Toward a standardized saliva proteome analysis methodology

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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 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 b

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

Whole saliva represents a complex mixture of different contributions such as major and minor salivary glands, crevicular fluid, serum, epithelial cells, bacteria and food debris. Besides water and salt, other components including proteins, peptides, hormones, lipids and sugars are part of the composition of whole saliva. Among the predominant proteins and peptides are amylase, mucins (MUC5B and MUC7), carbonic anhydrase, cystatins, proline-rich proteins (PRPs — acid and basic), histatins (1 and 3) and statherin [1]. This large array of peptides and proteins covers different molecular weight ranges, which complexity is further increased by the presence of posttranslational modifications (PTMs) and interactions with high molecular glycoproteins, turning difficult the analytical use of this easily accessible bodily fluid for clinical purposes [2–6]. To tentatively reduce sample complexity and to remove bacteria and cellular debris, a centrifugation step is generally

introduced in saliva treatment for proteomic analysis (reviewed in [7]). Nonetheless, no standardized procedure for saliva handling has yet been established albeit the technological improvements achieved in proteomics applied to the characterization of saliva composition, which allowed the identification of more than 3000 different species in this biological fluid [8–11]. According to Chevalier et al. [7] the best results in the characterization of saliva are obtained by gel electrophoresis (SDS-PAGE and 2DE) when sample collection is performed in ice, anti-protease added and then sample is centrifuged and stored between −20 °C to −80 °C. Regarding salivary peptide analysis, Castagnola and colleagues [4,12] perform, by routine, saliva precipitation with trifluoroacetic acid for peptide extraction while other groups prefer cut-off filter devices [13–15]. Through these efforts, issues respecting saliva pre-treatment, in particular the centrifugation step, have not been considered in most 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: [email protected] (R. Vitorino). 1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2012.05.045

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essential step forward is the analysis of this pre-treatment influence in saliva proteome studies. Indeed, for standardization of saliva handling, the following issues should be considered (i) samples should be representative of saliva, salivary aggregates, cell and bacteria; (ii) be suitable for analysis by different methods and platforms; (iii) be compatible with protein and peptide extraction and allow its maximum solubilization; and (iv) enable comparison of data sets under different pathophysiological conditions. Taken these issues in consideration, in the present study, we report the contribution of saliva clearance performed in the pre-treatment and its extensive characterization by different platforms of a representative “normal” saliva sample obtained from healthy individuals.

2.

Material and methods

2.1.

Reagents

HPLC-grade acetonitrile (ACN, Riedel, Seelze, Germany), Milli-Q grade water were used. General chemical reagents such as triethylammonium bicarbonate (TEAB), trifluoroacetic acid (TFA), protease inhibitor cocktail, formic acid, α-cyano-4hydroxycinnamic acid (α-CHCA), urea and CHAPS, were purchased from Sigma (Karlsruhe, Germany). Sequencing grade modified trypsin (bovine) was from ABSciex (ABSciex, USA).

2.2.

Whole saliva collection

For this study a pool of whole saliva was constituted from the saliva collected from three healthy subjects (3 males aged 23 to 32) showing no evidence of oral pathologies or inflammatory processes. Unstimulated whole saliva (WS) was collected at 10:00 a.m. by direct draining into a saliva collection tube (kept on ice) from all subjects, who had previously refrained from eating and drinking for at least 2 h. Immediately after whole saliva collection, 10 μL of PMSF 0.1 M, 1 μL of pepstatin 1 mM and 20 μL of anti-protease cocktail (Sigma P2714) including aprotinin, E-64, EDTA, AEBSF and leupeptin, were added to each mL of saliva. Saliva sample was divided in three different aliquots for different treatments.

3.

Sample preparation

3.1.

Total salivary protein extraction

3.1.1.

Procedure A (CS)

Whole saliva was centrifuged at 12,000 ×g, 30 min, 4 °C. The supernatant and pellet were separated and saved for analysis.

3.1.3.

Procedure C (CDS)

A similar approach to Procedure B was performed but in this case incubation at RT was replaced by 2 cycles of 5 s each of sonication. The pellet was discarded and the supernatant saved for analysis.

3.2.

Pellet protein extraction

Pellets obtained from 1 mL of whole saliva centrifugation (from Procedure A) were treated with 200 μL of solubilization buffer (7 M urea, 2 M thiourea, 1% (w/v) CHAPS, 1% (w/v) Triton X-100, 1% (v/v) ampholytes (3–10) and 1 mM TCEP) incubated for 10 min at room temperature (RT) under agitation 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 similar procedure to PCD with the addition of 2 sonication cycles of 5 s each.

3.3.

Peptide extraction

3.3.1.

Procedure D

Salivary peptides were extracted according to Castagnola and coll. [12]. Thus, a solution of 0.2% of TFA was mixed in the proportion of 1:1 with whole saliva, incubated in ice for 5 min and centrifuged for 5 min at 8000 × g and 4 °C. The supernatant containing enriched peptides was saved for analysis.

3.3.2.

Procedure E

Salivary peptides were extracted from each supernatant obtained with Procedures A, B and C. For this purpose, ice cold acetone was added drop by drop to 200 μL of each supernatant in the proportion of 9:1. Following this, the solution was mixed under constant agitation in ice for 1 h and then centrifuged at 19,000×g for 15 min at 4 °C. The supernatant (S1) was collected and saved. Pellet was resuspended in 200 μL of ACN/12 mM HCl, incubated under agitation for 1 h in ice and centrifuged again at 19,000×g for 15 min at 4 °C. The obtained supernatant was mixed with the first (S1) and lyophilized in a SpeedVac (Thermo).

3.4.

Quantification of protein and peptides

Peptides were quantified using the 2,4,6-trinitrobenzenesulfonic acid assay as previously described [16]. A standard curve was generated with glycine solution (20, 35, 50, 70, 90 μM) and the absorbance at 335 nm by 2,4,6-trinitrobenzenesulfonic acid (TNBS). Protein quantification was performed using RC DC Protein Assay kit (BioRad lab, Hercules, USA). A bovine serum albumin was used to generate the standard curve and the absorbance at 750 nm was measured.

3.5. 3.1.2.

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One-dimensional gel electrophoresis

Procedure B (CD)

Whole saliva was mixed with solubilization buffer (7 M urea, 2 M thiourea, 1% (w/v) CHAPS, 1% (w/v) Triton X-100, 1% (v/v) ampholytes (3–10) and 1 mM TCEP) in the proportion of 2:1. The mixture was then incubated for 10 min at room temperature (RT) under agitation being centrifuged at 12,000 ×g for 30 min at 4 °C. The pellet was discarded and the supernatant saved for analysis.

Thirty micrograms of protein was incubated with LDS sample buffer (0.4 mM EDTA, 8% (w/v) lauryl dodecyl sulfate (SDS), 50 mM Tris–HCl (pH 6.8), 4% glycerol, 0.075% Serva Blue G250 (w/v) and 0.025% Phenol Red (w/v) for 10 min at RT. Samples were loaded onto pre-cast 12% gels Bis-Tris (Novex, InVitrogen, Carlsbad, CA) and electrophoresis was carried out at a constant voltage of 150 V with running buffer (50 mM 2-(N-morpholino)

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ethane sulfonic acid (MES), 50 mM Tris base and 0.1% (w/v) SDS). Gels were stained with colloidal Coomassie [17,18].

3.6.

Two-dimensional gel electrophoresis

The first dimension was carried out using our previous conditions [17,18]. Briefly, 150 μg of protein was applied onto IPG strips (13 cm pH 3-10NL), containing immobilines pHs 3–10, thiourea, CHAPS and urea. After isoelectric focusing, the strip was applied on top of a 12% Bis-Tris home-made, as previously described [19], at constant voltage of 150 V and MES as running buffer. Spots were detected by colloidal Coomassie Brilliant blue G-250 for tryptic digestion and protein identification.

conventional reverse-phase LC. In the case of protein expression (from Procedures A, B and C), the obtained tryptic peptides, after iTRAQ labeling, were separated by a multidimensional approach based on a first dimension with high pH reverse phase (as previously described [20]) and a second dimension with the acidic reverse-phase system.

3.9.1.

High pH reverse-phase separation

3.7.

Digestion of proteins

Sample loading was performed at 200 μL/min with buffers (A) 72 mM TEA, 52 mM acetic acid in H2O, pH 10 and (B) 72 mM TEA, 52 mM acetic acid in ACN, pH 10 (98% A: 2% B). After 5 min of sample loading and washing, peptide fractionation was performed with linear gradient to 50% B over 35 min followed by a 100% B step. Sixteen fractions were collected, evaporated, and resuspended in 2% ACN and 0.1% TFA.

3.7.1.

In-gel

3.9.2.

Annotated spots or bands were manually excised and digested in-gel with trypsin. Briefly, gel pieces were washed three times with 25 mM ammonium bicarbonate/50% acetonitrile and further dried in a Speed-Vac. Then, 10 μL of 25 μg/mL modified bovine trypsin (ABSciex, USA) in 25 mM ammonium bicarbonate was added to the dried gel pieces and the samples were incubated overnight at 37 °C. Extraction of tryptic peptides was performed by three times addition of 10% formic acid/50% acetonitrile followed by lyophilization in a Speed-Vac. Tryptic peptides were resuspended in acetonitrile/formic acid solution and mixed (1:1) with a matrix consisting of α-cyano-4hydroxycinnamic acid. Aliquots of samples were spotted onto MALDI sample target plates.

3.7.2.

In-solution

An in-solution digestion was performed for iTRAQ labeling. Briefly, 100 μg of protein was used for digestion which was performed according to the protocol provided by the manufacturer (Applied Biosystems, Foster City, CA). Briefly, samples were mixed with triethyl ammonium bicarbonate buffer (TEAB) (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 5 mM tris(2-carboxyethyl) phosphine (TCEP) for 1 h at 37 °C and alkylated with 10 mM S-methyl methanethiosulfonate (MMTS) for 10 min at RT. Two micrograms of trypsin was added to each sample and the digestion was performed for 18 h at 37 °C. Samples were dried in a Speed-Vac.

3.8.

Labeling the protein digests with iTRAQ reagents

Digested samples and extracted peptides were labeled with the iTRAQ reagents (8-plex) following the protocol provided by the manufacturer (Applied Biosystems, Foster City, CA). In brief, peptides were reconstituted in 70% ethanol/30% TEAB 500 mM, added to each label and carried out for 2 h at room temperature. The reaction was stopped by adding water and the labeled digests corresponding to each of the four 8-plex experiments were combined and dried using Speed-Vac.

3.9.

Peptide separation

Salivary endogenous peptides obtained using Procedures D and E (with and without iTRAQ labeling) were separated by

Reverse-phase separation

Collected fractions and peptide fractions were separated as previously described [13]. Briefly, peptides loaded onto a C18 pre-column (5 μm particle size, 5 mm, from Dionex) connected to an RP column PepMap100 C18 (150 mm × 75 μm I.D., 3 μm particle size). The flow rate was set at 300 nL/min. The mobile phases A and B were 2% ACN 0.1% TFA in water and 95% ACN, 0.045% TFA, respectively. The gradient started at 10 min and ramped to 60% B till 50 min and 100% B at 55 min and retained at 100% B till 65 min. The column was equilibrated with solvent A for 20 min before the next sample was injected. The separation was monitored at 214 nm using a UV detector (Dionex/LC Packings, Sunnyvale, CA) equipped with a 3 nL flow cell. Using the micro-collector Probot (Dionex/ LC Packings) and, after a lag time of 5 min, peptides eluting from the capillary column that were mixed with a continuous flow of α-CHCA matrix solution (270 nL/min, 2 mg/mL in 70% ACN/0.3% TFA and internal standard Glu-Fib at 15 fmol) were directly deposited onto the LC-MALDI plates at 12 s intervals for each spot (150 nL/fraction). For every separation run, 208 fractions in total were collected.

3.10.

Gel-based ID

Peptide mass spectra were obtained on a MALDI-TOF/TOF mass spectrometer (4800 Proteomics Analyzer, Applied Biosystems, Foster City, CA, USA) in the positive ion reflector mode. Spectra were obtained in the mass range between 700 and 4500 Da with ca. 1500 laser shots. For each sample spot, a data dependent acquisition method was created to select the six most intense peaks, excluding those from the matrix, trypsin autolysis, or acrylamide peaks, for subsequent MS/MS data acquisition. Trypsin autolysis peaks were used for internal calibration of the mass spectra, allowing a routine mass accuracy of better than 20 ppm. Spectra were processed and analyzed by the Global Protein Server Workstation (Applied Biosystems, Foster City, CA, USA), which uses internal Mascot (v2.1.1. Matrix Science Ltd, UK) software for searching the peptide mass fingerprints and MS/MS data. Searches were performed against the SwissProt (release date 010111) under all taxonomic categories and the following parameters: (i) two missed cleavages by trypsin; (ii) mass tolerance of precursor ions 25 ppm and product ions 0.3 Da; and (iii) oxidation of methionine as variable modification.

