1 s2.0 S1874391912004228 Main

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 51 4 0 –51 6 5
Available online at www.sciencedirect.com
www.elsevier.com/locate/jprot
Toward a standardized saliva proteome analysis methodology
Rui Vitorinoa,⁎, Sofia Guedesa , Bruno Manadasc, d , Rita Ferreiraa , Francisco Amadoa, b
a
QOPNA, Mass spectrometry center, Department of Chemistry, University of Aveiro, Portugal
b
School of Health Sciences, University of Aveiro, Portugal
c
Biocant, 3060‐197 Cantanhede, Portugal
d
Center for Neuroscience and Cell Biology, University of Coimbra, 3004‐517 Coimbra, Portugal
AR TIC LE I N FO ABS TR ACT
Article history: The present study aimed the evaluation of saliva sample pre-treatment, in particular the
Received 3 April 2012 sample clearance usually performed by centrifugation, to the contribution of salivary proteome
Accepted 30 May 2012 and peptidome. Using in-gel and off-gel approaches, a large content of salivary proteins was
Available online 15 July 2012 detected in the pellet fraction that is usually discarded. In addition, chaotropic/detergent
treatment in combination with sonication, before the centrifugation step, resulted in salivary
Keywords: complex disruption and consequently in the extraction of high amounts of proteins. Based on
Whole saliva this data, we suggest the use of urea/detergent with sonication as a standard saliva sample
LC-MS/MS pre-treatment procedure. We also described a procedure to extract salivary peptides which can
Peptidomic be performed even after saliva sample treatment with chaotropic/detergents. In overall, we
PTM reported for the first time the contribution of the pellet fraction to the whole saliva proteome.
Proteomics iTRAQ analysis highlighted a higher number of different peptides as well as distinct quantities
of each protein class when after sample treatment with urea and sonication, acetone
precipitation followed by solubilization with acetonitrile/HCl was performed.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction introduced in saliva treatment for proteomic analysis (reviewed

in [7]). Nonetheless, no standardized procedure for saliva
Whole saliva represents a complex mixture of different handling has yet been established albeit the technological
contributions such as major and minor salivary glands, improvements achieved in proteomics applied to the charac-
crevicular fluid, serum, epithelial cells, bacteria and food debris. terization of saliva composition, which allowed the identifica-
Besides water and salt, other components including proteins, tion of more than 3000 different species in this biological fluid
peptides, hormones, lipids and sugars are part of the composi- [8–11].
tion of whole saliva. Among the predominant proteins and According to Chevalier et al. [7] the best results in the
peptides are amylase, mucins (MUC5B and MUC7), carbonic characterization of saliva are obtained by gel electrophoresis
anhydrase, cystatins, proline-rich proteins (PRPs — acid and (SDS-PAGE and 2DE) when sample collection is performed in ice,
basic), histatins (1 and 3) and statherin [1]. This large array of anti-protease added and then sample is centrifuged and stored
peptides and proteins covers different molecular weight ranges, between −20 °C to −80 °C. Regarding salivary peptide analysis,
which complexity is further increased by the presence of Castagnola and colleagues [4,12] perform, by routine, saliva
posttranslational modifications (PTMs) and interactions with precipitation with trifluoroacetic acid for peptide extraction
high molecular glycoproteins, turning difficult the analytical while other groups prefer cut-off filter devices [13–15]. Through
use of this easily accessible bodily fluid for clinical purposes these efforts, issues respecting saliva pre-treatment, in partic-
[2–6]. To tentatively reduce sample complexity and to remove ular the centrifugation step, have not been considered in most
bacteria and cellular debris, a centrifugation step is generally of those salivary proteome characterization works. Thus, an
⁎ Corresponding author at: Department of Chemistry, University of Aveiro, 3810‐193, Aveiro, Portugal. Fax: +351 234370084.
E-mail address: rvitorino@ua.pt (R. Vitorino).
1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.jprot.2012.05.045
J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 51 4 0 –5 16 5 5141
essential step forward is the analysis of this pre-treatment 3.1.3. Procedure C (CDS)
influence in saliva proteome studies. Indeed, for standardiza- A similar approach to Procedure B was performed but in this
tion of saliva handling, the following issues should be consid- case incubation at RT was replaced by 2 cycles of 5 s each of
ered (i) samples should be representative of saliva, salivary sonication. The pellet was discarded and the supernatant saved
aggregates, cell and bacteria; (ii) be suitable for analysis by for analysis.
different methods and platforms; (iii) be compatible with
protein and peptide extraction and allow its maximum solubi- 3.2. Pellet protein extraction
lization; and (iv) enable comparison of data sets under different
pathophysiological conditions. Pellets obtained from 1 mL of whole saliva centrifugation
Taken these issues in consideration, in the present study, (from Procedure A) were treated with 200 μL of solubilization
we report the contribution of saliva clearance performed in buffer (7 M urea, 2 M thiourea, 1% (w/v) CHAPS, 1% (w/v)
the pre-treatment and its extensive characterization by Triton X-100, 1% (v/v) ampholytes (3–10) and 1 mM TCEP)
different platforms of a representative “normal” saliva sample incubated for 10 min at room temperature (RT) under agita-
obtained from healthy individuals. tion and then centrifuged at 12,000 × g for 30 min at 4 °C. The
pellet was discarded and the supernatant saved for analysis
(assigned as PCD). Sample assigned as PCDS corresponds to a
2. Material and methods similar procedure to PCD with the addition of 2 sonication
cycles of 5 s each.
2.1. Reagents
3.3. Peptide extraction
HPLC-grade acetonitrile (ACN, Riedel, Seelze, Germany), Milli-Q
grade water were used. General chemical reagents such as 3.3.1. Procedure D
triethylammonium bicarbonate (TEAB), trifluoroacetic acid Salivary peptides were extracted according to Castagnola and
(TFA), protease inhibitor cocktail, formic acid, α-cyano-4- coll. [12]. Thus, a solution of 0.2% of TFA was mixed in the
hydroxycinnamic acid (α-CHCA), urea and CHAPS, were pur- proportion of 1:1 with whole saliva, incubated in ice for 5 min
chased from Sigma (Karlsruhe, Germany). Sequencing grade and centrifuged for 5 min at 8000 × g and 4 °C. The superna-
modified trypsin (bovine) was from ABSciex (ABSciex, USA). tant containing enriched peptides was saved for analysis.
2.2. Whole saliva collection 3.3.2. Procedure E

Salivary peptides were extracted from each supernatant
For this study a pool of whole saliva was constituted from the obtained with Procedures A, B and C. For this purpose, ice cold
saliva collected from three healthy subjects (3 males aged 23 acetone was added drop by drop to 200 μL of each supernatant in
to 32) showing no evidence of oral pathologies or inflamma- the proportion of 9:1. Following this, the solution was mixed
tory processes. Unstimulated whole saliva (WS) was collected under constant agitation in ice for 1 h and then centrifuged at
at 10:00 a.m. by direct draining into a saliva collection tube 19,000×g for 15 min at 4 °C. The supernatant (S1) was collected
(kept on ice) from all subjects, who had previously refrained and saved. Pellet was resuspended in 200 μL of ACN/12 mM HCl,
from eating and drinking for at least 2 h. Immediately after incubated under agitation for 1 h in ice and centrifuged again at
whole saliva collection, 10 μL of PMSF 0.1 M, 1 μL of pepstatin 19,000×g for 15 min at 4 °C. The obtained supernatant was
1 mM and 20 μL of anti-protease cocktail (Sigma P2714) mixed with the first (S1) and lyophilized in a SpeedVac (Thermo).
including aprotinin, E-64, EDTA, AEBSF and leupeptin, were
added to each mL of saliva. Saliva sample was divided in three 3.4. Quantification of protein and peptides
different aliquots for different treatments.
Peptides were quantified using the 2,4,6-trinitrobenzenesulfonic
acid assay as previously described [16]. A standard curve was
3. Sample preparation generated with glycine solution (20, 35, 50, 70, 90 μM) and the
absorbance at 335 nm by 2,4,6-trinitrobenzenesulfonic acid
3.1. Total salivary protein extraction (TNBS). Protein quantification was performed using RC DC
Protein Assay kit (BioRad lab, Hercules, USA). A bovine serum
3.1.1. Procedure A (CS) albumin was used to generate the standard curve and the
Whole saliva was centrifuged at 12,000 ×g, 30 min, 4 °C. The absorbance at 750 nm was measured.
supernatant and pellet were separated and saved for analysis.
3.5. One-dimensional gel electrophoresis
3.1.2. Procedure B (CD)
Whole saliva was mixed with solubilization buffer (7 M urea, Thirty micrograms of protein was incubated with LDS sample
2 M thiourea, 1% (w/v) CHAPS, 1% (w/v) Triton X-100, 1% (v/v) buffer (0.4 mM EDTA, 8% (w/v) lauryl dodecyl sulfate (SDS),
ampholytes (3–10) and 1 mM TCEP) in the proportion of 2:1. 50 mM Tris–HCl (pH 6.8), 4% glycerol, 0.075% Serva Blue G250
The mixture was then incubated for 10 min at room temper- (w/v) and 0.025% Phenol Red (w/v) for 10 min at RT. Samples
ature (RT) under agitation being centrifuged at 12,000 ×g for were loaded onto pre-cast 12% gels Bis-Tris (Novex, InVitrogen,
30 min at 4 °C. The pellet was discarded and the supernatant Carlsbad, CA) and electrophoresis was carried out at a constant
saved for analysis. voltage of 150 V with running buffer (50 mM 2-(N-morpholino)
5142 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 51 4 0 –51 6 5
ethane sulfonic acid (MES), 50 mM Tris base and 0.1% (w/v) SDS). conventional reverse-phase LC. In the case of protein expres-
Gels were stained with colloidal Coomassie [17,18]. sion (from Procedures A, B and C), the obtained tryptic peptides,
after iTRAQ labeling, were separated by a multidimensional
3.6. Two-dimensional gel electrophoresis approach based on a first dimension with high pH reverse
phase (as previously described [20]) and a second dimension
The first dimension was carried out using our previous with the acidic reverse-phase system.
conditions [17,18]. Briefly, 150 μg of protein was applied onto
IPG strips (13 cm pH 3-10NL), containing immobilines pHs 3–10, 3.9.1. High pH reverse-phase separation
thiourea, CHAPS and urea. After isoelectric focusing, the strip Sample loading was performed at 200 μL/min with buffers (A)
was applied on top of a 12% Bis-Tris home-made, as previously 72 mM TEA, 52 mM acetic acid in H2O, pH 10 and (B) 72 mM
described [19], at constant voltage of 150 V and MES as running TEA, 52 mM acetic acid in ACN, pH 10 (98% A: 2% B). After
buffer. Spots were detected by colloidal Coomassie Brilliant blue 5 min of sample loading and washing, peptide fractionation
G-250 for tryptic digestion and protein identification. was performed with linear gradient to 50% B over 35 min
followed by a 100% B step. Sixteen fractions were collected,
3.7. Digestion of proteins evaporated, and resuspended in 2% ACN and 0.1% TFA.
3.7.1. In-gel 3.9.2. Reverse-phase separation

