOF. In samples with appropriate signal (>10
counts), tandem MS data was obtained with the QoTOF and resultant
fragmentation data were screened using the PepSea and Mascot data
bases. The validity of protein matches was confirmed by either
additional tandem data or a careful examination of all MS data
available for that particular sample, and total coverage for each
result was calculated.
 |
RESULTS |
EP formed in vivo from a pooled sample derived from 20 subjects (100 µg) showed a characteristic 2-DE pattern using a
focusing range of pH 3-10 (Fig. 1,
panel A). Visual inspection of the gel revealed
that most of the proteins/peptides were distributed into three zones
dictated by molecular mass. The high molecular mass region I, spanning
between 36 and 97 kDa, contained many detectable spots clustered
together, which primarily focused between pI 5 and 6. The middle
molecular mass region II, ranging between 21 and 36 kDa, again showed
clustering of many spots focused between pI 5 and 7. The low molecular
mass region III, covering the area below 21 kDa, contained many spots
focusing in the range of pI 4-8. It is interesting to note that pI
ranges for the major spots in the three zones expand as their molecular
masses decrease. A 2-DE gel of proteins from a WS pool prepared using
the same electrophoretic conditions revealed a different pattern from
that of EP (Fig. 1, panel B). Although proteins
were also mainly distributed into zones I-III in the vertical
dimension, they were focused horizontally into wider pI ranges in zone
I and II with major spots shifted to a more basic pI region than was
observed with EP. To our knowledge, this is the first time that well
resolved EP and WS samples are shown in 2-DE gels with a full view (pI range 3-10 and molecular mass range 0-200 kDa). However, the
relatively similar pI values among some components in both biological
samples clearly pointed to the necessity of further resolving these
proteins/peptides. Because most of the major components were contained
in the pI 5-8 range, narrow range IPG strips (pH 5-8) were
subsequently applied in the IEF phase of the 2-DE.

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Fig. 1.
Two-dimensional gel electrophoresis using
isoelectric focusing with pH range 3-10 in the horizontal dimension
and SDS-PAGE (10%) in the vertical dimension. Panel
A, 100 µg of EP, stained with Sypro-Ruby and shown in
inverted form for better contrast. Panel B, 100 µg of WS, stained with silver. Gel spots that were cut out for in-gel
trypsin digestion and analyzed by MS were labeled P1-P13
and S1-S3 (P denotes EP, and S
denotes WS). Horizontal lines divide the gels
into zones I, II, and III, representing high, middle, and low molecular
mass regions, respectively.
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The pattern of spots visualized by 2-DE of proteins in WS and EP (Fig.
1) was next compared with those in PS and SMSL because the latter
secretions are thought to contain primarily intact proteins which can
serve as precursors to those in WS and the EP. The electrophoretogram
of protein (100 µg) from pooled PS showed a profile of proteins with
well defined separation (Fig. 2,
panel A). Using the same zoning criteria, zone I
contained ~30 detectable spots, which largely focused between pI 6 and 7. Zone II consisted of at least 10 distinguishable spots, which were focused between pI 5 and 6. Zone III was divided mainly into two
regions. The acidic region contained 3 heavily stained spots between pI
5 and 5.5 and several spots that migrated below pI 5. The basic region
(pI higher than 7) contained ~12 spots that were clearly discernible.
A similar, but not identical, electrophoretic pattern was observed with
SMSL (Fig. 2, panel B). The differences are
likely caused by the presence of some proteins in SMSL such as mucins
and cystatins, which are absent in PS. The tailing effect in the region
above zone I (Fig. 2, panel B) may be caused by migration of MUC-7 glycoforms (42). Additional spots seen in the basic
region of zone III (Fig. 2, panel B) likely
represent cystatin molecules. Although the data in Fig. 2 were obtained using pooled samples from two subjects, very similar electrophoretic patterns were obtained with samples from several other subjects.

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Fig. 2.
Two-dimensional gel electrophoresis of
salivary glandular secretions by isoelectric focusing with pH range
5-8 in the horizontal dimension and SDS-PAGE (10%) in the vertical
dimension. Panel A, proteins from PS (100 µg); panel B, proteins from SMSL (100 µg).