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3.11.

LC-based ID and quantification

Acquisition parameters were similar to Gel-based ID using Glu-Fib for internal calibration. The spectra were processed and analyzed by the ProteinPilot software (v4.0 AB Sciex, USA), which uses paragon algorithm for protein/peptide identification based on MS/MS data against the SwissProt protein database (release date 01012011, all taxonomic categories). Default search parameters were used: specifying trypsin as the digestion enzyme, fixed modification of Methylthio on Cysteine residue and iTRAQ 8Plex, biological modification with emphasis on phosphorylation and urea denaturation as the variable modification setting. Mass tolerances for precursor and fragments were default values for ProteinPilot®. Cut-off score value for accepting protein identification for ProteinPilot® was a ProteoScore of 1.3 (95% confidence). Data was normalized for loading error by bias correction, which is an algorithm in ProteinPilot that corrects for unequal mixing when combining the labeled samples of one experiment. It does so by calculating the median protein ratio for all proteins reported in each sample, adjusted to unity and assigning an autobias factor to it. Nevertheless, the quantification results were reviewed manually for all proteins found to be differentially expressed (iTRAQ ratio > 1.3 or < 0.7 according to [21]).

3.12.

Western blotting of salivary amylase

Briefly, salivary proteins were separated by SDS-PAGE (12% in Laemmli conditions) and electroblotted onto nitrocellulose membranes (Amersham, Pharmacia Biotech, Buckinghamshire, UK). The immunoblots were probed with 1:1000 dilution of monoclonal anti-amylase (A8273, Sigma, St. Louis, USA) and with 1:1000 dilution of the secondary antibody (antimouse IgG peroxidase conjugate, Amersham Pharmacia Biotech, Buckinghamshire, UK). The bands were visualized

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by treating the immunoblots with ECL chemiluminescence reagents (Amersham, Pharmacia Biotech, Buckinghamshire, UK), according to the supplier's instructions, followed by exposure to X-ray films (Sigma, Kodak Biomax Light Film, St. Louis, USA).

4.

Results and discussion

Currently, there are numerous reports characterizing saliva composition in different pathological/physiological status. The majority of these studies start with a clearance step to remove the large aggregates (food and cell debris), producing a clear extract for subsequent analysis, particularly relevant when unstimulated saliva is analyzed. Although easier collected in higher volumes, stimulated saliva is predominantly derived from the parotid gland and consists mainly of water whereas the major component of unstimulated saliva is derived from the submandibular gland and is mainly composed of mucins and cystatins, which largely contributes to the aggregation of salivary components [22]. This pre-treatment has been empirically accepted as a general procedure and widely adopted. However, its effects on proteome data were not tested despite the different contents of bacteria, glycoprotein or squamous cells present in distinct salivary samples [23–25]. Consequently, different amounts of aggregates are expected to be lost during the initial clearance step and with this, several other salivary components, with the subsequent effects in the whole saliva proteome profile. Thus, this study aimed the evaluation of alterations in the proteome composition of salivary samples caused by this clearance treatment, without taking in consideration the inter-individual variability reported in previous studies [13,26]. To this end, several methodological approaches were tested with pools of saliva samples and are summarized in Fig. 1.

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

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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 are separated in terms of their molecular weight. In spite the excellent performances given for proteins above 15 kDa (through different acrylamide gel pore sizes), SDS-PAGE lacks resolution to efficiently separate small proteins (<20 kDa). By using Bis-Tris as the trailing ion, the system allows high moving boundary speeds at reasonable pH values, which is therefore 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 a first approach, all extracts of saliva samples were loaded in 12% Bis-Tris gels. As expected, gel image analysis (Fig. 2) evidenced several bands covering a broad range of molecular weight in extracts corresponding to saliva (Procedure A-CS) (lane 2), saliva treated with urea (Procedure B-CD) (lane 3), treated with urea/sonication (Procedure C-CDS) (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 approximately 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 components 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), specifically 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 31, 205 ± 8% for CD and 232 ± 7% for CDS). Further western blot analysis of salivary amylase in samples prepared with Procedures B and C (Fig. 3) showed the same tendency observed

Fig. 2 – Representative Bis-Tris-PAGE gel. M refers to the molecular weight marker and remaining lanes to different saliva procedures: lane 1 — Procedure A, lane 2 — Procedure B, lane 3 — Procedure C, lane 4 — Procedure F, lane 5 — Procedure D, lane 6 — Procedure E, lane 7 — Procedure G from A, lane 8 — Procedure G from B and lane 9 — Procedure G from C (with correspondence to Fig. 1).

in Bis-Tris gel (38% vs 48% for CD-to-CS and CDS-to-CS, respectively). Those identified proteins have been previously identified as forming salivary heterotypic complexes as showed by Iontcheva et al. [34]. Faint bands corresponding to the GP340 (Fig. 2, bands 32 and 33) and MUC5B (Fig. 2, bands 32 and 33) were still observed after saliva treatment in CD (Procedure B) and CDS samples (Procedure C) and in the pellet

Fig. 3 – Western blot analysis of salivary amylase for saliva treatments (Procedures A–E). A representative immunoblot image is presented above the histogram. (*p < 0.05 vs CS; ***p < 0.001 vs CS).

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Table 1 – Protein identified in each 2D-BisTris-PAGE spot from Procedure E. Spot no. 1 2 5 11 12 13 14 18 21 24 25 25 26 27 28 29 30 31 31 32 33 34 37 38 39 40 41 42 43 45 46 47 48 49 49 50 51 51 51 51 52 52 53 54 54 55 55 55 55 55 55 56 56 56 57 57 57 58

Protein name

Keratin, type I cytoskeletal 13 Keratin, type II cytoskeletal 6A Keratin, type II cytoskeletal 6B Polymeric immunoglobulin receptor Alpha-amylase 2B Alpha-amylase 2B Polymeric immunoglobulin receptor Keratin, type II cytoskeletal 4 Polymeric immunoglobulin receptor Keratin, type II cytoskeletal 4 Polymeric immunoglobulin receptor Keratin, type II cytoskeletal 4 Polymeric immunoglobulin receptor Polymeric immunoglobulin receptor Polymeric immunoglobulin receptor Polymeric immunoglobulin receptor Polymeric immunoglobulin receptor Polymeric immunoglobulin receptor Keratin, type I cytoskeletal 16 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 4 Keratin, type I cytoskeletal 15 Keratin, type I cytoskeletal 15 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 15 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 15 Keratin, type II cytoskeletal 4 Ig alpha-2 chain C region Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 4 Protein-glutamine gamma-glutamyltransferase E Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 73 Protein-glutamine gamma-glutamyltransferase E Protein-glutamine gamma-glutamyltransferase E Keratin, type II cytoskeletal 4 Protein-glutamine gamma-glutamyltransferase E Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 1 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 3 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 73 Keratin, type II cytoskeletal 3 Keratin, type II cytoskeletal 6B Ig alpha-2 chain C region Keratin, type II cytoskeletal 4 Cornulin Cornulin Ig alpha-2 chain C region Keratin, type II cytoskeletal 6B Keratin, type II cytoskeletal 4

Accession name

Protein Protein MW PI

Peptide count

Total Total ion Best Best ion ion score C.I. ion score C.I. score % score %

K1C13_HUMAN K2C6A_HUMAN K2C6B_HUMAN PIGR_HUMAN AMY2B_HUMAN AMY2B_HUMAN PIGR_HUMAN K2C4_HUMAN PIGR_HUMAN K2C4_HUMAN PIGR_HUMAN K2C4_HUMAN PIGR_HUMAN PIGR_HUMAN PIGR_HUMAN PIGR_HUMAN PIGR_HUMAN PIGR_HUMAN K1C16_HUMAN K1C13_HUMAN K1C13_HUMAN K1C13_HUMAN K2C4_HUMAN K2C4_HUMAN K2C4_HUMAN K2C4_HUMAN K1C15_HUMAN K1C15_HUMAN K1C13_HUMAN K1C15_HUMAN K1C13_HUMAN K1C15_HUMAN K2C4_HUMAN IGHA2_HUMAN K2C4_HUMAN K2C4_HUMAN TGM3_HUMAN

49,557 60,008 60,030 83,232 57,673 57,673 83,232 57,250 83,232 57,250 83,232 57,250 83,232 83,232 83,232 83,232 83,232 83,232 51,236 49,557 49,557 49,557 57,250 57,250 57,250 57,250 49,181 49,181 49,557 49,181 49,557 49,181 57,250 36,503 57,250 57,250 76,584

4.91 8.09 8.09 5.58 6.64 6.64 5.58 6.25 5.58 6.25 5.58 6.25 5.58 5.58 5.58 5.58 5.58 5.58 4.99 4.91 4.91 4.91 6.25 6.25 6.25 6.25 4.71 4.71 4.91 4.71 4.91 4.71 6.25 5.71 6.25 6.25 5.62

1 1 13 2 15 11 18 3 11 1 2 1 1 2 3 3 2 3 1 20 11 26 12 2 1 1 2 1 3 1 17 1 1 1 1 2 3

78 76 191 114 228 162 435 243 396 59 156 68 52 177 288 277 139 273 85 185 168 183 72 112 93 66 81 70 190 50 112 64 76 88 60 90 228

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

78 76 79 66 106 102 157 85 130 59 104 68 52 104 143 108 76 106 85 77 98 67 45 77 93 66 50 70 77 50 59 64 76 88 60 61 99

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 44 11 100 100 100 100 100 100 100 19 100 100 100 100 100 100

K2C4_HUMAN K2C73_HUMAN TGM3_HUMAN

57,250 58,887 76,584

6.25 6.93 5.62

23 2 18

198 103 100

100 100 100

83 74 100

100 100 100

TGM3_HUMAN

76,584

5.62

3

181

100

80

100

K2C4_HUMAN TGM3_HUMAN

57,250 76,584

6.25 5.62

1 2

65 181

100 100

65 120

100 100

K2C4_HUMAN K2C1_HUMAN K2C4_HUMAN K2C3_HUMAN K2C4_HUMAN K2C73_HUMAN K2C3_HUMAN K2C6B_HUMAN IGHA2_HUMAN K2C4_HUMAN CRNN_HUMAN CRNN_HUMAN IGHA2_HUMAN K2C6B_HUMAN K2C4_HUMAN

57,250 65,999 57,250 64,378 57,250 58,887 64,378 60,030 36,503 57,250 53,502 53,502 36,503 60,030 57,250

6.25 8.15 6.25 6.12 6.25 6.93 6.12 8.09 5.71 6.25 5.73 5.73 5.71 8.09 6.25

3 1 3 23 19 2 2 20 1 1 1 17 1 3 2

220 35 179 112 104 102 98 94 101 90 75 115 115 106 130

100 99 100 100 100 100 100 100 100 100 100 100 100 100 100

93 35 81 78 46 81 75 48 101 90 75 78 115 58 92

100 99 100 23 21 100 100 23 100 100 100 14 100 100 100

(continued on next page)

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Table 1 (continued) Spot no. 58 58 59 59 59 59 59 59 60 60 61 62 63 64 65 66 67 68 69 70 70 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 95 96 96 97 99 100 101 102 103 103 104 105 106 107 108 109

Protein name

Keratin, type II cytoskeletal 6B Serum albumin Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 73 Keratin, type II cytoskeletal 73 Keratin, type II cytoskeletal 1 Keratin, type II cytoskeletal 6B Keratin, type II cytoskeletal 1 Keratin, type II cytoskeletal 8 Keratin, type II cytoskeletal 1 Heat shock 70 kDa protein 1A/1B Heat shock 70 kDa protein 1A/1B Heat shock cognate 71 kDa protein Heat shock cognate 71 kDa protein Cornulin Cornulin Cornulin 78 kDa glucose-regulated protein Keratin, type I cytoskeletal 15 Keratin, type II cytoskeletal 6B Cornulin Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type II cytoskeletal 6B Keratin, type II cytoskeletal 6B Keratin, type II cytoskeletal 6B Keratin, type II cytoskeletal 6B Keratin, type II cytoskeletal 2 oral Keratin, type II cytoskeletal 6B Keratin, type II cytoskeletal 1 Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6B Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6A Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6A Keratin, type II cytoskeletal 5 Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6B Keratin, type II cytoskeletal 4 Alpha-amylase 1 Keratin, type II cytoskeletal 6A Alpha-amylase 2B Alpha-amylase 2B Alpha-amylase 2B Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6B Ig alpha-2 chain C region Keratin, type II cytoskeletal 4 Ig alpha-2 chain C region Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 5 Keratin, type II cytoskeletal 4