Annotated spots or bands were manually excised and digested Collected fractions and peptide fractions were separated
in-gel with trypsin. Briefly, gel pieces were washed three times as previously described [13]. Briefly, peptides loaded onto a
with 25 mM ammonium bicarbonate/50% acetonitrile and C18 pre-column (5 μm particle size, 5 mm, from Dionex)
further dried in a Speed-Vac. Then, 10 μL of 25 μg/mL modified connected to an RP column PepMap100 C18 (150 mm × 75 μm
bovine trypsin (ABSciex, USA) in 25 mM ammonium bicarbon- I.D., 3 μm particle size). The flow rate was set at 300 nL/min.
ate was added to the dried gel pieces and the samples were The mobile phases A and B were 2% ACN 0.1% TFA in water
incubated overnight at 37 °C. Extraction of tryptic peptides was and 95% ACN, 0.045% TFA, respectively. The gradient started
performed by three times addition of 10% formic acid/50% at 10 min and ramped to 60% B till 50 min and 100% B at
acetonitrile followed by lyophilization in a Speed-Vac. Tryptic 55 min and retained at 100% B till 65 min. The column was
peptides were resuspended in acetonitrile/formic acid solution equilibrated with solvent A for 20 min before the next sample
and mixed (1:1) with a matrix consisting of α-cyano-4- was injected. The separation was monitored at 214 nm using
hydroxycinnamic acid. Aliquots of samples were spotted onto a UV detector (Dionex/LC Packings, Sunnyvale, CA) equipped
MALDI sample target plates. with a 3 nL flow cell. Using the micro-collector Probot (Dionex/
LC Packings) and, after a lag time of 5 min, peptides eluting
3.7.2. In-solution from the capillary column that were mixed with a continuous
An in-solution digestion was performed for iTRAQ labeling. flow of α-CHCA matrix solution (270 nL/min, 2 mg/mL in 70%
Briefly, 100 μg of protein was used for digestion which was ACN/0.3% TFA and internal standard Glu-Fib at 15 fmol) were
performed according to the protocol provided by the manufac- directly deposited onto the LC-MALDI plates at 12 s intervals
turer (Applied Biosystems, Foster City, CA). Briefly, samples for each spot (150 nL/fraction). For every separation run, 208
were mixed with triethyl ammonium bicarbonate buffer (TEAB) fractions in total were collected.
(1 M, pH 8.5) and RapiGest (Waters) to a final concentration of
0.5 M and 0.1%, respectively. Samples were then reduced with 3.10. Gel-based ID
5 mM tris(2-carboxyethyl) phosphine (TCEP) for 1 h at 37 °C and
alkylated with 10 mM S-methyl methanethiosulfonate (MMTS) Peptide mass spectra were obtained on a MALDI-TOF/TOF
for 10 min at RT. Two micrograms of trypsin was added to each mass spectrometer (4800 Proteomics Analyzer, Applied
sample and the digestion was performed for 18 h at 37 °C. Biosystems, Foster City, CA, USA) in the positive ion reflector
Samples were dried in a Speed-Vac. mode. Spectra were obtained in the mass range between 700
and 4500 Da with ca. 1500 laser shots. For each sample spot, a
3.8. Labeling the protein digests with iTRAQ reagents data dependent acquisition method was created to select the
six most intense peaks, excluding those from the matrix,
Digested samples and extracted peptides were labeled with trypsin autolysis, or acrylamide peaks, for subsequent MS/MS
the iTRAQ reagents (8-plex) following the protocol provided by data acquisition. Trypsin autolysis peaks were used for
the manufacturer (Applied Biosystems, Foster City, CA). In internal calibration of the mass spectra, allowing a routine
brief, peptides were reconstituted in 70% ethanol/30% TEAB mass accuracy of better than 20 ppm. Spectra were processed
500 mM, added to each label and carried out for 2 h at room and analyzed by the Global Protein Server Workstation
temperature. The reaction was stopped by adding water and (Applied Biosystems, Foster City, CA, USA), which uses
the labeled digests corresponding to each of the four 8-plex internal Mascot (v2.1.1. Matrix Science Ltd, UK) software for
experiments were combined and dried using Speed-Vac. searching the peptide mass fingerprints and MS/MS data.
Searches were performed against the SwissProt (release date
3.9. Peptide separation 010111) under all taxonomic categories and the following
parameters: (i) two missed cleavages by trypsin; (ii) mass
Salivary endogenous peptides obtained using Procedures D tolerance of precursor ions 25 ppm and product ions 0.3 Da;
and E (with and without iTRAQ labeling) were separated by and (iii) oxidation of methionine as variable modification.
3.11. LC-based ID and quantification by treating the immunoblots with ECL chemiluminescence
reagents (Amersham, Pharmacia Biotech, Buckinghamshire,
Acquisition parameters were similar to Gel-based ID using UK), according to the supplier's instructions, followed by
Glu-Fib for internal calibration. The spectra were processed exposure to X-ray films (Sigma, Kodak Biomax Light Film, St.
and analyzed by the ProteinPilot software (v4.0 AB Sciex, USA), Louis, USA).
which uses paragon algorithm for protein/peptide identifica-
tion based on MS/MS data against the SwissProt protein
database (release date 01012011, all taxonomic categories).
Default search parameters were used: specifying trypsin as 4. Results and discussion
the digestion enzyme, fixed modification of Methylthio on
Cysteine residue and iTRAQ 8Plex, biological modification Currently, there are numerous reports characterizing saliva
with emphasis on phosphorylation and urea denaturation as composition in different pathological/physiological status. The
the variable modification setting. Mass tolerances for precur- majority of these studies start with a clearance step to remove
sor and fragments were default values for ProteinPilot®. the large aggregates (food and cell debris), producing a clear
Cut-off score value for accepting protein identification for extract for subsequent analysis, particularly relevant when
ProteinPilot® was a ProteoScore of 1.3 (95% confidence). unstimulated saliva is analyzed. Although easier collected in
Data was normalized for loading error by bias correction, higher volumes, stimulated saliva is predominantly derived
which is an algorithm in ProteinPilot that corrects for unequal from the parotid gland and consists mainly of water whereas
mixing when combining the labeled samples of one experi- the major component of unstimulated saliva is derived from the
ment. It does so by calculating the median protein ratio for all submandibular gland and is mainly composed of mucins and
proteins reported in each sample, adjusted to unity and cystatins, which largely contributes to the aggregation of
assigning an autobias factor to it. Nevertheless, the quantifi- salivary components [22]. This pre-treatment has been empir-
cation results were reviewed manually for all proteins found ically accepted as a general procedure and widely adopted.
to be differentially expressed (iTRAQ ratio > 1.3 or < 0.7 However, its effects on proteome data were not tested despite
according to [21]). the different contents of bacteria, glycoprotein or squamous
cells present in distinct salivary samples [23–25]. Consequently,
3.12. Western blotting of salivary amylase different amounts of aggregates are expected to be lost during
the initial clearance step and with this, several other salivary
Briefly, salivary proteins were separated by SDS-PAGE (12% in components, with the subsequent effects in the whole saliva
Laemmli conditions) and electroblotted onto nitrocellulose proteome profile. Thus, this study aimed the evaluation of
membranes (Amersham, Pharmacia Biotech, Buckingham- alterations in the proteome composition of salivary samples
shire, UK). The immunoblots were probed with 1:1000 dilution caused by this clearance treatment, without taking in consid-
of monoclonal anti-amylase (A8273, Sigma, St. Louis, USA) eration the inter-individual variability reported in previous
and with 1:1000 dilution of the secondary antibody (anti- studies [13,26]. To this end, several methodological approaches
mouse IgG peroxidase conjugate, Amersham Pharmacia were tested with pools of saliva samples and are summarized in
Biotech, Buckinghamshire, UK). The bands were visualized Fig. 1.
Fig. 1 – Schematization of sample preparation using different methodological approaches.