Both gels were stained with Sypro-Ruby. Zones I, II, and III correspond
to high, middle, and low molecular mass regions, respectively.
*, zone I 5-protein spot series.
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Given the relatively simple electrophoretograms from both PS and SMSL,
one may imagine that WS, representing mostly a mixture of glandular
secretions with only minor contributions from gingival crevicular
fluid, would show an electrophoretic pattern that would nearly be a
summation of the two protein patterns (Fig. 2, A and B). Therefore, it was surprising to find that there were
significant differences between the protein patterns observed for
glandular secretions (Fig. 2, A and B) and WS
(Fig. 3) in the pI range 5-8. First, the
number of detectable protein spots in WS was dramatically greater than
those in either PS (Fig. 2A) or SMSL (Fig. 2B). There were
approximately 65 spots in zone I, 43 spots in zone II, and 30 in zone
III. Second, proteins/peptides in WS seemed to be more distributed
evenly throughout the pH range 5-8, in contrast to the clustering of
spots in specific pH ranges that was observed in glandular secretions.
Some proteins in glandular secretions such as the 5-protein spot series
in the 40-45-kDa region (zone I, indicated by * in Fig. 2,
A and B) were absent in WS, whereas more
proteins/peptides were detectable in zone II of WS. These observations
lend support to the finding that WS represents a mixture of proteins
not only derived from different sources but, more importantly, proteins
that have undergone significant processing and modification upon
entering the oral cavity.

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Fig. 3.
Two-dimensional gel electrophoresis of WS
(100 µg) by isoelectric focusing with pH range
5-8 in the horizontal dimension and SDS-PAGE (10%) in the vertical
dimension. The gel was stained with Sypro-Ruby. Gel spots that
were cut out for in-gel trypsin digestion and analyzed by MS were
labeled S4-S17. Zones I, II, and III indicate high, middle,
and low molecular mass regions, respectively. Framed
area contains eight protein doublets representing amylase
isoforms.
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EP was also analyzed using the narrow focusing range pH 5-8 (Fig.
4). A more refined pattern of spots was
observed when EP was separated by this range in comparison to that
shown in Fig. 1. This is particularly pronounced in the high (zone I)
and middle (zone II) molecular mass zones because of greater resolution
of the proteins/peptides separated in a narrower pH range exhibiting very close pI values. When components had greater pI variance, adequate
separation could be obtained in both pH ranges as seen in zone III of
Figs. 1 and 4. More than 40 spots were observed in each of the three
zones, showing an overall distribution pattern that was essentially the
same as that observed with the broad pH range gels (Fig. 1). The
majority of the spots in zone I (Fig. 4) were focused between pI values
of 5 and 6. This range expanded to pI 5-7 for zone II and to 5-8 for
zone III. Spots with high staining intensity appeared in the same
location for both gels and represent proteins/peptides of relatively
high abundance. Despite of the fact that the EP analyzed over the pH
range 3-10 was derived from a pool of pellicle proteins obtained from
different subjects than the EP analyzed over pH range 5-8, there are
considerable similarities in the overall EP patterns. These
similarities point toward a consistency in EP composition and suggest
that the generation of its constituents is dictated by a common
mechanism.

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Fig. 4.
Two-dimensional gel electrophoresis of EP
(100 µg) by isoelectric focusing with pH range
5-8 in the horizontal dimension and SDS-PAGE (10%) in the vertical
dimension. The gel was stained with Sypro-Ruby. Gel spots that
were cut out for in-gel trypsin digestion and analyzed by MS were
labeled P14-P23. Zones I, II, and III indicate high,
middle, and low molecular mass regions, respectively.
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The difference between the electrophoretic patterns of EP and WS (Fig.
1, panels A and B) became more obvious
when these proteins were separated in the focusing range of pH 5-8
(Figs. 3 and 4). In zone I, few of the spots observed in the pI range
of 5-6 in EP were observed in WS, whereas the staining intensity for
the protein series between pI 6 and 7 was significantly reduced in EP
compared with WS. In zone II, very few of the spots observed in EP
seemed to relate to those in WS. Although a few EP proteins in zone III
were comparable with those in WS with respect to spot location, they
varied in staining intensity. To identify specific protein/peptide
components in EP and WS, MS methods were employed. Selected protein
spots from WS and EP 2-DE gels were subjected to in-gel trypsin
digestion (43) and analyzed using a MALDI-TOF and ESI- and MALDI-QoTOF
mass spectrometers. Data were acquired with several types of mass
spectrometers to ensure more complete coverage of identified spots.