Accession name K2C6B_HUMAN ALBU_HUMAN K2C4_HUMAN K2C4_HUMAN K2C73_HUMAN K2C73_HUMAN K2C1_HUMAN K2C6B_HUMAN K2C1_HUMAN K2C8_HUMAN K2C1_HUMAN HSP71_HUMAN HSP71_HUMAN HSP7C_HUMAN HSP7C_HUMAN CRNN_HUMAN CRNN_HUMAN CRNN_HUMAN GRP78_HUMAN K1C15_HUMAN K2C6B_HUMAN CRNN_HUMAN K1C13_HUMAN K1C13_HUMAN K1C13_HUMAN K2C6B_HUMAN K2C6B_HUMAN K2C6B_HUMAN K2C6B_HUMAN K22O_HUMAN K2C6B_HUMAN K2C1_HUMAN K2C6C_HUMAN K2C6C_HUMAN K2C6C_HUMAN K2C6B_HUMAN K2C6C_HUMAN K2C6A_HUMAN K2C6C_HUMAN K2C6C_HUMAN K2C6C_HUMAN K2C6C_HUMAN K2C6A_HUMAN K2C5_HUMAN K2C6C_HUMAN K2C6B_HUMAN K2C4_HUMAN AMY1_HUMAN K2C6A_HUMAN AMY2B_HUMAN AMY2B_HUMAN AMY2B_HUMAN K2C6C_HUMAN K2C4_HUMAN K2C4_HUMAN K2C6C_HUMAN K2C6B_HUMAN IGHA2_HUMAN K2C4_HUMAN IGHA2_HUMAN K2C4_HUMAN K2C5_HUMAN K2C4_HUMAN

Protein Protein MW PI 60,030 69,321 57,250 57,250 58,887 58,887 65,999 60,030 65,999 53,671 65,999 70,009 70,009 70,854 70,854 53,502 53,502 53,502 72,288 49,181 60,030 53,502 49,557 49,557 49,557 60,030 60,030 60,030 60,030 65,800 60,030 65,999 59,988 59,988 59,988 60,030 59,988 60,008 59,988 59,988 59,988 59,988 60,008 62,340 59,988 60,030 57,250 57,731 60,008 57,673 57,673 57,673 59,988 57,250 57,250 59,988 60,030 36,503 57,250 36,503 57,250 62,340 57,250

8.09 5.92 6.25 6.25 6.93 6.93 8.15 8.09 8.15 5.52 8.15 5.48 5.48 5.37 5.37 5.73 5.73 5.73 5.07 4.71 8.09 5.73 4.91 4.91 4.91 8.09 8.09 8.09 8.09 8.38 8.09 8.15 8.09 8.09 8.09 8.09 8.09 8.09 8.09 8.09 8.09 8.09 8.09 7.59 8.09 8.09 6.25 6.47 8.09 6.64 6.64 6.64 8.09 6.25 6.25 8.09 8.09 5.71 6.25 5.71 6.25 7.59 6.25

Peptide count 2 16 4 3 2 2 2 2 13 2 3 16 3 3 2 13 1 1 3 2 2 1 25 27 22 3 14 4 3 2 3 2 4 17 23 35 35 43 39 36 24 31 34 29 4 3 4 18 2 13 24 18 19 28 28 4 35 2 32 2 25 3 29

Total Total ion Best Best ion ion score C.I. ion score C.I. score % score % 105 98 239 200 95 86 82 76 248 71 141 223 94 177 66 73 75 78 194 106 84 72 180 206 177 209 134 248 226 99 171 101 234 287 297 318 314 355 316 313 327 301 243 157 208 217 321 122 139 114 437 313 219 307 324 299 234 224 331 216 316 184 323

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

67 70 85 106 60 62 52 44 78 58 67 75 57 97 50 56 75 78 85 61 54 72 67 43 67 79 73 80 95 71 65 56 68 88 96 106 103 68 109 100 103 89 45 67 74 94 97 79 76 114 140 112 66 97 104 89 73 150 113 150 96 75 96

100 33 100 100 100 100 100 100 100 100 100 100 100 100 100 11 100 100 100 100 100 100 55 54 49 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

5147

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 51 4 0 –5 16 5

Table 1 (continued) Spot no. 110 112 113 114 115 116 116 116 117 118 119 120 121 123 124 125 126 127 127 128 129 130 131 132 132 132 133 134 135 136 137 138 139 140 141 142 143 143 144 145 146 148 149 150 151 153 154 155 156 157 158 158 159 159 161 162 163 163 164 165 166

Protein name

Accession name

Protein Protein MW PI

Peptide count

Total Total ion Best Best ion ion score C.I. ion score C.I. score % score %

Keratin, type II cytoskeletal 5 Keratin, type II cytoskeletal 5 Ig alpha-2 chain C region Keratin, type II cytoskeletal 5 Keratin, type II cytoskeletal 4 Ig alpha-2 chain C region Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 6B Beta-actin-like protein 2 Ig alpha-2 chain C region Ig alpha-2 chain C region Ig alpha-2 chain C region Keratin, type I cytoskeletal 10 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 15 Keratin, type I cytoskeletal 13 Keratin, type II cytoskeletal 6A Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 6B Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 16 Keratin, type I cytoskeletal 14 ATP synthase subunit beta, mitochondrial Keratin, type I cytoskeletal 14 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 19 Alpha-enolase Beta-enolase Phosphoglycerate kinase 1 Putative elongation factor 1-alpha-like 3 Actin, cytoplasmic 2 Actin, cytoplasmic 2 Actin, cytoplasmic 2 Actin, cytoplasmic 2 Actin, cytoplasmic 2 Serpin B13 Zinc-alpha-2-glycoprotein Serpin B13 Zinc-alpha-2-glycoprotein Keratin, type II cytoskeletal 4 Annexin A3 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 73 F-actin-capping protein subunit alpha-1 Serpin B5 Short PLUNC2

K2C5_HUMAN K2C5_HUMAN IGHA2_HUMAN K2C5_HUMAN K2C4_HUMAN IGHA2_HUMAN K2C4_HUMAN K2C6B_HUMAN ACTBL_HUMAN IGHA2_HUMAN IGHA2_HUMAN IGHA2_HUMAN K1C10_HUMAN K1C13_HUMAN K1C13_HUMAN K1C13_HUMAN K1C13_HUMAN K1C15_HUMAN K1C13_HUMAN K2C6A_HUMAN K2C4_HUMAN K2C4_HUMAN K2C4_HUMAN K2C4_HUMAN K2C4_HUMAN K2C6B_HUMAN K1C13_HUMAN K1C13_HUMAN K1C13_HUMAN K1C13_HUMAN K1C13_HUMAN K1C13_HUMAN K1C13_HUMAN K1C13_HUMAN K1C13_HUMAN K1C16_HUMAN K1C14_HUMAN ATPB_HUMAN

62,340 62,340 36,503 62,340 57,250 36,503 57,250 60,030 41,976 36,503 36,503 36,503 58,792 49,557 49,557 49,557 49,557 49,181 49,557 60,008 57,250 57,250 57,250 57,250 57,250 60,030 49,557 49,557 49,557 49,557 49,557 49,557 49,557 49,557 49,557 51,236 51,529 56,525

7.59 7.59 5.71 7.59 6.25 5.71 6.25 8.09 5.39 5.71 5.71 5.71 5.13 4.91 4.91 4.91 4.91 4.71 4.91 8.09 6.25 6.25 6.25 6.25 6.25 8.09 4.91 4.91 4.91 4.91 4.91 4.91 4.91 4.91 4.91 4.99 5.09 5.26

3 23 2 21 23 1 2 2 1 2 2 2 10 21 18 3 31 2 16 1 1 3 3 3 3 2 17 12 14 27 27 3 29 30 27 12 32 19

223 279 155 212 287 135 132 130 78 180 191 166 238 176 135 296 281 130 112 60 81 133 164 223 185 120 151 128 138 272 332 304 249 269 206 389 230 140

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

101 89 94 88 98 135 87 86 78 101 120 95 99 50 38 161 57 67 35 60 81 51 72 100 77 65 45 53 52 136 152 176 52 66 82 155 38 98

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

K1C14_HUMAN K1C13_HUMAN K1C19_HUMAN ENOA_HUMAN ENOB_HUMAN PGK1_HUMAN EF1A3_HUMAN ACTG_HUMAN ACTG_HUMAN ACTG_HUMAN ACTG_HUMAN ACTG_HUMAN SPB13_HUMAN ZA2G_HUMAN SPB13_HUMAN ZA2G_HUMAN K2C4_HUMAN ANXA3_HUMAN K2C4_HUMAN K2C73_HUMAN CAZA1_HUMAN SPB5_HUMAN SPLC2_HUMAN

51,529 49,557 44,079 47,139 46,902 44,586 50,153 41,766 41,766 41,766 41,766 41,766 44,248 34,237 44,248 34,237 57,250 36,353 57,250 58,887 32,902 42,073 26,995

5.09 4.91 5.04 7.01 7.59 8.3 9.15 5.31 5.31 5.31 5.31 5.31 5.48 5.71 5.48 5.71 6.25 5.63 6.25 6.93 5.45 5.72 5.35

36 34 23 22 1 3 8 11 11 16 14 17 1 1 5 7 11 11 3 2 6 8 1

397 296 212 191 65 243 148 303 310 309 300 256 83 78 85 55 201 184 181 100 137 92 135

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

174 67 43 47 65 104 90 130 128 108 114 111 83 78 85 55 102 92 78 78 80 92 135

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

(continued on next page)

5148

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 51 4 0 –51 6 5

Table 1 (continued) Spot no. 167 168 169 170 171 172 173 174 175 177 178 179 180 180 181 181 182 183 184 185 186 187 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 204 205 206 206 207 207 208 209 210 210 211 212 213 213 214 214 214 215 216

Protein name

Accession name

Protein Protein MW PI

Peptide count

Total Total ion Best Best ion ion score C.I. ion score C.I. score % score %

Zymogen granule protein 16 homolog B Annexin A5 Keratin, type I cytoskeletal 13 Keratin, type I cytoskeletal 13 Keratin, type II cytoskeletal 4 Annexin A1 Annexin A1 Leukocyte elastase inhibitor Carbonic anhydrase 6 Annexin A1 Carbonic anhydrase 6 Carbonic anhydrase 6 Annexin A1 Carbonic anhydrase 6 Serpin B3 Carbonic anhydrase 6 Annexin A1 Annexin A1 Annexin A1 Annexin A1 Annexin A2 Annexin A2 Annexin A2 Annexin A2 Annexin A2 Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Fructose-bisphosphate aldolase A Fructose-bisphosphate aldolase A Annexin A2 Voltage-dependent anion-selective channel protein 1 Annexin A1 Annexin A1 Annexin A1 Annexin A1 Annexin A1 Ig alpha-2 chain C region Lysozyme C Small proline-rich protein 3 Ig kappa chain C region Ig kappa chain C region Ig lambda-3 chain C regions Ig lambda-1 chain C regions Peroxiredoxin-1 Ig kappa chain V–III region Ti Ig kappa chain V–III region Ti Cystatin-B Phosphatidylethanolamine-binding protein 1 Glyceraldehyde-3-phosphate dehydrogenase Alpha-crystallin B chain Cystatin-B Ig lambda-3 chain C regions Phosphoglycerate mutase 1 Triosephosphate isomerase Ig alpha-2 chain C region Ig kappa chain C region

ZG16B_HUMAN ANXA5_HUMAN K1C13_HUMAN K1C13_HUMAN K2C4_HUMAN ANXA1_HUMAN ANXA1_HUMAN ILEU_HUMAN CAH6_HUMAN ANXA1_HUMAN CAH6_HUMAN CAH6_HUMAN ANXA1_HUMAN CAH6_HUMAN SPB3_HUMAN CAH6_HUMAN ANXA1_HUMAN ANXA1_HUMAN ANXA1_HUMAN ANXA1_HUMAN ANXA2_HUMAN ANXA2_HUMAN ANXA2_HUMAN ANXA2_HUMAN ANXA2_HUMAN G3P_HUMAN