To evaluate the potential losses related with whole saliva

centrifugation (Procedure A), the resultant pellet was further
solubilized with a buffer containing chaotropic, surfactant
and denaturating agents like urea, thiourea, CHAPS and
Triton X-100, which promotes protein complex dissociation
and a better protein solubilization [27–29]. In parallel, saliva
solubilization prior to centrifugation was tested, with or
without sonication (Procedures C and B, respectively), which
is known to promote a good protein solubilization in biological
samples [30,31]. As a positive control of peptidome extraction,
whole saliva samples were treated with trifluoroacetic acid
(TFA) to induce protein precipitation with the remaining
peptides in the supernatant obtained after centrifugation.
The effect of all these procedures in saliva proteome and/or
peptidome analysis was evaluated with in-gel and off-gel
approaches.
4.1. Gel-based electrophoresis
Among the widely methodological approaches employed in

the characterization of saliva composition are conventional
techniques, such as SDS-PAGE and 2DE. In both cases, proteins
Fig. 2 – Representative Bis-Tris-PAGE gel. M refers to the
are separated in terms of their molecular weight. In spite
molecular weight marker and remaining lanes to different
the excellent performances given for proteins above 15 kDa
saliva procedures: lane 1 — Procedure A, lane 2 — Procedure B,
(through different acrylamide gel pore sizes), SDS-PAGE lacks
lane 3 — Procedure C, lane 4 — Procedure F, lane 5 — Procedure
resolution to efficiently separate small proteins (<20 kDa). By
D, lane 6 — Procedure E, lane 7 — Procedure G from A, lane
using Bis-Tris as the trailing ion, the system allows high moving
8 — Procedure G from B and lane 9 — Procedure G from C (with
boundary speeds at reasonable pH values, which is therefore
correspondence to Fig. 1).
very convenient for the analysis of low-molecular-weight
proteins and peptides [19,32]. Thus, we adopted this separation
system as our choice for the simultaneous analyses of low and
high molecular salivary components. in Bis-Tris gel (38% vs 48% for CD-to-CS and CDS-to-CS,
In a first approach, all extracts of saliva samples were respectively). Those identified proteins have been previously
loaded in 12% Bis-Tris gels. As expected, gel image analysis identified as forming salivary heterotypic complexes as
(Fig. 2) evidenced several bands covering a broad range showed by Iontcheva et al. [34]. Faint bands corresponding to
of molecular weight in extracts corresponding to saliva the GP340 (Fig. 2, bands 32 and 33) and MUC5B (Fig. 2, bands 32
(Procedure A-CS) (lane 2), saliva treated with urea (Procedure and 33) were still observed after saliva treatment in CD
B-CD) (lane 3), treated with urea/sonication (Procedure C-CDS) (Procedure B) and CDS samples (Procedure C) and in the pellet
(lane 4) as well as in pellet extracts (Procedure D-PCD and
Procedure E-PCDS) (lane 6 and 7). However, 7 intense bands
detected below 15 kDa are of notice representing small
proteins/peptides, which are expected to contribute in ap-
proximately 30% for saliva proteome [33]. MS/MS analysis of
these bands resulted in the identification of salivary peptides
such as histatin 1 (band 31), P–B peptide (also known as
SMR3B; band 30) and PRP1 (band 29). High molecular compo-
nents such as MUC5B and DMBT1, also assigned as salivary
agglutinin or GP340, were identified in bands 1, 33 and 53
(Fig. 2).
Comparing detected bands in Bis-Tris gel (Fig. 2), specifi-
cally CD (lane 2) and CDS (lane 3) with CS (lane 1), a raise is
observable in optical density of those bands later identified as
amylase (Fig. 2, band 5, 43 ± 4% raise for CD and 68 ± 5% for
CDS), cystatins (Fig. 2, band 14, 11 ± 1% for CD and 19 ± 3%
CDUS), polymeric Ig (Fig. 2, band 1, 73 ± 6% for CD and 98 ± 7%
for CDS), in the Ig light chain variable region (Fig. 2, band 24,
50 ± 3% for CD and 53 ± 2% for CDS) and histatin 1 (Fig. 2, band Fig. 3 – Western blot analysis of salivary amylase for saliva
31, 205 ± 8% for CD and 232 ± 7% for CDS). Further western blot treatments (Procedures A–E). A representative immunoblot
analysis of salivary amylase in samples prepared with Pro- image is presented above the histogram. (*p < 0.05 vs CS;
cedures B and C (Fig. 3) showed the same tendency observed ***p < 0.001 vs CS).
Table 1 – Protein identified in each 2D-BisTris-PAGE spot from Procedure E.

Spot Protein name Accession Protein Protein Peptide Total Total ion Best Best ion
no. name MW PI count ion score C.I. ion score C.I.
score % score %
1 Keratin, type I cytoskeletal 13 K1C13_HUMAN 49,557 4.91 1 78 100 78 100

2 Keratin, type II cytoskeletal 6A K2C6A_HUMAN 60,008 8.09 1 76 100 76 100
5 Keratin, type II cytoskeletal 6B K2C6B_HUMAN 60,030 8.09 13 191 100 79 100
11 Polymeric immunoglobulin receptor PIGR_HUMAN 83,232 5.58 2 114 100 66 100
12 Alpha-amylase 2B AMY2B_HUMAN 57,673 6.64 15 228 100 106 100
18 Keratin, type II cytoskeletal 4 K2C4_HUMAN 57,250 6.25 3 243 100 85 100
49 Ig alpha-2 chain C region IGHA2_HUMAN 36,503 5.71 1 88 100 88 100
51 Protein-glutamine TGM3_HUMAN 76,584 5.62 3 228 100 99 100
gamma-glutamyltransferase E
56 Cornulin CRNN_HUMAN 53,502 5.73 1 75 100 75 100
(continued on next page)

Table 1 (continued)
score % score %

58 Serum albumin ALBU_HUMAN 69,321 5.92 16 98 100 70 33
62 Heat shock 70 kDa protein 1A/1B HSP71_HUMAN 70,009 5.48 16 223 100 75 100
63 Heat shock 70 kDa protein 1A/1B HSP71_HUMAN 70,009 5.48 3 94 100 57 100
64 Heat shock cognate 71 kDa protein HSP7C_HUMAN 70,854 5.37 3 177 100 97 100
65 Heat shock cognate 71 kDa protein HSP7C_HUMAN 70,854 5.37 2 66 100 50 100
69 78 kDa glucose-regulated protein GRP78_HUMAN 72,288 5.07 3 194 100 85 100
78 Keratin, type II cytoskeletal 2 oral K22O_HUMAN 65,800 8.38 2 99 100 71 100
81 Keratin, type II cytoskeletal 6C K2C6C_HUMAN 59,988 8.09 4 234 100 68 100
95 Alpha-amylase 1 AMY1_HUMAN 57,731 6.47 18 122 100 79 100
Table 1 (continued)
score % score %
117 Beta-actin-like protein 2 ACTBL_HUMAN 41,976 5.39 1 78 100 78 100
143 ATP synthase subunit beta, ATPB_HUMAN 56,525 5.26 19 140 100 98 100
mitochondrial
148 Alpha-enolase ENOA_HUMAN 47,139 7.01 22 191 100 47 100
149 Beta-enolase ENOB_HUMAN 46,902 7.59 1 65 100 65 100
150 Phosphoglycerate kinase 1 PGK1_HUMAN 44,586 8.3 3 243 100 104 100
151 Putative elongation factor 1-alpha-like 3 EF1A3_HUMAN 50,153 9.15 8 148 100 90 100
153 Actin, cytoplasmic 2 ACTG_HUMAN 41,766 5.31 11 303 100 130 100
158 Serpin B13 SPB13_HUMAN 44,248 5.48 1 83 100 83 100
158 Zinc-alpha-2-glycoprotein ZA2G_HUMAN 34,237 5.71 1 78 100 78 100
162 Annexin A3 ANXA3_HUMAN 36,353 5.63 11 184 100 92 100
164 F-actin-capping protein subunit alpha-1 CAZA1_HUMAN 32,902 5.45 6 137 100 80 100
166 Short PLUNC2 SPLC2_HUMAN 26,995 5.35 1 135 100 135 100

Table 1 (continued)
score % score %
167 Zymogen granule protein 16 homolog B ZG16B_HUMAN 22,725 6.74 1 43 100 43 100
174 Leukocyte elastase inhibitor ILEU_HUMAN 42,715 5.9 11 433 100 141 100
175 Carbonic anhydrase 6 CAH6_HUMAN 35,345 6.51 11 157 100 85 100
190 Glyceraldehyde-3-phosphate G3P_HUMAN 36,030 8.57 1 87 100 87 100
dehydrogenase
dehydrogenase
dehydrogenase
193 Fructose-bisphosphate aldolase A ALDOA_HUMAN 39,395 8.3 12 116 100 46 100
194 Fructose-bisphosphate aldolase A ALDOA_HUMAN 39,395 8.3 7 59 97 35 100
196 Voltage-dependent anion-selective VDAC1_HUMAN 30,754 8.62 10 166 100 67 100
channel protein 1
204 Lysozyme C LYSC_HUMAN 16,526 9.38 8 80 100 80 100
205 Small proline-rich protein 3 SPRR3_HUMAN 18,142 8.86 7 136 100 52 100
206 Ig kappa chain C region IGKC_HUMAN 11,602 5.58 1 54 100 54 100
207 Ig lambda-3 chain C regions LAC3_HUMAN 11,231 6.92 2 110 100 61 100
208 Peroxiredoxin-1 PRDX1_HUMAN 22,096 8.27 2 131 100 68 100
209 Ig kappa chain V–III region Ti KV304_HUMAN 11,781 8.72 1 104 100 104 100
210 Ig kappa chain V–III region Ti KV304_HUMAN 11,781 8.72 1 97 100 97 100
210 Cystatin-B CYTB_HUMAN 11,133 6.96 1 96 100 96 100
211 Phosphatidylethanolamine-binding PEBP1_HUMAN 21,044 7.01 2 104 100 56 100
protein 1
dehydrogenase
213 Alpha-crystallin B chain CRYAB_HUMAN 20,146 6.76 13 141 100 34 100
214 Phosphoglycerate mutase 1 PGAM1_HUMAN 28,786 6.67 1 71 100 71 100
214 Triosephosphate isomerase TPIS_HUMAN 26,653 6.45 9 65 99 38 100
Table 1 (continued)
score % score %
217 Peroxiredoxin-6 PRDX6_HUMAN 25,019 6 2 105 100 57 100