Analyzed protein spots are labeled numerically in Figs. 1, 3, and 4
(S for WS and P for EP). Results for individual
proteins identified are summarized in Table
I. In general, the experimentally
observed pI values were in good agreement with values reported in
protein data bases. Exceptions are lysozyme, histatin 3, and
calgranulin B. The discrepancy could be related in part to covalent
modifications occurring during the EP formation and in part to steric
influences and structural/conformational contributions. Details with
respect to matching peptide masses as well as observed modifications
(oxidation, pyroglutamination, phosphorylation, and alkylation) for
each protein are given in Table II.
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Table II
Summary of MS data used for protein assignment
Superscript CAM indicates that the peptide contains
carboxyamidomethylation of a cysteine residue. Superscript O
indicates that the peptide contains an oxidized methionine residue.
Superscript q indicates that the peptide contains a
pyroglutamine. Superscript P indicates that the peptide
contains a phosphorylated residue. Asterisk denotes the tryptic
fragments from which tandem MS data were obtained.
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Proteins identified in WS include cystatin SA, cystatin SA-III,
statherin, cystatin SN, calgranulin, salivary amylase, and serum
albumin. Fig. 5 contains representative
MALDI-QoTOF MS (panel A) and MS/MS
(panel B) spectra from spot S4. The MALDI-QoTOF
MS spectrum (Fig. 5A) contains several major ions at
m/z 842.48, 1292.67, 1897.90, 1914.95, 2109.98, and 2270.05. Some of these peaks correspond to trypsin autolysis
products (e.g. m/z 842.48). Peptide
ion masses not coincident with trypsin autolysis products were selected
for collision-induced dissociation (CID) tandem MS experiments.

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Fig. 5.
MS analysis of tryptic peptides from spot
S4. Panel A, MALDI-QoTOF mass spectrum.
Peaks matching cystatin SN tryptic fragments are labeled
with their N- and C-terminal amino acid residue number in
parentheses. Inset shows pyroglutamination ([M + H]+; m/z 1897.90) of the peptide
corresponding to residues 47-63 ([M + H]+;
m/z 1914.95) with a loss of 17 daltons (amine
group). Panel B, MALDI-QoTOF tandem MS spectrum
of tryptic peptide [M + H]+; m/z
1292.67 corresponding to residues 19-29 of cystatin SN. This confirmed
the identification of S4 as cystatin SN. Superscript
q indicates pyroglutamination. Spectra were accumulated for
30-60 s; MALDI matrix was DHB; laser power, 50 µJ.
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Fragmentation of ions by CID has been shown to occur primarily at and
around the peptide bond (44). Scheme 1 is
a diagram of the designations for peptide fragments from a theoretical
peptide (45, 46). The most common fragments observed using low energy CID are b ions (containing the N-terminal amino acid) and
y (containing the C-terminal amino acid). The other ions
(a, c, x, and z) are also
observed in low energy CID experiments but appear less frequently. Single amino acids are also observed in tandem experiments in the form
of immonium ions.
An example of such a CID tandem MS experiment is shown in Fig.
5B, where the parent ion with m/z
1292.67 (Fig. 5A) was subjected to a range of collision
voltages (50-110 V) resulting in multiple fragment ions as well as the
parent ion 1292.65 (Fig. 5B). Because all ions in the MALDI
MS spectrum (Fig. 5A) are singly charged (M + H)+, more energy is required for fragmentation than is
needed for multiply charged ions generated by ESI (see below). It is
also harder to control fragmentation, as evident by the large number of
low mass ions, internal ions (loss of both N- and C-terminal amino
acids), and immonium ions. Data base searching (PepSea) using the
fragmentation data obtained from these experiments matched to the
protein, cystatin SN. Fragment ions from the tandem MS spectrum (Fig.