22,725 35,914 49,557 49,557 57,250 38,690 38,690 42,715 35,345 38,690 35,345 35,345 38,690 35,345 44,537 35,345 38,690 38,690 38,690 38,690 38,580 38,580 38,580 38,580 38,580 36,030

6.74 4.94 4.91 4.91 6.25 6.57 6.57 5.9 6.51 6.57 6.51 6.51 6.57 6.51 6.35 6.51 6.57 6.57 6.57 6.57 7.57 7.57 7.57 7.57 7.57 8.57

1 5 21 20 3 15 11 11 11 18 12 2 3 2 18 7 22 13 15 14 18 4 21 21 12 1

43 88 172 159 249 289 314 433 157 413 165 144 279 152 243 71 451 275 390 326 255 265 226 348 192 87

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

43 51 46 49 103 101 124 141 85 121 108 91 111 101 124 71 145 121 115 117 95 98 83 118 80 87

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

G3P_HUMAN

36,030

8.57

7

109

100

109

100

G3P_HUMAN

36,030

8.57

8

118

100

118

100

ALDOA_HUMAN ALDOA_HUMAN ANXA2_HUMAN VDAC1_HUMAN

39,395 39,395 38,580 30,754

8.3 8.3 7.57 8.62

12 7 13 10

116 59 187 166

100 97 100 100

46 35 90 67

100 100 100 100

ANXA1_HUMAN ANXA1_HUMAN ANXA1_HUMAN ANXA1_HUMAN ANXA1_HUMAN IGHA2_HUMAN LYSC_HUMAN SPRR3_HUMAN IGKC_HUMAN IGKC_HUMAN LAC3_HUMAN LAC1_HUMAN PRDX1_HUMAN KV304_HUMAN KV304_HUMAN CYTB_HUMAN PEBP1_HUMAN

38,690 38,690 38,690 38,690 38,690 36,503 16,526 18,142 11,602 11,602 11,231 11,341 22,096 11,781 11,781 11,133 21,044

6.57 6.57 6.57 6.57 6.57 5.71 9.38 8.86 5.58 5.58 6.92 7.89 8.27 8.72 8.72 6.96 7.01

15 14 19 16 14 11 8 7 1 1 2 1 2 1 1 1 2

398 434 345 388 187 227 80 136 54 47 110 78 131 104 97 96 104

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

140 129 127 128 114 150 80 52 54 47 61 78 68 104 97 96 56

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

G3P_HUMAN

36,030

8.57

1

101

100

101

100

CRYAB_HUMAN CYTB_HUMAN LAC3_HUMAN PGAM1_HUMAN TPIS_HUMAN IGHA2_HUMAN IGKC_HUMAN

20,146 11,133 11,231 28,786 26,653 36,503 11,602

6.76 6.96 6.92 6.67 6.45 5.71 5.58

13 1 2 1 9 2 2

141 76 158 71 65 190 203

100 100 100 100 99 100 100

34 76 88 71 38 129 141

100 100 100 100 100 100 100

5149

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 51 4 0 –5 16 5

Table 1 (continued) Spot no. 217 218 219 220 221 221 222 223 223 224 225 226 227 227 228 229 229 229 230 231 232 232 233 233 233 234 234 235 236 237 238 239 240 241 242 243 244 244 244 245 245 246 247 248 248 249 250 251 252 253 254 255 256 257 259 259 260 260 260 261 261

Protein name

Accession name

Protein Protein MW PI

Peptide count

Total Total ion Best Best ion ion score C.I. ion score C.I. score % score %

Peroxiredoxin-6 Chloride intracellular channel protein 3 Heat shock protein beta-1 Ig kappa chain C region Protein S100-A9 Heat shock protein beta-1 Heat shock protein beta-1 Heat shock protein beta-1 Zymogen granule protein 16 homolog B Protein S100-A9 Heat shock protein beta-1 Heat shock protein beta-1 Heat shock protein beta-1 Zymogen granule protein 16 homolog B Heat shock protein beta-1 Heat shock protein beta-1 Heat shock protein beta-1 Zymogen granule protein 16 homolog B Heat shock protein beta-1 Heat shock protein beta-1 Heat shock protein beta-1 14-3-3 protein sigma Zymogen granule protein 16 homolog B Heat shock protein beta-1 Keratin, type I cytoskeletal 15 Zymogen granule protein 16 homolog B Keratin, type I cytoskeletal 15 Immunoglobulin J chain Keratin, type I cytoskeletal 14 Glutathione S-transferase P Glutathione S-transferase P Interleukin-1 receptor antagonist protein Prolactin-inducible protein Prolactin-inducible protein Prolactin-inducible protein Prolactin-inducible protein Prolactin-inducible protein Heat shock protein beta-1 14-3-3 protein sigma Histone H2A type 1-C Keratin, type I cytoskeletal 14 Calmodulin Calmodulin-like protein 3 Protein S100-A9 Cystatin-S Cystatin-S Cystatin-SA Protein S100-A14 Protein S100-A14 Thioredoxin Prolactin-inducible protein Prolactin-inducible protein Protein S100-A9 Protein S100-A9 Protein S100-A14 Lysozyme C Cystatin-S Cystatin-B Lysozyme C Protein S100-A9 Cystatin-A

PRDX6_HUMAN CLIC3_HUMAN HSPB1_HUMAN IGKC_HUMAN S10A9_HUMAN HSPB1_HUMAN HSPB1_HUMAN HSPB1_HUMAN ZG16B_HUMAN S10A9_HUMAN HSPB1_HUMAN HSPB1_HUMAN HSPB1_HUMAN ZG16B_HUMAN HSPB1_HUMAN HSPB1_HUMAN HSPB1_HUMAN ZG16B_HUMAN HSPB1_HUMAN HSPB1_HUMAN HSPB1_HUMAN 1433S_HUMAN ZG16B_HUMAN HSPB1_HUMAN K1C15_HUMAN ZG16B_HUMAN K1C15_HUMAN IGJ_HUMAN K1C14_HUMAN GSTP1_HUMAN GSTP1_HUMAN IL1RA_HUMAN

25,019 26,632 22,768 11,602 13,234 22,768 22,768 22,768 22,725 13,234 22,768 22,768 22,768 22,725 22,768 22,768 22,768 22,725 22,768 22,768 22,768 27,757 22,725 22,768 49,181 22,725 49,181 18,087 51,529 23,341 23,341 20,042

6 5.99 5.98 5.58 5.71 5.98 5.98 5.98 6.74 5.71 5.98 5.98 5.98 6.74 5.98 5.98 5.98 6.74 5.98 5.98 5.98 4.68 6.74 5.98 4.71 6.74 4.71 5.12 5.09 5.43 5.43 5.83

2 3 4 1 10 2 3 9 2 2 7 9 8 2 6 3 3 1 6 8 2 1 2 2 1 1 1 5 1 9 3 6

105 240 343 45 184 159 256 165 121 168 140 151 246 111 204 187 167 57 162 205 149 102 113 75 56 56 52 139 53 343 236 428

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

57 95 109 45 99 97 100 101 77 86 77 92 100 68 88 104 79 57 73 90 91 102 65 48 56 56 52 81 53 134 137 119

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

PIP_HUMAN PIP_HUMAN PIP_HUMAN PIP_HUMAN PIP_HUMAN HSPB1_HUMAN 1433S_HUMAN H2A1C_HUMAN K1C14_HUMAN CALM_HUMAN CALL3_HUMAN S10A9_HUMAN CYTS_HUMAN CYTS_HUMAN CYTT_HUMAN S10AE_HUMAN S10AE_HUMAN THIO_HUMAN PIP_HUMAN PIP_HUMAN S10A9_HUMAN S10A9_HUMAN S10AE_HUMAN LYSC_HUMAN CYTS_HUMAN CYTB_HUMAN LYSC_HUMAN S10A9_HUMAN CYTA_HUMAN

16,562 16,562 16,562 16,562 16,562 22,768 27,757 14,097 51,529 16,827 16,880 13,234 16,204 16,204 16,434 11,655 11,655 11,730 16,562 16,562 13,234 13,234 11,655 16,526 16,204 11,133 16,526 13,234 11,000

8.26 8.26 8.26 8.26 8.26 5.98 4.68 11.05 5.09 4.09 4.3 5.71 4.95 4.95 4.85 5.16 5.16 4.82 8.26 8.26 5.71 5.71 5.16 9.38 4.95 6.96 9.38 5.71 5.38

4 6 3 4 1 1 11 1 1 2 8 2 7 9 7 6 6 5 7 7 6 7 1 8 5 5 8 2 1

285 317 241 299 89 74 72 104 55 185 140 194 181 377 215 158 190 153 179 93 196 192 111 89 164 86 74 183 114

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

93 96 93 88 89 74 36 104 55 108 140 105 100 163 107 97 133 96 105 93 104 106 111 89 95 86 74 92 114

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

(continued on next page)

5150

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 51 4 0 –51 6 5

Table 1 (continued) Spot no. 262 263 264 265 266 267 270 271 271 272 272 273 273 274 275 276 277 277 278 278 279 280 281 282 283 284 284 284 285 285 285 286 286 225 226 227 227 228 229 229 229 230 231 232 232 233 233 233 234 234 235 236 237 238 239 240 241 242 243 244 244 244

Protein name

Protein S100-A9 Protein S100-A9 Protein S100-A9 Protein S100-A9 Protein S100-A11 Protein S100-A12 Lysozyme C Cystatin-SN Lysozyme C Cystatin-B Lysozyme C Cystatin-B Lysozyme C Cystatin-B Histatin-1 Protein S100-A10 Cystatin-SN Cystatin-B Profilin-1 Cystatin-C Peptidyl-prolyl cis-trans isomerase A Lysozyme C Lysozyme C Lysozyme C Lysozyme C Cystatin-SN Cystatin-B Lysozyme C Cystatin-B Cystatin-SN Lysozyme C Cystatin-SN Cystatin-B Heat shock protein beta-1 Heat shock protein beta-1 Heat shock protein beta-1 Zymogen granule protein 16 homolog Heat shock protein beta-1 Heat shock protein beta-1 Heat shock protein beta-1 Zymogen granule protein 16 homolog Heat shock protein beta-1 Heat shock protein beta-1 Heat shock protein beta-1 14-3-3 protein sigma Zymogen granule protein 16 homolog Heat shock protein beta-1 Keratin, type I cytoskeletal 15 Zymogen granule protein 16 homolog Keratin, type I cytoskeletal 15 Immunoglobulin J chain Keratin, type I cytoskeletal 14 Glutathione S-transferase P Glutathione S-transferase P Interleukin-1 receptor antagonist protein Prolactin-inducible protein Prolactin-inducible protein Prolactin-inducible protein Prolactin-inducible protein Prolactin-inducible protein Heat shock protein beta-1 14-3-3 protein sigma

Accession name

B

B

B

B

Protein Protein MW PI

Peptide count

Total Total ion Best Best ion ion score C.I. ion score C.I. score % score %

S10A9_HUMAN S10A9_HUMAN S10A9_HUMAN S10A9_HUMAN S10AB_HUMAN S10AC_HUMAN LYSC_HUMAN CYTN_HUMAN LYSC_HUMAN CYTB_HUMAN LYSC_HUMAN CYTB_HUMAN LYSC_HUMAN CYTB_HUMAN HIS1_HUMAN S10AA_HUMAN CYTN_HUMAN CYTB_HUMAN PROF1_HUMAN CYTC_HUMAN PPIA_HUMAN LYSC_HUMAN LYSC_HUMAN LYSC_HUMAN LYSC_HUMAN CYTN_HUMAN CYTB_HUMAN LYSC_HUMAN CYTB_HUMAN CYTN_HUMAN LYSC_HUMAN CYTN_HUMAN CYTB_HUMAN HSPB1_HUMAN HSPB1_HUMAN HSPB1_HUMAN ZG16B_HUMAN HSPB1_HUMAN HSPB1_HUMAN HSPB1_HUMAN ZG16B_HUMAN HSPB1_HUMAN HSPB1_HUMAN HSPB1_HUMAN 1433S_HUMAN ZG16B_HUMAN HSPB1_HUMAN K1C15_HUMAN ZG16B_HUMAN K1C15_HUMAN IGJ_HUMAN K1C14_HUMAN GSTP1_HUMAN GSTP1_HUMAN IL1RA_HUMAN