218 Chloride intracellular channel protein 3 CLIC3_HUMAN 26,632 5.99 3 240 100 95 100
219 Heat shock protein beta-1 HSPB1_HUMAN 22,768 5.98 4 343 100 109 100
221 Protein S100-A9 S10A9_HUMAN 13,234 5.71 10 184 100 99 100
232 14-3-3 protein sigma 1433S_HUMAN 27,757 4.68 1 102 100 102 100
235 Immunoglobulin J chain IGJ_HUMAN 18,087 5.12 5 139 100 81 100
237 Glutathione S-transferase P GSTP1_HUMAN 23,341 5.43 9 343 100 134 100
239 Interleukin-1 receptor antagonist IL1RA_HUMAN 20,042 5.83 6 428 100 119 100
protein
240 Prolactin-inducible protein PIP_HUMAN 16,562 8.26 4 285 100 93 100
245 Histone H2A type 1-C H2A1C_HUMAN 14,097 11.05 1 104 100 104 100
246 Calmodulin CALM_HUMAN 16,827 4.09 2 185 100 108 100
247 Calmodulin-like protein 3 CALL3_HUMAN 16,880 4.3 8 140 100 140 100
248 Cystatin-S CYTS_HUMAN 16,204 4.95 7 181 100 100 100
250 Cystatin-SA CYTT_HUMAN 16,434 4.85 7 215 100 107 100
251 Protein S100-A14 S10AE_HUMAN 11,655 5.16 6 158 100 97 100
253 Thioredoxin THIO_HUMAN 11,730 4.82 5 153 100 96 100
261 Cystatin-A CYTA_HUMAN 11,000 5.38 1 114 100 114 100

Table 1 (continued)
score % score %
266 Protein S100-A11 S10AB_HUMAN 11,733 6.56 6 68 100 34 100
267 Protein S100-A12 S10AC_HUMAN 10,569 5.83 5 162 100 116 100
271 Cystatin-SN CYTN_HUMAN 16,377 6.73 5 309 100 124 100
275 Histatin-1 HIS1_HUMAN 6958 9.11 1 73 100 73 100
276 Protein S100-A10 S10AA_HUMAN 11,196 6.82 2 137 100 81 100
278 Profilin-1 PROF1_HUMAN 15,045 8.44 1 80 100 80 100
278 Cystatin-C CYTC_HUMAN 15,789 9 1 69 100 69 100
279 Peptidyl-prolyl cis-trans isomerase A PPIA_HUMAN 18,001 7.68 6 269 100 107 100
239 Interleukin-1 receptor antagonist IL1RA_HUMAN 20,042 5.83 6 428 100 119 100
protein
Table 1 (continued)
score % score %
245 Histone H2A type 1-C H2A1C_HUMAN 14,097 11.05 1 104 100 104 100
246 Calmodulin CALM_HUMAN 16,827 4.09 2 185 100 108 100
247 Calmodulin-like protein 3 CALL3_HUMAN 16,880 4.3 8 140 100 140 100
250 Cystatin-SA CYTT_HUMAN 16,434 4.85 7 215 100 107 100
253 Thioredoxin THIO_HUMAN 11,730 4.82 5 153 100 96 100
261 Cystatin-A CYTA_HUMAN 11,000 5.38 1 114 100 114 100
266 Protein S100-A11 S10AB_HUMAN 11,733 6.56 6 68 100 34 100
267 Protein S100-A12 S10AC_HUMAN 10,569 5.83 5 162 100 116 100
276 Protein S100-A10 S10AA_HUMAN 11,196 6.82 2 137 100 81 100
278 Profilin-1 PROF1_HUMAN 15,045 8.44 1 80 100 80 100
278 Cystatin-C CYTC_HUMAN 15,789 9 1 69 100 69 100
279 Peptidyl-prolyl cis-trans isomerase A PPIA_HUMAN 18,001 7.68 6 269 100 107 100
287 Galectin-7 LEG7_HUMAN 15,066 7.03 11 200 100 107 100
290 Histone H4 H4_HUMAN 11,360 11.36 2 125 100 69 100
295 Neutrophil defensin 3 DEF3_HUMAN 10,238 5.71 1 39 100 39 100
296 Neutrophil defensin 3 DEF3_HUMAN 10,238 5.71 1 33 99 33 99

Table 1 (continued)
score % score %

305 Protein FAM25 FAM25_HUMAN 9314 5.78 1 89 100 89 100
307 Keratin, type II cuticular Hb3 KRT83_HUMAN 54,161 5.54 21 84 100 32 99
332 Alpha-amylase 1 AMY1_HUMAN 57,731 6.47 21 180 100 42 100
Table 1 (continued)
score % score %

344 Deleted in malignant brain tumors 1 DMBT1_HUMAN 260,569 5.18 1 93 100 97 100
protein
344 Mucin-5B MUC5B_HUMAN 595,960 6.2 1 58 100 58 100
protein
345 Mucin-5B MUC5B_HUMAN 595,960 6.2 2 40 100 39 100
protein
fractions (Procedures D and E). Regarding the pellet fractions, proteins' relative abundance across sample sets has been
several salivary proteins were identified such as cystatin SN, hampered by several factors associated to gel-to-gel variation
cystatin B and lysozyme (Fig. 2, band 48), Ig alpha-2 (Fig. 2, including the reproducibility in protein focusing, staining
band 53). In addition, several forms of keratin (bands 35–39) sensitivity, gel distortions and normalization of spot OD
were also identified in those extracts and presented a across gel sets. Thus and as previously reported [39], it is
significant contribution in terms of optical densities. Com- reasonable to assume a coefficient of variation (CV) of 20–30%
paring optical densities of Bis-Tris gel bands between PCD in 2-DE [39], which is higher than those associated to typically
(Procedure D) and PCDS (Procedure E), only a slight increase one-dimension (1D) SDS-PAGE where all samples are loaded
(10%), in the band OD corresponding to amylase was observed in a single gel. In fact, as observed in 1D-PAGE, we obtained
(Fig. 2), further confirmed by western blot analysis (Fig. 3). variations within CV below 8% whereas in the case of 2-DE,
Since this result was the most prominent, we considered this our data showed a CV range of 25–35%. Nevertheless, a similar
extract (PCDS) for further analysis by 2DE. trend to Bis-Tris gels was observed if the sum of all spots OD
Transposing to 2DE, as shown in Fig. S1, and similarly to with similar molecular weight in 2DE maps is considered.”
previous studies [11,35–38], around 180 different spots were The most outstanding result was the detection of about
detected in 2DE profiles, independently of the treatment (Fig. 480 different spots (including all detected spots in salivary 2DE
S1A — CS, B — CD and C — CDS). Comparing the different profiles) in the pellet fraction after Procedure E (Fig. S1-
treatments regarding optical densities of each detected spot, a D-PCDS). From these, 366 spots were excised and 322 were
lack of statistical differences was observed. However, it positively identified by mass spectrometry (Table 1). Several
should be pointed out that 2DE use for the comparison of salivary proteins such as amylase (spots 95–99 in Fig. S2,
Fig. 4 – Pie chart showing the biological function category as a percentage of the 95 distinct identified proteins based on the
PANTHER classification system.
Supplementary data) or cystatins (S 248–249, SA 250, A 260, B categories are metabolic process (17%), response to stimulus
261, SN 271, C 278 in Fig. S2) were identified in this fraction. (11%), immune response (12%) and cellular processes (17.6%). In
Besides salivary proteins, others belonging to distinct sources these top four categories salivary proteins are included such as Ig
such as calmodulin (spots 246–247 in Fig. S2), peroxiredoxin alpha-2 chain C or cystatins A, B, C, SN and S, which comprise the
(spot 208 in Fig. S2) or cornulin (spots 56–57 in Fig. S2), a body's first line of defense against pathogens [33].
squamous epithelia cell-specific protein, were also identified. Despite limitations of 2DE in the separation of high molecular
In order to clarify the role of the identified proteins, a weight proteins, using Bis-Tris system it is possible to increase
qualitative analysis was performed in terms of functional the molecular weight separation range of proteins. Indeed, this
clusters. As can be observed in Fig. 4, according to the PANTHER system yielded the identification of low molecular weight
classification system (http://www.pantherdb.org), identified pro- components that are usually lost in second dimension separa-
teins in 2DE maps are distributed in 14 different biological tion with conventional systems (SDS-PAGE), such as histatin 1
function categories being the top four biological function (spots 275, 278 in Fig. S2), Protein FAM25 (spot 305 in Fig. S2) and
Fig. 5 – Pie chart showing the biological function category as a percentage of the 440 distinct identified proteins based on the
PANTHER classification system.
Fig. 6 – Venn diagram representing the overlap of proteins