5B) are labeled reflecting nearly complete b and y ion series. Additionally, a, c,
x, z, and immonium ions were also detected, as
well as many amine and water losses. Reexamination of the MS spectrum
(Fig. 5A) yielded other matching peptides confirming the
protein identification with 81% sequence coverage. Several tryptic
peptides that do not appear to match with theoretical digestion
products for cystatin SN (e.g. m/z
1897.90) can be assigned by noting their relationship to the series of
peptides with N-terminal glutamine that is observed at higher mass of
17.05 daltons (Fig. 5A, see inset). The
difference of 17 daltons (NH3) is consistent with the
formation of pyroglutamine and has been previously reported as a side
reaction during in-gel digestion (47).
Table I also lists the proteins identified from 2-DE gels of EP. These
proteins include lysozyme, histatin 3, histatin 1, statherin,
cytokeratins, calgranulin B, and phosphodiesterase. The corresponding
gel and spot number as well as relevant electrophoresis data (apparent
pI and molecular mass) are also listed. Not surprisingly, the samples
analyzed from the narrow range gels tended to contain less mixed
protein spectra that the broad range gels. Quality results were,
however, still obtained from the broad range gels. For example, Fig.
6 shows representative ESI-QoTOF MS
(panel A) and ESI-QoTOF MS/MS (panel
B) spectra from P12 (shown in Fig. 1). Resultant data base
searches allowed for the identification of this protein as cytokeratin
15, an oral epithelial specific type I keratin. Although a number of
ions corresponding to cytokeratin 15 are detectable, there are many
peaks present that do not correspond to this protein, indicating that
an unidentified protein is also present in the spectrum.

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Fig. 6.
MS analysis of tryptic peptides from spot
P12. Panel A, ESI-QoTOF mass spectrum. Peaks
matching cytokeratin 15 tryptic fragments are labeled with the charge
state as well as their N- and C-terminal amino acid residue number in
parentheses. Panel B, ESI-QoTOF tandem
MS spectrum of the tryptic peptide ([M + 2H]2+;
m/z 651.35) corresponding to residues 125-136 of
cytokeratin 15. This confirmed the identification of P12 as cytokeratin
15. Spectra were accumulated for 30-60 s.
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Fig. 7 contains representative MALDI-TOF
MS (panel A), ESI-QoTOF MS (panel
B), and ESI-QoTOF MS/MS (panel C)
spectra from P17. The data from the MALDI-TOF MS (Fig. 7A)
and the ESI-QoTOF MS (Fig. 7B) are comparable, with the
exception that ions are detected at higher m/z
values in the MALDI-TOF mass spectrum. This is a result of the
difference between MALDI ionization, which produces singly charged
species (z = 1), as compared with a nano- or
electrospray source, which produces primarily multiply charged ions
(z > 1). Additionally, the use of different ionization
methods yielded different distributions of peptide ions with some only present in the MALDI spectrum (e.g.
m/z 3180.3), whereas other species are only
visible in the ESI spectrum. These may represent peptides for which
analysis reveals the presence of additional components in the mixture,
the signals of which are suppressed in the MALDI or nano-ESI mass
spectrum.

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Fig. 7.
MS analysis of tryptic peptides from spot
P17. Panel A, MALDI-TOF spectrum.
Panel B, ESI-QoTOF mass spectrum. Peaks matching
calgranulin B tryptic fragments are labeled with the charge state, as
well as their N- and C-terminal amino acid residue number in
parentheses. Inset shows pyroglutamination ([M + 2H]2+; m/z 815.38) of the methionine
oxidized peptide 73-85 ([M + 2H]2+;
m/z 823.89). Panel C,
ESI-QoTOF tandem MS spectra of the tryptic peptides ([M + 3H]3+; m/z 602.98) corresponding to
residues 11-25 of calgranulin B. This confirmed the identification of
P17 as calgranulin B. Superscript q,
pyroglutamination; superscript O, oxidized
methionine. Spectra were accumulated for 30-60 s; MALDI matrix was
DHB; laser power, 50 µJ.