13,234 13,234 13,234 13,234 11,733 10,569 16,526 16,377 16,526 11,133 16,526 11,133 16,526 11,133 6958 11,196 16,377 11,133 15,045 15,789 18,001 16,526 16,526 16,526 16,526 16,377 11,133 16,526 11,133 16,377 16,526 16,377 11,133 22,768 22,768 22,768 22,725 22,768 22,768 22,768 22,725 22,768 22,768 22,768 27,757 22,725 22,768 49,181 22,725 49,181 18,087 51,529 23,341 23,341 20,042

5.71 5.71 5.71 5.71 6.56 5.83 9.38 6.73 9.38 6.96 9.38 6.96 9.38 6.96 9.11 6.82 6.73 6.96 8.44 9 7.68 9.38 9.38 9.38 9.38 6.73 6.96 9.38 6.96 6.73 9.38 6.73 6.96 5.98 5.98 5.98 6.74 5.98 5.98 5.98 6.74 5.98 5.98 5.98 4.68 6.74 5.98 4.71 6.74 4.71 5.12 5.09 5.43 5.43 5.83

9 8 9 11 6 5 4 5 8 2 8 6 8 5 1 2 4 1 1 1 6 5 6 7 1 2 2 10 6 1 8 2 6 7 9 8 2 6 3 3 1 6 8 2 1 2 2 1 1 1 5 1 9 3 6

291 200 194 297 68 162 40 309 86 140 86 248 91 256 73 137 159 98 80 69 269 77 77 93 94 144 118 88 210 112 84 229 205 140 151 246 111 204 187 167 57 162 205 149 102 113 75 56 56 52 139 53 343 236 428

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

106 114 109 115 34 116 40 124 86 71 86 92 91 93 73 81 100 98 80 69 107 77 77 93 94 87 66 88 107 112 84 121 122 77 92 100 68 88 104 79 57 73 90 91 102 65 48 56 56 52 81 53 134 137 119

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

PIP_HUMAN PIP_HUMAN PIP_HUMAN PIP_HUMAN PIP_HUMAN HSPB1_HUMAN 1433S_HUMAN

16,562 16,562 16,562 16,562 16,562 22,768 27,757

8.26 8.26 8.26 8.26 8.26 5.98 4.68

4 6 3 4 1 1 11

285 317 241 299 89 74 72

100 100 100 100 100 100 100

93 96 93 88 89 74 36

100 100 100 100 100 100 100

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Table 1 (continued) Spot no. 245 245 246 247 248 248 249 250 251 252 253 254 255 256 257 259 259 260 260 260 261 261 262 263 264 265 266 267 270 271 271 272 272 273 273 274 275 276 277 277 278 278 279 280 281 282 283 284 284 284 285 285 285 286 286 286 287 287 288 290 295 296

Protein name

Histone H2A type 1-C Keratin, type I cytoskeletal 14 Calmodulin Calmodulin-like protein 3 Protein S100-A9 Cystatin-S Cystatin-S Cystatin-SA Protein S100-A14 Protein S100-A14 Thioredoxin Prolactin-inducible protein Prolactin-inducible protein Protein S100-A9 Protein S100-A9 Protein S100-A14 Lysozyme C Cystatin-S Cystatin-B Lysozyme C Protein S100-A9 Cystatin-A Protein S100-A9 Protein S100-A9 Protein S100-A9 Protein S100-A9 Protein S100-A11 Protein S100-A12 Lysozyme C Cystatin-SN Lysozyme C Cystatin-B Lysozyme C Cystatin-B Lysozyme C Cystatin-B Histatin-1 Protein S100-A10 Cystatin-SN Cystatin-B Profilin-1 Cystatin-C Peptidyl-prolyl cis-trans isomerase A Lysozyme C Lysozyme C Lysozyme C Lysozyme C Cystatin-SN Cystatin-B Lysozyme C Cystatin-B Cystatin-SN Lysozyme C Cystatin-SN Cystatin-B Lysozyme C Cystatin-SN Galectin-7 Protein S100-A8 Histone H4 Neutrophil defensin 3 Neutrophil defensin 3

Accession name H2A1C_HUMAN K1C14_HUMAN CALM_HUMAN CALL3_HUMAN S10A9_HUMAN CYTS_HUMAN CYTS_HUMAN CYTT_HUMAN S10AE_HUMAN S10AE_HUMAN THIO_HUMAN PIP_HUMAN PIP_HUMAN S10A9_HUMAN S10A9_HUMAN S10AE_HUMAN LYSC_HUMAN CYTS_HUMAN CYTB_HUMAN LYSC_HUMAN S10A9_HUMAN CYTA_HUMAN S10A9_HUMAN S10A9_HUMAN S10A9_HUMAN S10A9_HUMAN S10AB_HUMAN S10AC_HUMAN LYSC_HUMAN CYTN_HUMAN LYSC_HUMAN CYTB_HUMAN LYSC_HUMAN CYTB_HUMAN LYSC_HUMAN CYTB_HUMAN HIS1_HUMAN S10AA_HUMAN CYTN_HUMAN CYTB_HUMAN PROF1_HUMAN CYTC_HUMAN PPIA_HUMAN LYSC_HUMAN LYSC_HUMAN LYSC_HUMAN LYSC_HUMAN CYTN_HUMAN CYTB_HUMAN LYSC_HUMAN CYTB_HUMAN CYTN_HUMAN LYSC_HUMAN CYTN_HUMAN CYTB_HUMAN LYSC_HUMAN CYTN_HUMAN LEG7_HUMAN S10A8_HUMAN H4_HUMAN DEF3_HUMAN DEF3_HUMAN

Protein Protein MW PI 14,097 51,529 16,827 16,880 13,234 16,204 16,204 16,434 11,655 11,655 11,730 16,562 16,562 13,234 13,234 11,655 16,526 16,204 11,133 16,526 13,234 11,000 13,234 13,234 13,234 13,234 11,733 10,569 16,526 16,377 16,526 11,133 16,526 11,133 16,526 11,133 6958 11,196 16,377 11,133 15,045 15,789 18,001 16,526 16,526 16,526 16,526 16,377 11,133 16,526 11,133 16,377 16,526 16,377 11,133 16,526 16,377 15,066 10,828 11,360 10,238 10,238

11.05 5.09 4.09 4.3 5.71 4.95 4.95 4.85 5.16 5.16 4.82 8.26 8.26 5.71 5.71 5.16 9.38 4.95 6.96 9.38 5.71 5.38 5.71 5.71 5.71 5.71 6.56 5.83 9.38 6.73 9.38 6.96 9.38 6.96 9.38 6.96 9.11 6.82 6.73 6.96 8.44 9 7.68 9.38 9.38 9.38 9.38 6.73 6.96 9.38 6.96 6.73 9.38 6.73 6.96 9.38 6.73 7.03 6.51 11.36 5.71 5.71

Peptide count 1 1 2 8 2 7 9 7 6 6 5 7 7 6 7 1 8 5 5 8 2 1 9 8 9 11 6 5 4 5 8 2 8 6 8 5 1 2 4 1 1 1 6 5 6 7 1 2 2 10 6 1 8 2 6 1 2 11 10 2 1 1

Total Total ion Best Best ion ion score C.I. ion score C.I. score % score % 104 55 185 140 194 181 377 215 158 190 153 179 93 196 192 111 89 164 86 74 183 114 291 200 194 297 68 162 40 309 86 140 86 248 91 256 73 137 159 98 80 69 269 77 77 93 94 144 118 88 210 112 84 229 205 87 239 200 261 125 39 33

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99

104 55 108 140 105 100 163 107 97 133 96 105 93 104 106 111 89 95 86 74 92 114 106 114 109 115 34 116 40 124 86 71 86 92 91 93 73 81 100 98 80 69 107 77 77 93 94 87 66 88 107 112 84 121 122 87 148 107 82 69 39 33

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99

(continued on next page)

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Table 1 (continued) Spot no. 298 302 303 303 303 304 305 306 307 308 309 309 309 309 310 310 311 311 312 312 312 313 313 313 313 314 315 316 317 318 319 320 321 322 323 324 325 325 326 326 326 327 327 329 330 331 331 331 332 332 333 335 335 336 336 337 337 338 339 339 339 340 341

Protein name

Histatin-1 Protein S100-A14 Protein S100-A9 Protein S100-A9 Lysozyme C Lysozyme C Protein FAM25 Lysozyme C Keratin, type II cuticular Hb3 Protein S100-A9 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 73 Keratin, type II cytoskeletal 6B Keratin, type II cytoskeletal 6B Ig alpha-2 chain C region Ig alpha-2 chain C region Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6B Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6C Keratin, type II cytoskeletal 6B Ig alpha-2 chain C region Ig alpha-2 chain C region Polymeric immunoglobulin receptor Ig alpha-1 chain C region Ig alpha-2 chain C region Alpha-amylase 2B Polymeric immunoglobulin receptor Alpha-amylase 2B Alpha-amylase 2B Alpha-amylase 2B Zinc-alpha-2-glycoprotein Carbonic anhydrase 6 Zymogen granule protein 16 homolog Zymogen granule protein 16 homolog Zymogen granule protein 16 homolog Prolactin-inducible protein Lysozyme C Cystatin-SN Cystatin-SN Lysozyme C Cystatin-SN Cystatin-S Ig alpha-2 chain C region Polymeric immunoglobulin receptor Alpha-amylase 2B Ig alpha-2 chain C region Polymeric immunoglobulin receptor Ig alpha-2 chain C region Alpha-amylase 1 Alpha-amylase 2B Zinc-alpha-2-glycoprotein Carbonic anhydrase 6 Zymogen granule protein 16 homolog Ig lambda-1 chain C regions Zymogen granule protein 16 homolog Immunoglobulin J chain Prolactin-inducible protein Cystatin-SN Cystatin-SN Cystatin-S Cystatin-S Lysozyme C

Accession name

B B B

B B

HIS1_HUMAN S10AE_HUMAN S10A9_HUMAN S10A9_HUMAN LYSC_HUMAN LYSC_HUMAN FAM25_HUMAN LYSC_HUMAN KRT83_HUMAN S10A9_HUMAN K2C4_HUMAN K2C73_HUMAN K2C6B_HUMAN K2C6B_HUMAN IGHA2_HUMAN IGHA2_HUMAN K2C6C_HUMAN K2C6B_HUMAN K2C6C_HUMAN K2C6C_HUMAN K2C6B_HUMAN IGHA2_HUMAN IGHA2_HUMAN PIGR_HUMAN IGHA1_HUMAN IGHA2_HUMAN AMY2B_HUMAN PIGR_HUMAN AMY2B_HUMAN AMY2B_HUMAN AMY2B_HUMAN ZA2G_HUMAN CAH6_HUMAN ZG16B_HUMAN ZG16B_HUMAN ZG16B_HUMAN PIP_HUMAN LYSC_HUMAN CYTN_HUMAN CYTN_HUMAN LYSC_HUMAN CYTN_HUMAN CYTS_HUMAN IGHA2_HUMAN PIGR_HUMAN AMY2B_HUMAN IGHA2_HUMAN PIGR_HUMAN IGHA2_HUMAN AMY1_HUMAN AMY2B_HUMAN ZA2G_HUMAN CAH6_HUMAN ZG16B_HUMAN LAC1_HUMAN ZG16B_HUMAN IGJ_HUMAN PIP_HUMAN CYTN_HUMAN CYTN_HUMAN CYTS_HUMAN CYTS_HUMAN LYSC_HUMAN

Protein Protein MW PI 6958 11,655 13,234 13,234 16,526 16,526 9314 16,526 54,161 13,234 57,250 58,887 60,030 60,030 36,503 36,503 59,988 60,030 59,988 59,988 60,030 36,503 36,503 83,232 37,631 36,503 57,673 83,232 57,673 57,673 57,673 34,237 35,345 22,725 22,725 22,725 16,562 16,526 16,377 16,377 16,526 16,377 16,204 36,503 83,232 57,673 36,503 83,232 36,503 57,731 57,673 34,237 35,345 22,725 11,341 22,725 18,087 16,562 16,377 16,377 16,204 16,204 16,526

9.11 5.16 5.71 5.71 9.38 9.38 5.78 9.38 5.54 5.71 6.25 6.93 8.09 8.09 5.71 5.71 8.09 8.09 8.09 8.09 8.09 5.71 5.71 5.58 6.08 5.71 6.64 5.58 6.64 6.64 6.64 5.71 6.51 6.74 6.74 6.74 8.26 9.38 6.73 6.73 9.38 6.73 4.95 5.71 5.58 6.64 5.71 5.58 5.71 6.47 6.64 5.71 6.51 6.74 7.89 6.74 5.12 8.26 6.73 6.73 4.95 4.95 9.38