(center) and unique proteins identified by off-gel (left) and
gel-based (right) approach. Numbers in parenthesis
represent the number of proteins identified.
the first time detected defensin 3 in 2DE maps (295–296, Fig. S2),
only achieved by the analysis of the pellet fraction. In addition,
after MS analysis of the large spot (293–297 in Fig. S2), several ions
were detected suggesting a large contribution of typical salivary
peptide fragments in face of their characteristic low molecular
weight and low isoelectric point.
It should also be noted that in several spots more than one
protein was identified and with different annotated sources Fig. 7 – Comparison of obtained individual ratio values (log2)
besides saliva, like serum and squamous cells (Table 1). for 61 significantly expressed proteins (p < 0.05) between two
Regarding the contribution of bacteria, none of the excised independent iTRAQ experiments: CDS:CS (A) and CD:CS (B).
spots was positively identified when searches were performed
against bacteria taxonomy.
4.2. Off-gel-based approach treatment [39]. In another example, cystatin S presents three
phosphorylated sites [1,11,17,40], which justify the appearance
In order to get high proteome coverage, a RP–RP system was of three spots (Fig. S2). Behind these specific examples, in 2DE it
used with the first RP column at pH 10 and the second at is also possible to monitor multiple spots identified as the same
pH 2.6 [20], and two independent runs were carried out. The protein as showed for cystatin SN or cystatin B (Table 1).
merge of both datasets led to the identification of a total of 489 Although relative quantification analysis by Protein-Pilot
distinct proteins with a score higher than 1.3 (95%), covering a 4.0 software comes with statistical analysis, since most
high molecular weight and isoelectric point ranges. It should methods are prone to technical variation, we included an
be pointed out that, with this approach and searching against additional 1.3-fold (log2 < ± 0.38) change cutoff for iTRAQ
the SwissProt human and bacteria protein database, 440 ratios. In addition, the analysis of the ratios of each individual
human proteins and 49 proteins from lactobacilli were variation within the first and second duplicate evidenced a
identified (Supplemental Table 2). These 440 human proteins significant positive correlation, which indicates a good
could be assigned into 17 functional categories using the technical sample preparation and a good analytical reproduc-
PANTHER classification system (Fig. 5), being the top three ibility (Fig. 7). Thus, out of all identified proteins only 61 (12%)
biological functions are cellular processes (16.8%), metabolic differentially expressed proteins (p < 0.05) in CD, CDS and
processes (16%) and developmental processes (11%). pellet (PCDS) versus CS (Table 2) were considered in this
A considerable higher number of proteins (489) were analysis, ensuring that only proteins with low individual
obtained using the off-gel approach when comparing with variability have been selected.
the gel-based approach (95 proteins). Analyzing the identified The real contribution of each protein to the pellet fraction is
proteins in both approaches (Fig. 6), it is possible to notice that unknown since, to the best of our knowledge, pellet influence to
only 47 distinct proteins are common. Nevertheless, salivary whole saliva protein was never evaluated. Moreover, individual
analysis by 2DE is advantageous facing the complexity of sample variability should be taken in consideration once it
saliva in terms of post-translational modified proteins. Indeed, might lead to different pellet amounts. Nevertheless, treating
several proteins present in their composition different glycans saliva with chaotropic/detergent agents, as well as with
which could modify their migration in focusing step as well ultrasounds (Table 2), relative increments in protein amounts
as in separation based on molecular weight as exemplified were observed for CD and CDS suggesting an improvement in
by others in salivary amylase before and after glycosidase protein solubilization before the clearance step. In fact,
Table 2 – iTRAQ evaluation of identified proteins in Procedures A, B, C and E.

Protscore % Cov Accession Name Species Peptides CD:CS CDS:CS PCDS:
(95%) CS
99.99 77.7 P04745 AMY1_HUMAN Alpha-amylase 1 HUMAN 184 2.1562 2.8913 2.0335
86.41 82.5 P13646 K1C13_HUMAN Keratin, type I cytoskeletal 13 HUMAN 151 1.7756 0.9301 9.4285
60.03 65.5 P19013 K2C4_HUMAN Keratin, type II cytoskeletal 4 HUMAN 94 1.5272 0.7632 7.8792
39.84 61 P04259 K2C6B_HUMAN Keratin, type II cytoskeletal 6B HUMAN 58 1.3891 1.1306 9.0563
33.97 74.3 P61626 LYSC_HUMAN Lysozyme C HUMAN 38 2.8864 2.4838 5.5283
25.72 58.1 P01876 IGHA1_HUMAN Ig alpha-1 chain C region HUMAN 38 2.0699 4.1348 1.2838
4.8 49.7 P01877 IGHA2_HUMAN Ig alpha-2 chain C region HUMAN 36 7.5834 12.8725 5.1703
22.7 80.1 P01036 CYTS_HUMAN Cystatin-S HUMAN 31 2.1457 2.8323 3.0343
16.35 46.9 P08779 K1C16_HUMAN Keratin, type I cytoskeletal 16 HUMAN 29 2.2557 1.5636 16.9849
18.17 24.5 P01833 PIGR_HUMAN Polymeric immunoglobulin receptor HUMAN 25 1.6366 2.0754 0.9161
27.7 67.1 P02814 SMR3B_HUMAN Submaxillary gland androgen-regulated HUMAN 24 4.5817 6.589 0.8619
protein 3B
16.04 38.4 P04083 ANXA1_HUMAN Annexin A1 HUMAN 24 1.6448 0.5327 8.1994
6.93 86.5 P01037 CYTN_HUMAN Cystatin-SN HUMAN 22 8.0051 13.4003 3.1145
14 64.9 P06702 S10A9_HUMAN Protein S100-A9 HUMAN 20 1.9323 1.5185 5.3145
18.97 13.2 Q9HC84 MUC5B_HUMAN Mucin-5B HUMAN 17 3.8191 5.3122 2.8833
12.78 27.1 P25311 ZA2G_HUMAN Zinc-alpha-2-glycoprotein HUMAN 16 2.2279 3.8949 1.4113
6.63 34.9 P01834 IGKC_HUMAN Ig kappa chain C region HUMAN 12 4.4615 7.9137 2.9365
12.15 26 Q9UGM3 DMBT1_HUMAN Deleted in malignant brain tumors 1 HUMAN 11 2.8 3.9388 2.7417
protein
11.75 22 Q8TAX7 MUC7_HUMAN Mucin-7 HUMAN 10 1.9952 3.9834 1.2777
10 18.8 P23280 CAH6_HUMAN Carbonic anhydrase 6 HUMAN 9 1.4861 1.9648 0.8507
12.14 57.9 P15515 HIS1_HUMAN Histatin-1 HUMAN 9 1.2303 2.0994 0.7723
8 16.2 Q9UBG3 CRNN_HUMAN Cornulin HUMAN 9 0.8509 1.0002 3.8163
10.62 32.2 Q96DA0 YP003_HUMAN Uncharacterized protein UNQ773/PRO1567 HUMAN 9 0.8244 1.3477 0.6185
11.31 16.1 P02768 ALBU_HUMAN Serum albumin HUMAN 7 2.2848 4.373 1.0323
8.63 22.4 Q6P5S2 CF058_HUMAN Uncharacterized protein C6orf58 HUMAN 6 1.8868 3.8141 1.6103
6 16.5 P07355 ANXA2_HUMAN Annexin A2 HUMAN 6 1.4452 0.6493 5.7016
6 25.8 P05109 S10A8_HUMAN Protein S100-A8 HUMAN 6 1.4121 1.4172 2.8812
4.03 31.9 P59666 DEF3_HUMAN Neutrophil defensin 3 HUMAN 5 4.1915 6.3838 4.5262
4 12.5 P04406 G3P_HUMAN Glyceraldehyde-3-phosphate dehydrogenase HUMAN 5 2.6956 3.0106 7.9326
1.72 49 P15516 HIS3_HUMAN Histatin-3 HUMAN 5 2.0857 6.8662 1.9177
4.01 32.7 P04792 HSPB1_HUMAN Heat shock protein beta-1 HUMAN 5 1.6489 1.3245 6.3496
2 8.5 P19652 A1AG2_HUMAN Alpha-1-acid glycoprotein 2 HUMAN 4 7.9877 12.1331 2.6351
4 10.9 P68363 TBA1B_HUMAN Tubulin alpha-1B chain HUMAN 4 5.2601 4.0849 18.5078
4 11 P48594 SPB4_HUMAN Serpin B4 HUMAN 4 3.6065 3.983 4.6398
2.18 14.3 P54108 CRIS3_HUMAN Cysteine-rich secretory protein 3 HUMAN 4 2.8733 4.546 1.7155
4 17.7 P68871 HBB_HUMAN Hemoglobin subunit beta HUMAN 4 2.5314 3.7634 1.2827
5 21.9 P01591 IGJ_HUMAN Immunoglobulin J chain HUMAN 4 1.0755 1.3623 1.5198
2.01 12.4 Q9BVA1 TBB2B_HUMAN Tubulin beta-2B chain HUMAN 4 0.7371 0.3647 2.6531
2.02 18.7 Q5SSG8 MUC21_HUMAN Mucin-21 HUMAN 3 4.7927 4.6435 13.4754
2 7.9 P06744 G6PI_HUMAN Glucose-6-phosphate isomerase HUMAN 3 3.6801 5.4776 5.9701
6 5 A8K2U0 A2ML1_HUMAN Alpha-2-macroglobulin-like protein 1 HUMAN 3 1.207 1.7187 2.9448
4.04 13.8 Q8N4F0 BPIL1_HUMAN Bactericidal/permeability-increasing HUMAN 3 1.1526 1.5726 0.8253
protein-like 1
6.09 16.3 P22079 PERL_HUMAN Lactoperoxidase HUMAN 3 1.0905 1.9304 0.8348
4.67 15.2 Q9BYB0 SHAN3_HUMAN SH3 and multiple ankyrin repeat domains HUMAN 3 0.7886 0.7 0.5071
protein 3
2 19.1 P01842 LAC_HUMAN Ig lambda chain C regions HUMAN 2 1.38034 2.54565 4.6211
4.6 7 P12814 ACTN1_HUMAN Alpha-actinin-1 HUMAN 2 2.9476 7.137 6.7622
2 9.9 P06870 KLK1_HUMAN Kallikrein-1 HUMAN 2 2.6012 4.5539 2.2044
2 16.8 P24158 PRTN3_HUMAN Myeloblastin HUMAN 2 2.5041 3.7409 3.1117
3.7 35.8 Q16378 PROL4_HUMAN Proline-rich protein 4 HUMAN 2 2.3323 3.5505 0.5033
2.02 8.5 P14618 KPYM_HUMAN Pyruvate kinase isozymes M1/M2 HUMAN 2 1.9549 2.1548 7.0789
2.24 23.1 P08311 CATG_HUMAN Cathepsin G HUMAN 2 0.9751 1.0086 3.9612
3.05 4.5 P13797 PLST_HUMAN Plastin-3 HUMAN 1 1.1042 1.4565 1.5078
2 16 Q6MZM9 CD040_HUMAN Uncharacterized protein C4orf40 HUMAN 1 4.4808 5.5179 3.079
2 7.9 P08246 ELNE_HUMAN Leukocyte elastase HUMAN 1 2.7032 2.8111 6.0081
2.03 17.9 P01871 IGHM_HUMAN Ig mu chain C region HUMAN 1 2.2607 2.757 1.1295
2 9.4 P06733 ENOA_HUMAN Alpha-enolase HUMAN 1 1.7561 2.2912 3.83
2 6.4 Q96DR5 SPLC2_HUMAN Short palate, lung and nasal epithelium HUMAN 1 0.8752 1.9618 0.8895
carcinoma-associated protein 2
1.94 7.3 P23229 ITA6_HUMAN Integrin alpha-6 HUMAN 1 0.5924 0.4179 0.8463
3.67 7.6 Q9NYQ6 CELR1_HUMAN HUMAN 1 0.4802 0.3591 0.6157
Table 2 (continued)
Protscore % Cov Accession Name Species Peptides CD:CS CDS:CS PCDS:
(95%) CS
Cadherin EGF LAG seven-pass G-type