|
|
The triply charged ion at m/z 602.98 (M + 3H)3+ shown in Fig. 7B was subjected to a range
of collision voltages (18-50 V), and the resultant data were summed
(Fig. 7C). In this ESI-QoTOF tandem MS spectra the parent
ion was m/z 602.97 (M + 3H)3+. Data
base searching (PepSea) using the fragmentation data obtained from
these experiments matched to the protein, calgranulin B. Fragment ions
from the MS/MS spectrum (Fig. 7C) are labeled, and a partial
b ion and y ion series is observed. As lower
collision energy was required to fragment this multiply charged
peptide, not as many immonium and internal ions, and amine and water
losses, were observed, making the spectrum less complex than the CID
spectrum of a singly charged peptide (Fig. 5B). However, it
should be noted that, when running nano-ESI CID experiments, one also
must be mindful of multiply charged fragment ions. For example, in the spectrum shown in Fig. 7C, both doubly and triply charged
fragment ions are observed (m/z 790.38;
y132+ and 564.96;
y143+). The resolution of the QoTOF
makes determination of peptide charge states straightforward (see
insets). Reexamination of the MS spectra (Fig. 7,
A and B) yielded other matching peptides
confirming the protein identification with 61% sequence coverage.
Again, several ions are observed that do not appear to correspond to the theoretical digest of calgranulin B. Some of these ions can be
accounted for when the oxidation of methionine residues is taken into
account. Furthermore, pyroglutamination is also observed. The
inset of Fig. 7B shows tryptic peptide 73-85
with a methionine oxidized (m/z 823.89) and with
pyroglutamination (m/z 815.38).
 |
DISCUSSION |
Data generated in these experiments using 2-DE provided
qualitative comparison of the proteins present in EP, WS, and major glandular secretions, as well as definitive identification of selected proteins/peptides. It demonstrates that the combination of
2-DE coupled with MS techniques is a powerful tool to resolve individual components from complex protein/peptide mixtures and to
characterize them with subpicomolar sensitivity.
Both 2-DE and mass spectrometry have unique characteristics
and advantages for studies on complex mixtures such as EP and WS. The
utilization of 2-DE allows for separation of not only different
molecules with similar molecular masses, but also different modification patterns or isoforms of the same molecule. This resolution is exemplified by
-amylase, of which eight isoforms were detected with identical molecular mass ranging in pI from 5.9 to 7.2 (Fig. 3).
Eight additional isoforms in the same pI range were found, each
exhibiting a mobility change of 3 kDa in apparent molecular mass.
Although this 3-kDa difference between glycosylated and non-glycosylated amylase was originally reported using a
one-dimensional electrophoresis system (48, 49), the resolution into
eight different isoforms was made possible through the use of this 2-DE approach.
The utilization of MS allows for unambiguous identification of
proteins/peptides whether they already exist in the current data base
or are hitherto unidentified molecules. This aspect is extremely
important for studies of complex biological samples such as WS and EP.
These samples comprise both known and novel proteins subject to
modifications in the oral environment. The advantage of the technique
employed is well demonstrated in the current study for the
identification of calgranulin and cytokeratin family members, which
represent novel EP constituents originating from non-salivary glandular
sources. These discoveries were made feasible by our initial approach
of obtaining tandem MS for selected tryptic fragments observed in the
MS spectra to generate sequence-related product ions, which were then
matched to theoretical data from proteins in a data base. Once a
protein match with reasonable probability was obtained, the initial MS
data were reevaluated to determine whether other tryptic peptides and
known modifications from the proposed protein could be observed.
The combined utilization of 2-DE and MS provides complementary
information on proteins/peptides. In MS experiments, we obtained a low
coverage from some of the selected spots after trypsin digestion. This
difficulty could be the result of resistance to proteolytic digestion
of some proteins, incomplete recovery of digested peptides from the gel
as a result of large size/hydrophobicity of some fragments, and/or low
efficiency in peptide ionization. If only a minimal amount of data was
acquired and no corroborating data (tandem MS or detection of a known
modification) was obtained, protein identification was not reported.