Peptide count 1 3 2 2 1 7 1 8 21 2 2 2 2 2 2 2 2 1 3 11 11 2 2 13 8 2 25 9 15 22 16 12 11 4 4 4 3 1 3 3 1 3 9 2 24 16 1 17 2 21 24 11 11 3 1 3 1 3 3 3 2 3 1

Total Total ion Best Best ion ion score C.I. ion score C.I. score % score % 78 103 175 153 86 95 89 111 84 170 94 91 79 50 169 140 133 47 123 63 62 243 235 63 48 216 275 216 214 240 198 118 157 348 336 275 206 70 306 274 97 298 84 155 472 131 93 71 209 180 389 92 76 191 69 199 83 224 319 309 214 232 63

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99 99 100 100 99 67 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

78 103 101 82 86 95 89 83 32 91 57 57 43 49 115 97 69 47 44 32 32 166 161 34 30 151 116 111 92 96 126 118 103 122 139 112 74 70 126 97 97 134 36 120 162 72 93 36 135 42 126 92 34 78 69 92 83 101 157 137 141 88 63

100 100 100 100 100 100 100 100 99 100 100 100 100 100 100 100 100 100 100 99 99 100 100 99 98 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99 100 100 100 100 99 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

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Table 1 (continued) Spot no. 342 344 344 345 345 345 347 348 348 348 349 350 351 351 353 353 353 354 354 355 356 356 356 357 358 359 360 360 360 360 361 361 362 365 365 366

Protein name

Lysozyme C Deleted in malignant brain tumors 1 protein Mucin-5B Zymogen granule protein 16 homolog Deleted in malignant brain tumors 1 protein Mucin-5B Polymeric immunoglobulin receptor Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 6A Keratin, type II cytoskeletal 1 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 4 Keratin, type I cytoskeletal 13 Actin, cytoplasmic 2 Annexin A1 Keratin, type I cytoskeletal 13 Annexin A1 Zymogen granule protein 16 homolog Zymogen granule protein 16 homolog Zymogen granule protein 16 homolog Zymogen granule protein 16 homolog Lysozyme C Zymogen granule protein 16 homolog Zymogen granule protein 16 homolog Zymogen granule protein 16 homolog Protein S100-A9 Cystatin-SN Lysozyme C Cystatin-B Protein S100-A9 Cystatin-B Protein S100-A8 Ig alpha-2 chain C region Deleted in malignant brain tumors 1 protein Ig alpha-2 chain C region

Accession name

Protein Protein MW PI

Peptide count

Total Total ion Best Best ion ion score C.I. ion score C.I. score % score %

LYSC_HUMAN DMBT1_HUMAN

16,526 260,569

9.38 5.18

1 1

59 93

100 100

59 97

100 100

B

MUC5B_HUMAN ZG16B_HUMAN DMBT1_HUMAN

595,960 22,725 260,569

6.2 6.74 5.18

1 1 1

58 72 52

100 100 100

58 72 56

100 100 100

B B B B

MUC5B_HUMAN PIGR_HUMAN K2C4_HUMAN K2C6A_HUMAN K2C1_HUMAN K2C4_HUMAN K2C4_HUMAN K2C4_HUMAN K1C13_HUMAN ACTG_HUMAN ANXA1_HUMAN K1C13_HUMAN ANXA1_HUMAN ZG16B_HUMAN ZG16B_HUMAN ZG16B_HUMAN ZG16B_HUMAN LYSC_HUMAN ZG16B_HUMAN ZG16B_HUMAN ZG16B_HUMAN S10A9_HUMAN CYTN_HUMAN LYSC_HUMAN CYTB_HUMAN S10A9_HUMAN CYTB_HUMAN S10A8_HUMAN IGHA2_HUMAN DMBT1_HUMAN

595,960 83,232 57,250 60,008 65,999 57,250 57,250 57,250 49,557 41,766 38,690 49,557 38,690 22,725 22,725 22,725 22,725 16,526 22,725 22,725 22,725 13,234 16,377 16,526 11,133 13,234 11,133 10,828 36,503 260,569

6.2 5.58 6.25 8.09 8.15 6.25 6.25 6.25 4.91 5.31 6.57 4.91 6.57 6.74 6.74 6.74 6.74 9.38 6.74 6.74 6.74 5.71 6.73 9.38 6.96 5.71 6.96 6.51 5.71 5.18

2 12 3 1 2 21 29 4 24 3 14 14 2 2 4 3 3 1 3 3 2 1 1 9 1 2 1 3 2 1

40 315 236 99 79 250 318 297 138 195 80 74 220 153 299 191 187 82 188 172 113 109 98 92 70 184 86 175 222 84

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

39 124 103 99 58 101 149 103 32 113 43 32 125 94 102 87 83 82 98 98 59 109 98 92 70 102 86 82 145 88

100 100 100 100 100 100 100 100 100 100 99 99 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

IGHA2_HUMAN

36,503

5.71

2

243

100

166

100

B B B

fractions (Procedures D and E). Regarding the pellet fractions, several salivary proteins were identified such as cystatin SN, cystatin B and lysozyme (Fig. 2, band 48), Ig alpha-2 (Fig. 2, band 53). In addition, several forms of keratin (bands 35–39) were also identified in those extracts and presented a significant contribution in terms of optical densities. Comparing optical densities of Bis-Tris gel bands between PCD (Procedure D) and PCDS (Procedure E), only a slight increase (10%), in the band OD corresponding to amylase was observed (Fig. 2), further confirmed by western blot analysis (Fig. 3). Since this result was the most prominent, we considered this extract (PCDS) for further analysis by 2DE. Transposing to 2DE, as shown in Fig. S1, and similarly to previous studies [11,35–38], around 180 different spots were detected in 2DE profiles, independently of the treatment (Fig. S1A — CS, B — CD and C — CDS). Comparing the different treatments regarding optical densities of each detected spot, a lack of statistical differences was observed. However, it should be pointed out that 2DE use for the comparison of

proteins' relative abundance across sample sets has been hampered by several factors associated to gel-to-gel variation including the reproducibility in protein focusing, staining sensitivity, gel distortions and normalization of spot OD across gel sets. Thus and as previously reported [39], it is reasonable to assume a coefficient of variation (CV) of 20–30% in 2-DE [39], which is higher than those associated to typically one-dimension (1D) SDS-PAGE where all samples are loaded in a single gel. In fact, as observed in 1D-PAGE, we obtained variations within CV below 8% whereas in the case of 2-DE, our data showed a CV range of 25–35%. Nevertheless, a similar trend to Bis-Tris gels was observed if the sum of all spots OD with similar molecular weight in 2DE maps is considered.” The most outstanding result was the detection of about 480 different spots (including all detected spots in salivary 2DE profiles) in the pellet fraction after Procedure E (Fig. S1D-PCDS). From these, 366 spots were excised and 322 were positively identified by mass spectrometry (Table 1). Several salivary proteins such as amylase (spots 95–99 in Fig. S2,

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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 261, SN 271, C 278 in Fig. S2) were identified in this fraction. Besides salivary proteins, others belonging to distinct sources such as calmodulin (spots 246–247 in Fig. S2), peroxiredoxin (spot 208 in Fig. S2) or cornulin (spots 56–57 in Fig. S2), a squamous epithelia cell-specific protein, were also identified. In order to clarify the role of the identified proteins, a qualitative analysis was performed in terms of functional clusters. As can be observed in Fig. 4, according to the PANTHER classification system (http://www.pantherdb.org), identified proteins in 2DE maps are distributed in 14 different biological function categories being the top four biological function

categories are metabolic process (17%), response to stimulus (11%), immune response (12%) and cellular processes (17.6%). In these top four categories salivary proteins are included such as Ig alpha-2 chain C or cystatins A, B, C, SN and S, which comprise the body's first line of defense against pathogens [33]. Despite limitations of 2DE in the separation of high molecular weight proteins, using Bis-Tris system it is possible to increase the molecular weight separation range of proteins. Indeed, this system yielded the identification of low molecular weight components that are usually lost in second dimension separation with conventional systems (SDS-PAGE), such as histatin 1 (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.

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 51 4 0 –5 16 5

5155

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 besides saliva, like serum and squamous cells (Table 1). Regarding the contribution of bacteria, none of the excised spots was positively identified when searches were performed against bacteria taxonomy.

4.2.

Off-gel-based approach

In order to get high proteome coverage, a RP–RP system was used with the first RP column at pH 10 and the second at pH 2.6 [20], and two independent runs were carried out. The merge of both datasets led to the identification of a total of 489 distinct proteins with a score higher than 1.3 (95%), covering a high molecular weight and isoelectric point ranges. It should be pointed out that, with this approach and searching against the SwissProt human and bacteria protein database, 440 human proteins and 49 proteins from lactobacilli were identified (Supplemental Table 2). These 440 human proteins could be assigned into 17 functional categories using the PANTHER classification system (Fig. 5), being the top three biological functions are cellular processes (16.8%), metabolic processes (16%) and developmental processes (11%). A considerable higher number of proteins (489) were obtained using the off-gel approach when comparing with the gel-based approach (95 proteins). Analyzing the identified proteins in both approaches (Fig. 6), it is possible to notice that only 47 distinct proteins are common. Nevertheless, salivary analysis by 2DE is advantageous facing the complexity of saliva in terms of post-translational modified proteins. Indeed, several proteins present in their composition different glycans which could modify their migration in focusing step as well as in separation based on molecular weight as exemplified by others in salivary amylase before and after glycosidase

Fig. 7 – Comparison of obtained individual ratio values (log2) for 61 significantly expressed proteins (p < 0.05) between two independent iTRAQ experiments: CDS:CS (A) and CD:CS (B).

treatment [39]. In another example, cystatin S presents three phosphorylated sites [1,11,17,40], which justify the appearance of three spots (Fig. S2). Behind these specific examples, in 2DE it is also possible to monitor multiple spots identified as the same protein as showed for cystatin SN or cystatin B (Table 1). Although relative quantification analysis by Protein-Pilot 4.0 software comes with statistical analysis, since most methods are prone to technical variation, we included an additional 1.3-fold (log2 < ± 0.38) change cutoff for iTRAQ ratios. In addition, the analysis of the ratios of each individual variation within the first and second duplicate evidenced a significant positive correlation, which indicates a good technical sample preparation and a good analytical reproducibility (Fig. 7). Thus, out of all identified proteins only 61 (12%) differentially expressed proteins (p < 0.05) in CD, CDS and pellet (PCDS) versus CS (Table 2) were considered in this analysis, ensuring that only proteins with low individual variability have been selected. The real contribution of each protein to the pellet fraction is unknown since, to the best of our knowledge, pellet influence to whole saliva protein was never evaluated. Moreover, individual sample variability should be taken in consideration once it might lead to different pellet amounts. Nevertheless, treating saliva with chaotropic/detergent agents, as well as with ultrasounds (Table 2), relative increments in protein amounts were observed for CD and CDS suggesting an improvement in protein solubilization before the clearance step. In fact,

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

% Cov

Accession

Name Alpha-amylase 1 Keratin, type I cytoskeletal 13 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 6B Lysozyme C Ig alpha-1 chain C region Ig alpha-2 chain C region Cystatin-S Keratin, type I cytoskeletal 16 Polymeric immunoglobulin receptor Submaxillary gland androgen-regulated protein 3B Annexin A1 Cystatin-SN Protein S100-A9 Mucin-5B Zinc-alpha-2-glycoprotein Ig kappa chain C region Deleted in malignant brain tumors 1 protein Mucin-7 Carbonic anhydrase 6 Histatin-1 Cornulin Uncharacterized protein UNQ773/PRO1567 Serum albumin Uncharacterized protein C6orf58 Annexin A2 Protein S100-A8 Neutrophil defensin 3 Glyceraldehyde-3-phosphate dehydrogenase Histatin-3 Heat shock protein beta-1 Alpha-1-acid glycoprotein 2 Tubulin alpha-1B chain Serpin B4 Cysteine-rich secretory protein 3 Hemoglobin subunit beta Immunoglobulin J chain Tubulin beta-2B chain Mucin-21 Glucose-6-phosphate isomerase Alpha-2-macroglobulin-like protein 1 Bactericidal/permeability-increasing protein-like 1 Lactoperoxidase SH3 and multiple ankyrin repeat domains protein 3 Ig lambda chain C regions Alpha-actinin-1 Kallikrein-1 Myeloblastin Proline-rich protein 4 Pyruvate kinase isozymes M1/M2 Cathepsin G Plastin-3 Uncharacterized protein C4orf40 Leukocyte elastase Ig mu chain C region Alpha-enolase Short palate, lung and nasal epithelium carcinoma-associated protein 2 Integrin alpha-6