receptor 1
1.89 13.5 Q15772 SPEG_HUMAN Striated muscle preferentially expressed HUMAN 1 0.2772 0.3106 0.2458
protein kinase
1.53 8.1 Q8N7U6 EFHB_HUMAN EF-hand domain-containing family HUMAN 1 0.1962 0.132 0.5399
member B
glycoproteins such as MUC5B or MUC7, typically associated to no significant contributions of each procedure for bacterial
the salivary complex trapping of other components [34], present protein identification were observed with iTRAQ analysis.
relative increments after saliva treatment (in CD and CDS).
Supporting this evidence is the presence of these glycoproteins 4.3. Peptidome analysis
and salivary proteins such as histatin 1, histatin 3, carbonic
anhydrase 6, cystatin S and cystatin SN in relative significant Similar to other bodily fluids, saliva contains several protein
amounts in PCDS. It should be noticed that iTRAQ labeling in species of low molecular weight, comprising around 40–50% of
off-gel analysis allows the identification of many low concen- the total secreted proteins [1]. We have previously analyzed the
trated proteins by removing abundant salivary proteins when salivary peptidome [13,43] and identified several fragments
comparing to 2DE [41]. Moreover, saliva sample treatment with from the most abundant protein classes. Taking in consider-
chaotropic/detergent agents plus sonication (CDS) [30] resulted ation the importance of salivary peptides for oral cavity
in the solubilization of several proteins belonging to cytoskel- homeostasis, we also introduced a procedure to extract
eton or cellular signaling (Table 3). In fact, proteins from peptides after saliva treatment with chaotropic/detergents
cytoskeleton such as keratins and tubulin were identified in based on acetone precipitation/acid extraction (Procedure G,
2DE maps from the pellet fraction. Although these proteins Fig. 1) as previously described [43,44]. The evaluation of this
were not identified in 2DE maps of whole saliva (Fig. S1A) or in procedure started by the analysis of extracts by Bis-Tris gel and
CD and CDS treatments (Fig. S1B and S1C), when these samples was compared with the traditional peptide extraction proce-
were analyzed by iTRAQ those structural proteins were found dure involving the addition of TFA to saliva, which results in the
and with a relative protein increment in CD and CDS treat- enrichment of a large amount of salivary peptides [12]. As can
ments. Supporting 2DE data, iTRAQ analysis evidenced a be depicted in Fig. 2 (lane 4), a large contribution of high
significant relative contribution of these proteins in the pellet molecular weight components such as amylase (Fig. 2, lane 4,
fraction (PCDS). In opposition, proteins such as lactoperoxidade band 2), Ig alpha-1 chain C (Fig. 2, lane 4, band 1) remains soluble
and cathepsin G showed a non-significant variation in iTRAQ after TFA precipitation, although in lower amounts comparing
analysis, either regarding sample fractions or treatment to CS saliva (Fig. 2, lane 1) based on OD analysis. Although a
procedure. band corresponding to amylase was detected in extracts
Albeit the presence of bacteria in saliva is recognized [42], the obtained from CS, CD and CDS following Procedure G, a
identification of proteins belonging to bacteria using proteomic depletion of high molecular weight components was noticeable
approaches is less clear. As observed from our data, only a few (Fig. 2, lanes 7–9). Furthermore, an OD increment of 15% was
bacterial proteins were detected. This is in-line with a recent detected in the range of 5–15 kDa for procedure G.
paper devoted to the analysis of microbiota protein composition Each extract was subsequently analyzed by LC-MS/MS
in human saliva. To address it, Rudney et al. [23] used a resulting in the identification of more than 150 different
metaproteomic approach combining three different fraction- peptides belonging to the main salivary classes (statherin,
ations yielding in the identification of peptides belonging PRPs (aPRP, bPRP1, bPRP2, bPRP3, bPRP4), histatins 1 and 3 and
mainly to Firmicutes, in particular streptococci. Furthermore, P–B peptide (SMR3B)), as presented in Table 4. Procedure G
allowed the extraction of a higher number of peptides when
comparing to the TFA treatment. The most noticeable was
Table 3 – No. of identified salivary peptides by LC-MS/MS the increment in the number of peptides belonging to bPRP1
per class and procedure. and aPRP in CD and CDS. However, distribution of identified
Peptide class TFA Saliva Saliva (CD) Saliva (CDS) peptides based on Gravy score (Fig. 8) showed a similar
distribution among all extracts.
bPRP1 35 17 54 69
bPRP2 7 53 6 17 Despite the heterogeneity observed in the number of
bPRP3 7 13 10 9 peptides in each class (Table 3), with iTRAQ labeling, 136
bPRP4 13 10 13 9 different peptides were assigned as common to all extracts
aPRP 24 35 26 44 (Table 4). Comparing the number of peptides labeled with
histatin 1 27 26 39 37 iTRAQ with all the ones identified by LC-MS/MS, a decrease in
histatin 3 17 14 9 22
their number was observed which could be attributed to the fact
statherin 27 25 60 38
SMR3B 23 29 32 21
that few of them are over dynamic range of iTRAQ being not
considered for analysis. Furthermore, many other small
5158
Table 4 – iTRAQ peptide evaluation for Procedures F and G (CS, CD and CDS).
Protscore % cov Name Peptides Sequence Modifications ΔMass Theor CDS: CD: CS:
accession (95%) m/z TFA TFA TFA
conf
15.01 59.7 P02808 Statherin 13 96 FGYGY iTRAQ8plex@N-term −0.06 910.46 2.5312 1.9883 2.4538
99 FGYGYGPY iTRAQ8plex@N-term −0.11 1227.60 2.5548 1.8928 2.4437
99 GYGPYQPVPEQPL iTRAQ8plex@N-term −0.12 1748.92 2.1736 1.722 1.8916
97 GYGYGPY iTRAQ8plex@N-term −0.01 1080.53 2.54 1.3931 2.0718
99 GYGYGPYQPVPEQPL iTRAQ8plex@N-term 0.04 1969.00 2.8275 1.5496 2.0538
99 RFGYGYGPYQPVPEQPLYPQPYQPQ iTRAQ8plex@N-term −0.18 3273.63 3.8212 1.6225 3.1573
99 YQPVPEQPL iTRAQ8plex@N-term 0.01 1374.76 3.234 2.0957 2.0602
97 YQQYTF iTRAQ8plex@N-term 0.00 1153.58 5.6727 2.5643 4.3415
97 FLRR iTRAQ8plex@N-term 0.00 895.58 2.4507 2.5918 3.5961
98 IGRF iTRAQ8plex@N-term −0.01 796.50 3.9981 1.7334 2.6506
95 QYQQYTF iTRAQ8plex@N-term; 0.13 1280.66 1.8684 2.0125 2.5049
Amidated@C-term
96 QYTF iTRAQ8plex@N-term 0.00 862.46 6.7144 3.5633 4.2836
96 RIGRF iTRAQ8plex@N-term 0.00 952.60 1.6928 1.6457 2.1725
15.84 88.8 P04280 Basic 17 96 GGRPSRPPQ iTRAQ8plex@N-term 0.00 1255.72 7.8037 4.0943 5.087
PRP1 98 GPPAQGGSK iTRAQ8plex@N-term; −0.01 1406.82 12.846 2.7775 5.792
iTRAQ8plex(K)@9
98 GPPPPGKPQ iTRAQ8plex@N-term; 0.00 1482.89 6.0115 1.8266 2.6419
iTRAQ8plex(K)@7
97 GPPPPGKPQ Formyl@N-term; 0.01 1206.68 0.9416 1.2026 1.1877
iTRAQ8plex(K)@7
95 GPPPPPGKPQ No iTRAQ8plex@N-term; 0.01 1275.74 0.985 0.5997 0.6269
iTRAQ8plex(K)@8
96 GPPPPPGKPQ iTRAQ8plex@N-term; −0.01 1579.94 7.0423 2.2092 2.9095
iTRAQ8plex(K)@8
95 GPPPPPGKPQ Acetyl@N-term; −0.10 1317.75 0.5226 0.9464 1.4376
iTRAQ8plex(K)@8
96 GPPPPPGKPQ Carbamyl@N-term; 0.05 1318.74 11.3197 2.8913 6.9305
iTRAQ8plex(K)@8
97 GPPPQGDK iTRAQ8plex@N-term; 0.00 1403.81 12.2455 3.4189 5.0803
iTRAQ8plex(K)@8
99 GPPPQGGNQPQ iTRAQ8plex@N-term 0.00 1380.72 5.9458 2.9747 3.0907
99 GPPQQEGNNPQ iTRAQ8plex@N-term 0.00 1469.73 3.117 1.603 1.6115
98 GPPQQGGNRPQ iTRAQ8plex@N-term 0.00 1439.77 5.851 2.5332 2.5055
99 GPPRPPQGGRPSRPPQ iTRAQ8plex@N-term −0.01 1985.11 6.3212 2.1287 2.9221
98 PQQPQAPPAGQPQGPPRPPQGGRPSRPPQ iTRAQ8plex@N-term; −0.01 3310.75 9.6551 0.8354 1.0717
Deamidated(Q)@29
97 SPPGKPQ iTRAQ8plex@N-term; −0.01 1318.79 3.436 1.0214 2.2402
iTRAQ8plex(K)@5
99 SPPGKPQGPPPQGGNQPQ iTRAQ8plex@N-term; −0.02 2376.29 20.0082 4.5356 6.1751
iTRAQ8plex(K)@5
99 SPPGKPQGPPPQGGNQPQ No iTRAQ8plex@N-term; 0.00 2072.08 3.0186 1.9832 1.9713
iTRAQ8plex(K)@5
20.5 92.1 P02812 Basic 23 99 GPPPQGDNK iTRAQ8plex@N-term; −0.02 1517.85 18.2385 3.7761 6.157
PRP2 iTRAQ8plex(K)@9
99 GPPPQGGSK iTRAQ8plex@N-term; −0.01 1432.84 15.4593 3.503 4.9798
iTRAQ8plex(K)@9
96 GGRPSRPPQ iTRAQ8plex@N-term 0.00 1255.72 4.647 2.2432 2.8817
iTRAQ8plex(K)@7
iTRAQ8plex(K)@7
96 GPPPPGKPQGPPPQGDN iTRAQ8plex@N-term; 0.02 2259.23 12.2629 3.4656 3.4086
iTRAQ8plex(K)@7;
Methyl(Q)@14
iTRAQ8plex(K)@7;
Oxidation(P)@13;
J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 51 4 0 –5 16 5
Deamidated(Q)@14
iTRAQ8plex(K)@8
iTRAQ8plex(K)@8
iTRAQ8plex(K)@8
iTRAQ8plex(K)@8
iTRAQ8plex(K)@8
98 GPPPQGDNK iTRAQ8plex@N-term; 0.00 1517.85 8.4371 2.441 3.4611
iTRAQ8plex(K)@9
99 GPPPQGDNKSRSSR No iTRAQ8plex@N-term; 0.00 1866.91 2.6731 1.0182 0.565
iTRAQ8plex(K)@9;
Phospho(S)@13
iTRAQ8plex(K)@9
97 IAGNPQGAPPQGGN iTRAQ8plex@N-term; −0.01 1743.88 6.6196 8.2348 0.6849
Hex(N)@14
98 PQQPQAPPAGQPQGPPRPP iTRAQ8plex@N-term; −0.14 3310.75 9.6551 0.8354 1.0717
QGGRPSRPPQ Deamidated(Q)@29
iTRAQ8plex(K)@5
iTRAQ8plex(K)@5
99 SPPGKPQGPPPQGGNQPQ No iTRAQ8plex@N-term; −0.01 2072.08 10.0395 5.2778 6.3392
iTRAQ8plex(K)@5
20.5 92.1 P02812 Basic 23 99 GPPPQGDNK iTRAQ8plex@N-term; −0.02 1517.85 18.2385 3.7761 6.157
PRP2 iTRAQ8plex(K)@9
5159
5160
Table 4 (continued)
conf