Whether an identified protein is intact or is a proteolytic fragment
was determined by examining both MS and electrophoretic data. If an
identified protein migrates to a similar pI and molecular mass as the
reported intact form, it is likely that this protein is intact, even if not all peptides were observed in the MS spectrum. Using these criteria, we found evidence for the presence of intact statherin, amylase, albumin, cystatins SA-III, SA, and SN in WS, and intact histatin 1, statherin, and lysozyme in EP. The detection of intact cystatins, well known to be cysteine protease inhibitors, is consistent with previous studies where initial complete sequences were obtained on
cystatin SN and SA purified from WS (50, 51). Contrary to expectation,
these data reveal that other proteins can resist proteolysis and other
modifications and a portion of such molecules can survive unaltered in
the oral cavity. The two-dimensional electrophoretic positioning of
amylase and albumin in our study were similar to those described in PS
(52), suggesting that little, if any, modification occurred on these
proteins after exposure to the oral environment. Although statherin and
histatin 1 are known to be susceptible to bacterial degradation in the oral cavity, the mechanism by which a portion of these proteins remain
intact in EP is unknown. Mechanistic possibilities for this finding
include formation of covalent/non-covalent complexes with other
proteins making enzyme cleavage sites inaccessible or conformational
changes occurring after adsorption to enamel crystallites, rendering
them resistant to proteolysis (53).
The present investigation showed that the electrophoretic patterns of
proteins in glandular secretions were significantly different from
those of WS and EP. These results suggested that proteins originating
from non-glandular sources may contribute more significantly to WS and
EP than previously recognized or that proteins may have undergone
extensive proteolysis, cross-linking, and other modifications (54, 55).
Some interesting contributors to EP are members of the cytokeratin
family, cytokeratin 13 and 15 (56), pointing to oral epithelium as one
of the sources of proteins deposited on the tooth surface. The
cytokeratins identified in EP were distinct from those normally found
in skin keratinocytes or hair (e.g. cytokeratin 9 or
cuticular keratin), excluding the possibility that the proteins
identified stem from sample contamination. Another novel component
found in EP was calgranulin B, which has been previously identified in
WS and was shown to be a component of gingival crevicular fluid (37).
The calgranulin family contains a calcium-binding domain possibly
involved in enamel deposition.
This study also revealed that the protein/peptide components of WS
differed markedly from those of EP. This suggests that some EP
constituents may derive directly from glandular secretions and other
oral sources and that, contrary to expectations, the formation of EP is
not totally dependent on protein modification occurring in WS.
Comparison of WS and EP confirms the previously held notion that
pellicle formation is dictated by a selective protein/peptide
absorption process (5) and that the presence of phosphoproteins
histatin 1 and statherin (2, 57) is consistent with the ionic
interaction between proteins and enamel surfaces.
It is obvious that the identifications made in this study represent
only a fraction of major pellicle components. Nevertheless, the current
investigation showed the variety of novel as well as expected
components in WS and EP, which are both extremely important to oral
homeostasis. The use of proteomic technology overcame a number of
limitations imposed by classic protein isolation and characterization
methods. The results obtained open up a new avenue to directly
characterizing EP, which should ultimately lead to an understanding of
its three-dimensional structure and true functions.
 |
ACKNOWLEDGEMENTS |
We thank ThermoBioanalysis Corp. and Applied
Biosystems, Inc. for the loan of the Vision and QStarTM, respectively.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DE07652 from NIDCR (to F. G. O.), DE05672 (F. G. O.), P41-RR10888 from National Center of Research
Resources (to C. E. C.), and DE11691 from NIDCR (to
R. F. T.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
These authors made equal contributions to this work.
**
To whom correspondence should be addressed: 700 Albany St., CABR
Bldg., W-201, Boston, MA 02118. Tel.: 617-638-4942; Fax: 617-638-4924; E-mail: fropp@bu.edu.
Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M206333200
 |
ABBREVIATIONS |
The abbreviations used are:
WS, whole saliva;
SMSL, submandibular/sublingual secretion;
CID, collision-induced
dissociation;
ESI, electrospray ionization;
QoTOF, quadrupole
orthogonal time-of-flight;
EP, enamel pellicle;
MS, mass spectrometry;
MALDI, matrix-assisted laser desorption/ionization;
DHB, 2,5-dihydroxybenzoic acid;
TOF, time-of-flight;
2-DE, two-dimensional
electrophoresis;
J, joule(s);
PS, parotid secretion;
PVDF, polyvinylidene difluoride;
IPG, immobilized pH gradient;
IEF, isoelectric focusing.
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