99.99 86.41 60.03 39.84 33.97 25.72 4.8 22.7 16.35 18.17 27.7

77.7 82.5 65.5 61 74.3 58.1 49.7 80.1 46.9 24.5 67.1

P04745 P13646 P19013 P04259 P61626 P01876 P01877 P01036 P08779 P01833 P02814

AMY1_HUMAN K1C13_HUMAN K2C4_HUMAN K2C6B_HUMAN LYSC_HUMAN IGHA1_HUMAN IGHA2_HUMAN CYTS_HUMAN K1C16_HUMAN PIGR_HUMAN SMR3B_HUMAN

16.04 6.93 14 18.97 12.78 6.63 12.15

38.4 86.5 64.9 13.2 27.1 34.9 26

P04083 P01037 P06702 Q9HC84 P25311 P01834 Q9UGM3

ANXA1_HUMAN CYTN_HUMAN S10A9_HUMAN MUC5B_HUMAN ZA2G_HUMAN IGKC_HUMAN DMBT1_HUMAN

11.75 10 12.14 8 10.62 11.31 8.63 6 6 4.03 4 1.72 4.01 2 4 4 2.18 4 5 2.01 2.02 2 6 4.04

22 18.8 57.9 16.2 32.2 16.1 22.4 16.5 25.8 31.9 12.5 49 32.7 8.5 10.9 11 14.3 17.7 21.9 12.4 18.7 7.9 5 13.8

Q8TAX7 P23280 P15515 Q9UBG3 Q96DA0 P02768 Q6P5S2 P07355 P05109 P59666 P04406 P15516 P04792 P19652 P68363 P48594 P54108 P68871 P01591 Q9BVA1 Q5SSG8 P06744 A8K2U0 Q8N4F0

MUC7_HUMAN CAH6_HUMAN HIS1_HUMAN CRNN_HUMAN YP003_HUMAN ALBU_HUMAN CF058_HUMAN ANXA2_HUMAN S10A8_HUMAN DEF3_HUMAN G3P_HUMAN HIS3_HUMAN HSPB1_HUMAN A1AG2_HUMAN TBA1B_HUMAN SPB4_HUMAN CRIS3_HUMAN HBB_HUMAN IGJ_HUMAN TBB2B_HUMAN MUC21_HUMAN G6PI_HUMAN A2ML1_HUMAN BPIL1_HUMAN

6.09 4.67

16.3 P22079 15.2 Q9BYB0

PERL_HUMAN SHAN3_HUMAN

2 4.6 2 2 3.7 2.02 2.24 3.05 2 2 2.03 2 2

19.1 7 9.9 16.8 35.8 8.5 23.1 4.5 16 7.9 17.9 9.4 6.4

LAC_HUMAN ACTN1_HUMAN KLK1_HUMAN PRTN3_HUMAN PROL4_HUMAN KPYM_HUMAN CATG_HUMAN PLST_HUMAN CD040_HUMAN ELNE_HUMAN IGHM_HUMAN ENOA_HUMAN SPLC2_HUMAN

1.94 3.67

P01842 P12814 P06870 P24158 Q16378 P14618 P08311 P13797 Q6MZM9 P08246 P01871 P06733 Q96DR5

7.3 P23229 7.6 Q9NYQ6

ITA6_HUMAN CELR1_HUMAN

Species Peptides CD:CS CDS:CS PCDS: (95%) CS HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN

184 151 94 58 38 38 36 31 29 25 24

2.1562 1.7756 1.5272 1.3891 2.8864 2.0699 7.5834 2.1457 2.2557 1.6366 4.5817

2.8913 0.9301 0.7632 1.1306 2.4838 4.1348 12.8725 2.8323 1.5636 2.0754 6.589

2.0335 9.4285 7.8792 9.0563 5.5283 1.2838 5.1703 3.0343 16.9849 0.9161 0.8619

HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN

24 22 20 17 16 12 11

1.6448 8.0051 1.9323 3.8191 2.2279 4.4615 2.8

0.5327 13.4003 1.5185 5.3122 3.8949 7.9137 3.9388

8.1994 3.1145 5.3145 2.8833 1.4113 2.9365 2.7417

HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN

10 9 9 9 9 7 6 6 6 5 5 5 5 4 4 4 4 4 4 4 3 3 3 3

1.9952 1.4861 1.2303 0.8509 0.8244 2.2848 1.8868 1.4452 1.4121 4.1915 2.6956 2.0857 1.6489 7.9877 5.2601 3.6065 2.8733 2.5314 1.0755 0.7371 4.7927 3.6801 1.207 1.1526

3.9834 1.9648 2.0994 1.0002 1.3477 4.373 3.8141 0.6493 1.4172 6.3838 3.0106 6.8662 1.3245 12.1331 4.0849 3.983 4.546 3.7634 1.3623 0.3647 4.6435 5.4776 1.7187 1.5726

1.2777 0.8507 0.7723 3.8163 0.6185 1.0323 1.6103 5.7016 2.8812 4.5262 7.9326 1.9177 6.3496 2.6351 18.5078 4.6398 1.7155 1.2827 1.5198 2.6531 13.4754 5.9701 2.9448 0.8253

HUMAN HUMAN

3 3

1.0905 0.7886

1.9304 0.7

0.8348 0.5071

HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN

2 2 2 2 2 2 2 1 1 1 1 1 1

1.38034 2.9476 2.6012 2.5041 2.3323 1.9549 0.9751 1.1042 4.4808 2.7032 2.2607 1.7561 0.8752

2.54565 7.137 4.5539 3.7409 3.5505 2.1548 1.0086 1.4565 5.5179 2.8111 2.757 2.2912 1.9618

4.6211 6.7622 2.2044 3.1117 0.5033 7.0789 3.9612 1.5078 3.079 6.0081 1.1295 3.83 0.8895

HUMAN HUMAN

1 1

0.5924 0.4802

0.4179 0.3591

0.8463 0.6157

5157

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 51 4 0 –5 16 5

Table 2 (continued) Protscore

1.89 1.53

% Cov

13.5 Q15772 8.1 Q8N7U6

Accession

SPEG_HUMAN EFHB_HUMAN

Name Cadherin EGF LAG seven-pass G-type receptor 1 Striated muscle preferentially expressed protein kinase EF-hand domain-containing family member B

glycoproteins such as MUC5B or MUC7, typically associated to the salivary complex trapping of other components [34], present relative increments after saliva treatment (in CD and CDS). Supporting this evidence is the presence of these glycoproteins and salivary proteins such as histatin 1, histatin 3, carbonic anhydrase 6, cystatin S and cystatin SN in relative significant amounts in PCDS. It should be noticed that iTRAQ labeling in off-gel analysis allows the identification of many low concentrated proteins by removing abundant salivary proteins when comparing to 2DE [41]. Moreover, saliva sample treatment with chaotropic/detergent agents plus sonication (CDS) [30] resulted in the solubilization of several proteins belonging to cytoskeleton or cellular signaling (Table 3). In fact, proteins from cytoskeleton such as keratins and tubulin were identified in 2DE maps from the pellet fraction. Although these proteins were not identified in 2DE maps of whole saliva (Fig. S1A) or in CD and CDS treatments (Fig. S1B and S1C), when these samples were analyzed by iTRAQ those structural proteins were found and with a relative protein increment in CD and CDS treatments. Supporting 2DE data, iTRAQ analysis evidenced a significant relative contribution of these proteins in the pellet fraction (PCDS). In opposition, proteins such as lactoperoxidade and cathepsin G showed a non-significant variation in iTRAQ analysis, either regarding sample fractions or treatment procedure. Albeit the presence of bacteria in saliva is recognized [42], the identification of proteins belonging to bacteria using proteomic approaches is less clear. As observed from our data, only a few bacterial proteins were detected. This is in-line with a recent paper devoted to the analysis of microbiota protein composition in human saliva. To address it, Rudney et al. [23] used a metaproteomic approach combining three different fractionations yielding in the identification of peptides belonging mainly to Firmicutes, in particular streptococci. Furthermore,

Table 3 – No. of identified salivary peptides by LC-MS/MS per class and procedure. Peptide class bPRP1 bPRP2 bPRP3 bPRP4 aPRP histatin 1 histatin 3 statherin SMR3B

TFA 35 7 7 13 24 27 17 27 23

Saliva 17 53 13 10 35 26 14 25 29

Species Peptides CD:CS CDS:CS PCDS: (95%) CS

Saliva (CD)

Saliva (CDS)

54 6 10 13 26 39 9 60 32

69 17 9 9 44 37 22 38 21

HUMAN

1

0.2772

0.3106

0.2458

HUMAN

1

0.1962

0.132

0.5399

no significant contributions of each procedure for bacterial protein identification were observed with iTRAQ analysis.

4.3.

Peptidome analysis

Similar to other bodily fluids, saliva contains several protein species of low molecular weight, comprising around 40–50% of the total secreted proteins [1]. We have previously analyzed the salivary peptidome [13,43] and identified several fragments from the most abundant protein classes. Taking in consideration the importance of salivary peptides for oral cavity homeostasis, we also introduced a procedure to extract peptides after saliva treatment with chaotropic/detergents based on acetone precipitation/acid extraction (Procedure G, Fig. 1) as previously described [43,44]. The evaluation of this procedure started by the analysis of extracts by Bis-Tris gel and was compared with the traditional peptide extraction procedure involving the addition of TFA to saliva, which results in the enrichment of a large amount of salivary peptides [12]. As can be depicted in Fig. 2 (lane 4), a large contribution of high molecular weight components such as amylase (Fig. 2, lane 4, band 2), Ig alpha-1 chain C (Fig. 2, lane 4, band 1) remains soluble after TFA precipitation, although in lower amounts comparing to CS saliva (Fig. 2, lane 1) based on OD analysis. Although a band corresponding to amylase was detected in extracts obtained from CS, CD and CDS following Procedure G, a depletion of high molecular weight components was noticeable (Fig. 2, lanes 7–9). Furthermore, an OD increment of 15% was detected in the range of 5–15 kDa for procedure G. Each extract was subsequently analyzed by LC-MS/MS resulting in the identification of more than 150 different peptides belonging to the main salivary classes (statherin, PRPs (aPRP, bPRP1, bPRP2, bPRP3, bPRP4), histatins 1 and 3 and 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 the increment in the number of peptides belonging to bPRP1 and aPRP in CD and CDS. However, distribution of identified peptides based on Gravy score (Fig. 8) showed a similar distribution among all extracts. Despite the heterogeneity observed in the number of peptides in each class (Table 3), with iTRAQ labeling, 136 different peptides were assigned as common to all extracts (Table 4). Comparing the number of peptides labeled with iTRAQ with all the ones identified by LC-MS/MS, a decrease in their number was observed which could be attributed to the fact that few of them are over dynamic range of iTRAQ being not considered for analysis. Furthermore, many other small

Protscore

% cov accession

Name

15.01

59.7 P02808

Statherin

88.8 P04280

Basic PRP1

13

17

Sequence

96 99 99 97 99 99 99 97 97 98 95

FGYGY FGYGYGPY GYGPYQPVPEQPL GYGYGPY GYGYGPYQPVPEQPL RFGYGYGPYQPVPEQPLYPQPYQPQ YQPVPEQPL YQQYTF FLRR IGRF QYQQYTF

96 96 96 98

QYTF RIGRF GGRPSRPPQ GPPAQGGSK

98 GPPPPGKPQ 97 GPPPPGKPQ 95 GPPPPPGKPQ 96 GPPPPPGKPQ 95 GPPPPPGKPQ 96 GPPPPPGKPQ 97 GPPPQGDK 99 GPPPQGGNQPQ 99 GPPQQEGNNPQ 98 GPPQQGGNRPQ 99 GPPRPPQGGRPSRPPQ 98 PQQPQAPPAGQPQGPPRPPQGGRPSRPPQ 97 SPPGKPQ 99 SPPGKPQGPPPQGGNQPQ 99 SPPGKPQGPPPQGGNQPQ

ΔMass

Modifications

iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term; Amidated@C-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term; iTRAQ8plex(K)@9 iTRAQ8plex@N-term; iTRAQ8plex(K)@7 Formyl@N-term; iTRAQ8plex(K)@7 No iTRAQ8plex@N-term; iTRAQ8plex(K)@8 iTRAQ8plex@N-term; iTRAQ8plex(K)@8 Acetyl@N-term; iTRAQ8plex(K)@8 Carbamyl@N-term; iTRAQ8plex(K)@8 iTRAQ8plex@N-term; iTRAQ8plex(K)@8 iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term; Deamidated(Q)@29 iTRAQ8plex@N-term; iTRAQ8plex(K)@5 iTRAQ8plex@N-term; iTRAQ8plex(K)@5 No iTRAQ8plex@N-term; iTRAQ8plex(K)@5