iTRAQ8plex(K)@9
96 GGRPSRPPQ iTRAQ8plex@N-term 0.00 1255.72 4.647 2.2432 2.8817
iTRAQ8plex(K)@7
iTRAQ8plex(K)@7
iTRAQ8plex(K)@7;
Methyl(Q)@14
iTRAQ8plex(K)@7;
Oxidation(P)@13;
Deamidated(Q)@14
iTRAQ8plex(K)@8
iTRAQ8plex(K)@8
iTRAQ8plex(K)@8
iTRAQ8plex(K)@8
iTRAQ8plex(K)@8
98 GPPPQGDNK iTRAQ8plex@N-term; 0.00 1517.85 8.4371 2.441 3.4611
iTRAQ8plex(K)@9
99 GPPPQGDNKSRSSR No iTRAQ8plex@N-term; 0.00 1866.91 2.6731 1.0182 0.565
iTRAQ8plex(K)@9;
Phospho(S)@13
iTRAQ8plex(K)@9
97 IAGNPQGAPPQGGN iTRAQ8plex@N-term; −0.01 1743.88 6.6196 8.2348 0.6849
Hex(N)@14
98 PQQPQAPPAGQPQGPPRPPQ iTRAQ8plex@N-term; −0.14 3310.75 9.6551 0.8354 1.0717
GGRPSRPPQ Deamidated(Q)@29
iTRAQ8plex(K)@5
iTRAQ8plex(K)@5
99 SPPGKPQGPPPQGGNQPQ No iTRAQ8plex@N-term; −0.01 2072.08 10.0395 5.2778 6.3392
iTRAQ8plex(K)@5
10.07 33.7 Q04118 Basic 12 99 GGRPHRPPQGQPPQ iTRAQ8plex@N-term −0.01 1812.99 10.7251 3.4056 4.0274
PRP3 99 GPPPPPQGGRPHRPPQGQPPQ iTRAQ8plex@N-term −0.02 2483.33 14.5012 5.6332 5.3149
99 QSLNEDVSQEESPSVISGKPEGR Gln->pyro-Glu@N-term; 0.00 2759.36 6.1051 3.635 5.7312
iTRAQ8plex(K)@19
96 RPHRPPQGQPPQ iTRAQ8plex@N-term −0.01 1698.95 13.1281 3.0983 3.7095
95 GGRPHRPPQGQPPQ iTRAQ8plex@N-term −0.01 1812.99 2.1215 0.8669 1.4152
97 GKPEGR iTRAQ8plex@N-term; −0.01 1251.76 19.8941 5.0111 7.0254
iTRAQ8plex(K)@2
96 GPPPHPGKPQ iTRAQ8plex@N-term; −0.01 1619.95 8.811 1.8202 4.1798
iTRAQ8plex(K)@8
96 GRPHRPPQGQPPQ iTRAQ8plex@N-term −0.01 1755.97 7.2527 1.714 2.3976
96 PPPPQGGRPHRPP iTRAQ8plex@N-term 1.83 1693.96 0.729 0.5531 0.3584
98 QSLNEDVSQEESPSVISGKP Gln->pyro-Glu@N-term; 0.00 2455.15 1.0261 0.6851 0.7083
Cation:K(E)@11;
iTRAQ8plex(K)@19
−0.11
99 QSLNEDVSQEESPSVISGKPEGR Gln->pyro-Glu@N-term; 2741.35 8.1343 3.2673 6.2617
Dehydrated(S)@8;
iTRAQ8plex(K)@19
99 QSLNEDVSQEESPSVISGKPEGR Gln->pyro-Glu@N-term; 0.04 2839.33 6.3063 3.4361 6.4223
Phospho(S)@8;
iTRAQ8plex(K)@19
9.88 55.2 P10163 Basic 9 99 GGRPPRPAQGQQPPQ iTRAQ8plex@N-term 0.00 1875.03 8.0664 3.392 3.827
PRP4 99 GPPPPPQGGRPPRPAQGQQPPQ iTRAQ8plex@N-term −0.22 2545.37 8.0197 3.8326 4.6859
99 LISGKPEGR iTRAQ8plex@N-term; −0.02 1564.96 9.2707 0.8772 1.1029
iTRAQ8plex(K)@5
97 RPQGGNQPQR iTRAQ8plex@N-term 0.00 1441.79 7.6733 3.9161 5.7411
97 GKPEGR iTRAQ8plex@N-term; −0.01 1251.76 19.8941 5.0111 7.0254
iTRAQ8plex(K)@2
97 GPPPHPGKPE iTRAQ8plex@N-term; −0.01 1619.95 8.811 1.8202 4.1798
iTRAQ8plex(K)@8;
Amidated@C-term
iTRAQ8plex(K)@7
98 GPPPPGKPQ iTRAQ8plex@N-term; −0.01 1482.89 7.8598 2.1899 4.147
iTRAQ8plex(K)@7
iTRAQ8plex(K)@5
31.09 67.1 P02814 SMR3B 22 99 APPQPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term −0.09 2627.41 8.8238 3.0757 3.5905
97 FVPPPPPPPYGPGR iTRAQ8plex@N-term −0.02 1778.99 2.2737 1.3848 1.6401
97 GFVPPPPPPPYGPGR iTRAQ8plex@N-term −0.15 1836.01 2.7853 1.2446 1.4193
95 GIFPPPPPQP iTRAQ8plex@N-term −0.10 1350.77 1.3742 0.8587 0.5474
99 GPGFVPPPPPPPYGPGR iTRAQ8plex@N-term −0.02 1990.09 8.1009 2.7944 3.183
99 GPGIFPPPPPQP iTRAQ8plex@N-term −0.11 1504.85 2.6169 2.4418 2.5038
80 GPYPPGPL iTRAQ8plex@N-term 0.00 1101.62 5.7772 3.1118 3.5181
99 GPYPPGPLAPPQPF iTRAQ8plex@N-term −0.19 1738.95 1.156 0.8315 0.8515
99 GPYPPGPLAPPQPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term 0.00 3405.81 8.1062 4.3326 5.8779
95 GRIPPPPPAPY iTRAQ8plex@N-term −0.01 1465.85 2.0128 0.6659 0.8756
99 GRIPPPPPAPY iTRAQ8plex@N-term 0.00 1465.85 4.8553 1.461 1.7307
5161
5162
Table 4 (continued)
conf
99 QPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term −0.13 2362.26 1.866 0.9118 1.1063

97 RGPYPPGPLAPPQPF iTRAQ8plex@N-term −0.18 1895.05 2.9055 0.9968 1.2553
99 RGPYPPGPLAPPQPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term 0.01 3561.91 5.7167 2.5686 4.2356
99 YPPGPLAPPQPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term −0.08 3251.73 13.704 4.9346 8.7672
93 APPQPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term −1.10 2627.41 6.9453 2.6073 2.8396
99 GPLAPPQPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term −0.36 2894.57 10.7916 5.8341 5.7283
99 GPYPPGPLAPPQPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term −0.40 3405.81 9.6094 7.0175 9.648
99 LAPPQPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term −0.33 2740.49 2.7422 1.5213 1.4156
99 PGPLAPPQPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term −1.30 2991.62 0.3593 0.4315 0.343
99 RGPYPPGPLAPPQPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term −0.35 3561.91 5.6668 1.8944 2.896
99 YPPGPLAPPQPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term −0.39 3251.73 8.9695 3.1086 5.6263
64.41 95.8 P02810 aPRP 40 99 DGGDSEQFIDEER iTRAQ8plex@N-term; 0.00 1880.79 3.3568 1.991
Phospho(S)@5
99 GGDSEQFIDEER iTRAQ8plex@N-term −0.01 1685.79 3.5056 1.6872
99 GGDSEQFIDEER iTRAQ8plex@N-term; −0.01 1765.76 5.6359 2.4177
Phospho(S)@4
99 GGHPPPPQGRPQ iTRAQ8plex@N-term −0.01 1528.83 4.2839 2.1403
97 GGRPQGPPQGQSPQ iTRAQ8plex@N-term −0.01 1694.89 13.6596 5.3195
97 GPPPPPPGKPQ iTRAQ8plex@N-term; −0.01 1676.99 2.7131 0.784
iTRAQ8plex(K)@9
96 GPPPQGGRPQ iTRAQ8plex@N-term 0.00 1294.72 5.9031 3.5311
99 GPPPQGGRPQGPPQGQSPQ iTRAQ8plex@N-term 0.00 2171.13 7.6799 3.5452
98 GPPQGQSPQ iTRAQ8plex@N-term 0.00 1199.63 6.6635 2.8578
99 GPPQQGGHP iTRAQ8plex@N-term 0.00 1178.62 2.8621 2.1231
99 GPPQQGGHPPPPQGR iTRAQ8plex@N-term −0.02 1810.96 14.2761 3.2827
99 GPPQQGGHPPPPQGRPQ iTRAQ8plex@N-term −0.01 2036.07 5.6124 1.5873
99 GPPQQGGHPRPP iTRAQ8plex@N-term 0.00 1528.83 4.5761 2.6965
99 GPPQQGGHPRPPR iTRAQ8plex@N-term 0.00 1684.93 11.8249 3.7167
99 GPPQQGGHQQ iTRAQ8plex@N-term 0.00 1337.69 5.6506 2.968
99 GRPQGPPQQGGHQ iTRAQ8plex@N-term −0.01 1647.86 9.2082 3.658
99 GRPQGPPQQGGHQQ iTRAQ8plex@N-term 0.01 1775.92 4.6827 3.5427
99 GRPQGPPQQGGHQQGPPPP iTRAQ8plex@N-term; −0.02 3129.69 17.6073 3.7939
PPGKPQ iTRAQ8plex(K)@23
99 GRPQGPPQQGGHQQGPPPPP iTRAQ8plex@N-term; −0.02 4101.19 35.5568 7.5486
PGKPQGPPP iTRAQ8plex(K)@23
QGGRPQ
99 PQGPPQQGGHPRPPR iTRAQ8plex@N-term −0.01 1910.04 4.0669 1.0998
99 RGRPQGPPQQGGHQQ iTRAQ8plex@N-term −0.02 1932.02 9.5097 1.8093
95 RPQGPPQQGGHQQ iTRAQ8plex@N-term −0.01 1718.90 15.0219 1.8392
99 RPQGPPQQGGHQQ iTRAQ8plex@N-term 0.00 1718.90 9.6835 1.4374
96 DSEQFIDEER iTRAQ8plex@N-term; 0.00 1667.71 3.6536 1.3368
Oxidation(D)@1; Phospho(S)@2
97 FIDEER iTRAQ8plex@N-term 0.00 1112.59 6.4584 1.7087
98 GPPPPPPGKPQ iTRAQ8plex@N-term; 0.00 1676.99 10.6486 2.8207
iTRAQ8plex(K)@9
99 GPPPPPPGKPQGPPPQGGRPQ No iTRAQ8plex@N-term; 0.00 3220.69 9.4287 4.2216
GPPQGQSPQ iTRAQ8plex(K)@9
99 GPPPPPPGKPQGPPPQGGRPQ No iTRAQ8plex@N-term; −0.06 3220.69 10.9522 5.4427
GPPQGQSPQ iTRAQ8plex(K)@9
98 GPPQGQSPQ iTRAQ8plex@N-term 0.00 1199.63 1.7041 0.7278
95 GPPQGQSPQ iTRAQ8plex@N-term; 0.01 1198.65 8.9578 4.1687
Amidated@C-term
98 GRPQGPPQGQSP iTRAQ8plex@N-term; −0.19 1830.01 6.0046 3.1339
iTRAQ8plex(S)@11;
Oxidation(P)@12
97 GRPQGPPQQ iTRAQ8plex@N-term 0.00 1268.70 5.3657 3.385
97 GRPQGPPQQGGH iTRAQ8plex@N-term 0.00 1519.80 3.6905 2.6306
99 GRPQGPPQQGGHPRPPR iTRAQ8plex@N-term −0.32 2123.16 13.6373 4.2703
97 GRPQGPPQQGGHQQ iTRAQ8plex@N-term −0.01 1775.92 6.8084 3.2573
99 DGGDSEQFIDEER iTRAQ8plex@N-term; 0.00 1880.79 3.3568 1.991
Phospho(S)@5
99 GGDSEQFIDEER iTRAQ8plex@N-term −0.01 1685.79 3.5056 1.6872
99 GGDSEQFIDEER iTRAQ8plex@N-term; −0.01 1765.76 5.6359 2.4177
Phospho(S)@4
5163
Supplementary data related to this article can be found

online at http://dx.doi.org/10.1016/j.jprot.2012.05.045.
Acknowledgment
This work was supported by Portuguese Foundation for

Science and Technology (FCT) [grant numbers PEst-C/QUI/
UI0062/2011, PTDC/QUI/72683/2006].
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J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 51 4 0 –51 6 5

Available online at www.sciencedirect.com

Toward a standardized saliva proteome analysis methodology

AR TIC LE I N FO ABS TR ACT

1. Introduction introduced in saliva treatment for proteomic analysis (reviewed

2.2. Whole saliva collection 3.3.2. Procedure E

3.7.1. In-gel 3.9.2. Reverse-phase separation

Fig. 1 – Schematization of sample preparation using different methodological approaches.

To evaluate the potential losses related with whole saliva

4.1. Gel-based electrophoresis

Among the widely methodological approaches employed in

Table 1 – Protein identified in each 2D-BisTris-PAGE spot from Procedure E.

1 Keratin, type I cytoskeletal 13 K1C13_HUMAN 49,557 4.91 1 78 100 78 100

(continued on next page)

58 Keratin, type II cytoskeletal 6B K2C6B_HUMAN 60,030 8.09 2 105 100 67 100

(continued on next page)

217 Peroxiredoxin-6 PRDX6_HUMAN 25,019 6 2 105 100 57 100

(continued on next page)

(continued on next page)

298 Histatin-1 HIS1_HUMAN 6958 9.11 1 78 100 78 100

342 Lysozyme C LYSC_HUMAN 16,526 9.38 1 59 100 59 100

Fig. 6 – Venn diagram representing the overlap of proteins

Table 2 – iTRAQ evaluation of identified proteins in Procedures A, B, C and E.

Cadherin EGF LAG seven-pass G-type

99 GPPPQGGSK iTRAQ8plex@N-term; −0.01 1432.84 15.4593 3.503 4.9798

99 QPFGPGFVPPPPPPPYGPGR iTRAQ8plex@N-term −0.13 2362.26 1.866 0.9118 1.1063

Supplementary data related to this article can be found

This work was supported by Portuguese Foundation for

Fig. 8 – Box plot of Gravy score of identified peptides in

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