Theor m/z

CDS: TFA

CD: TFA

CS: TFA

−0.06 −0.11 −0.12 −0.01 0.04 −0.18 0.01 0.00 0.00 −0.01 0.13

910.46 1227.60 1748.92 1080.53 1969.00 3273.63 1374.76 1153.58 895.58 796.50 1280.66

2.5312 2.5548 2.1736 2.54 2.8275 3.8212 3.234 5.6727 2.4507 3.9981 1.8684

1.9883 1.8928 1.722 1.3931 1.5496 1.6225 2.0957 2.5643 2.5918 1.7334 2.0125

2.4538 2.4437 1.8916 2.0718 2.0538 3.1573 2.0602 4.3415 3.5961 2.6506 2.5049

0.00 0.00 0.00 −0.01

862.46 952.60 1255.72 1406.82

6.7144 1.6928 7.8037 12.846

3.5633 1.6457 4.0943 2.7775

4.2836 2.1725 5.087 5.792

0.00

1482.89

6.0115

1.8266

2.6419

0.01

1206.68

0.9416

1.2026

1.1877

0.01

1275.74

0.985

0.5997

0.6269

−0.01

1579.94

7.0423

2.2092

2.9095

−0.10

1317.75

0.5226

0.9464

1.4376

0.05

1318.74

11.3197

2.8913

6.9305

0.00

1403.81

12.2455

3.4189

5.0803

0.00 0.00 0.00 −0.01 −0.01

1380.72 1469.73 1439.77 1985.11 3310.75

5.9458 3.117 5.851 6.3212 9.6551

2.9747 1.603 2.5332 2.1287 0.8354

3.0907 1.6115 2.5055 2.9221 1.0717

−0.01

1318.79

3.436

1.0214

2.2402

−0.02

2376.29

20.0082

4.5356

6.1751

0.00

2072.08

3.0186

1.9832

1.9713

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 51 4 0 –51 6 5

15.84

Peptides (95%) conf

5158

Table 4 – iTRAQ peptide evaluation for Procedures F and G (CS, CD and CDS).

20.5

92.1 P02812

Basic PRP2

23

99 GPPPQGDNK 99 GPPPQGGSK 96 GGRPSRPPQ 96 GPPPPGKPQ 97 GPPPPGKPQ 96 GPPPPGKPQGPPPQGDN

95 GPPPPGKPQGPPPQGDN

97 GPPPPPGKPQ 98 GPPPPPGKPQ 95 GPPPPPGKPQ 99 GPPPQGDK 98 GPPPQGDNK 99 GPPPQGDNKSRSSR

99 GPPPQGGNQPQ 96 GPPPQGGSK 99 GPPQQEGNNPQ 99 GPPRPPQGGRPSRPPQ 97 IAGNPQGAPPQGGN 98 PQQPQAPPAGQPQGPPRPP QGGRPSRPPQ 98 SPPGKPQ 99 SPPGKPQGPPPQGGNQPQ 99 SPPGKPQGPPPQGGNQPQ 20.5

92.1 P02812

23

99 GPPPQGDNK

1517.85

18.2385

3.7761

6.157

−0.01

1432.84

15.4593

3.503

4.9798

0.00 0.01

1255.72 1206.68

4.647 0.9416

2.2432 1.2026

2.8817 1.1877

0.00

1482.89

15.8497

3.8873

6.6429

0.02

2259.23

12.2629

3.4656

3.4086

0.08

2262.20

44.4381

−0.10

1317.75

0.5226

0.9464

1.4376

0.05

1318.74

11.3197

2.8913

6.9305

−0.01

1579.94

6.6494

1.7084

2.7236

0.01

1275.74

0.985

0.5997

0.6269

0.00

1403.81

12.2455

3.4189

5.0803

0.00

1517.85

8.4371

2.441

3.4611

0.00

1866.91

2.6731

1.0182

0.565

0.00 −0.01

1380.72 1432.84

5.9458 8.1095

2.9747 2.1158

3.0907 3.4734

0.00 −0.01 −0.01

1469.73 1985.11 1743.88

3.117 6.3212 6.6196

1.603 2.1287 8.2348

1.6115 2.9221 0.6849

−0.14

3310.75

9.6551

0.8354

1.0717

−0.01

1318.79

11.3197

2.8913

6.9305

−0.02

2376.29

20.0082

4.5356

6.1751

−0.01

2072.08

10.0395

5.2778

6.3392

−0.02

1517.85

18.2385

3.7761

6.157

12.12

13.7576

(continued on next page)

5159

Basic PRP2

−0.02

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 51 4 0 –5 16 5

95 GPPPPPGKPQ

iTRAQ8plex@N-term; iTRAQ8plex(K)@9 iTRAQ8plex@N-term; iTRAQ8plex(K)@9 iTRAQ8plex@N-term Formyl@N-term; iTRAQ8plex(K)@7 iTRAQ8plex@N-term; iTRAQ8plex(K)@7 iTRAQ8plex@N-term; iTRAQ8plex(K)@7; Methyl(Q)@14 iTRAQ8plex@N-term; iTRAQ8plex(K)@7; Oxidation(P)@13; Deamidated(Q)@14 Acetyl@N-term; iTRAQ8plex(K)@8 Carbamyl@N-term; iTRAQ8plex(K)@8 iTRAQ8plex@N-term; iTRAQ8plex(K)@8 No iTRAQ8plex@N-term; iTRAQ8plex(K)@8 iTRAQ8plex@N-term; iTRAQ8plex(K)@8 iTRAQ8plex@N-term; iTRAQ8plex(K)@9 No iTRAQ8plex@N-term; iTRAQ8plex(K)@9; Phospho(S)@13 iTRAQ8plex@N-term iTRAQ8plex@N-term; iTRAQ8plex(K)@9 iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term; Hex(N)@14 iTRAQ8plex@N-term; Deamidated(Q)@29 iTRAQ8plex@N-term; iTRAQ8plex(K)@5 iTRAQ8plex@N-term; iTRAQ8plex(K)@5 No iTRAQ8plex@N-term; iTRAQ8plex(K)@5 iTRAQ8plex@N-term; iTRAQ8plex(K)@9

Protscore

% cov accession

Name

Peptides (95%) conf

Sequence

99 GPPPQGGSK 96 GGRPSRPPQ 96 GPPPPGKPQ 97 GPPPPGKPQ 96 GPPPPGKPQGPPPQGDN

95 GPPPPPGKPQ 97 GPPPPPGKPQ 98 GPPPPPGKPQ 95 GPPPPPGKPQ 99 GPPPQGDK 98 GPPPQGDNK 99 GPPPQGDNKSRSSR

99 GPPPQGGNQPQ 96 GPPPQGGSK 99 GPPQQEGNNPQ 99 GPPRPPQGGRPSRPPQ 97 IAGNPQGAPPQGGN 98 PQQPQAPPAGQPQGPPRPPQ GGRPSRPPQ 98 SPPGKPQ 99 SPPGKPQGPPPQGGNQPQ 99 SPPGKPQGPPPQGGNQPQ

iTRAQ8plex@N-term; iTRAQ8plex(K)@9 iTRAQ8plex@N-term Formyl@N-term; iTRAQ8plex(K)@7 iTRAQ8plex@N-term; iTRAQ8plex(K)@7 iTRAQ8plex@N-term; iTRAQ8plex(K)@7; Methyl(Q)@14 iTRAQ8plex@N-term; iTRAQ8plex(K)@7; Oxidation(P)@13; Deamidated(Q)@14 Acetyl@N-term; iTRAQ8plex(K)@8 Carbamyl@N-term; iTRAQ8plex(K)@8 iTRAQ8plex@N-term; iTRAQ8plex(K)@8 No iTRAQ8plex@N-term; iTRAQ8plex(K)@8 iTRAQ8plex@N-term; iTRAQ8plex(K)@8 iTRAQ8plex@N-term; iTRAQ8plex(K)@9 No iTRAQ8plex@N-term; iTRAQ8plex(K)@9; Phospho(S)@13 iTRAQ8plex@N-term iTRAQ8plex@N-term; iTRAQ8plex(K)@9 iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term; Hex(N)@14 iTRAQ8plex@N-term; Deamidated(Q)@29 iTRAQ8plex@N-term; iTRAQ8plex(K)@5 iTRAQ8plex@N-term; iTRAQ8plex(K)@5 No iTRAQ8plex@N-term; iTRAQ8plex(K)@5

Theor m/z

CDS: TFA

CD: TFA

CS: TFA

−0.01

1432.84

15.4593

3.503

4.9798

0.00 0.01

1255.72 1206.68

4.647 0.9416

2.2432 1.2026

2.8817 1.1877

0.00

1482.89

15.8497

3.8873

6.6429

0.02

2259.23

12.2629

3.4656

3.4086

0.08

2262.20

44.4381

−0.10

1317.75

0.5226

0.9464

1.4376

0.05

1318.74

11.3197

2.8913

6.9305

−0.01

1579.94

6.6494

1.7084

2.7236

0.01

1275.74

0.985

0.5997

0.6269

0.00

1403.81

12.2455

3.4189

5.0803

0.00

1517.85

8.4371

2.441

3.4611

0.00

1866.91

2.6731

1.0182

0.565

0.00 −0.01

1380.72 1432.84

5.9458 8.1095

2.9747 2.1158

3.0907 3.4734

0.00 −0.01 −0.01

1469.73 1985.11 1743.88

3.117 6.3212 6.6196

1.603 2.1287 8.2348

1.6115 2.9221 0.6849

−0.14

3310.75

9.6551

0.8354

1.0717

−0.01

1318.79

11.3197

2.8913

6.9305

−0.02

2376.29

20.0082

4.5356

6.1751

−0.01

2072.08

10.0395

5.2778

6.3392

12.12

13.7576

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 51 4 0 –51 6 5

95 GPPPPGKPQGPPPQGDN

ΔMass

Modifications

5160

Table 4 (continued)

10.07

33.7 Q04118

Basic PRP3

12

99 GGRPHRPPQGQPPQ 99 GPPPPPQGGRPHRPPQGQPPQ 99 QSLNEDVSQEESPSVISGKPEGR 96 RPHRPPQGQPPQ 95 GGRPHRPPQGQPPQ 97 GKPEGR 96 GPPPHPGKPQ 96 GRPHRPPQGQPPQ 96 PPPPQGGRPHRPP 98 QSLNEDVSQEESPSVISGKP

99 QSLNEDVSQEESPSVISGKPEGR

9.88

55.2 P10163

Basic PRP4

9

99 GGRPPRPAQGQQPPQ 99 GPPPPPQGGRPPRPAQGQQPPQ 99 LISGKPEGR 97 RPQGGNQPQR 97 GKPEGR 97 GPPPHPGKPE

96 GPPPPGKPQ 98 GPPPPGKPQ 98 SPPGKPQ 31.09

67.1 P02814

SMR3B

22

1812.99 2483.33 2759.36

10.7251 14.5012 6.1051

3.4056 5.6332 3.635

4.0274 5.3149 5.7312

−0.01 −0.01 −0.01

1698.95 1812.99 1251.76

13.1281 2.1215 19.8941

3.0983 0.8669 5.0111

3.7095 1.4152 7.0254

−0.01

1619.95

8.811

1.8202

4.1798

−0.01 1.83 0.00

1755.97 1693.96 2455.15

7.2527 0.729 1.0261

1.714 0.5531 0.6851

2.3976 0.3584 0.7083

−0.11

2741.35

8.1343

3.2673

6.2617

0.04

2839.33

6.3063

3.4361

6.4223

0.00 −0.22 −0.02

1875.03 2545.37 1564.96

8.0664 8.0197 9.2707

3.392 3.8326 0.8772

3.827 4.6859 1.1029

0.00 −0.01

1441.79 1251.76

7.6733 19.8941

3.9161 5.0111

5.7411 7.0254

−0.01

1619.95

8.811

1.8202

4.1798

0.01

1206.68

0.9416

1.2026

1.1877

−0.01

1482.89

7.8598

2.1899

4.147

−0.01

1318.79

11.3197

2.8913

6.9305

−0.09 −0.02 −0.15 −0.10 −0.02 −0.11 0.00 −0.19 0.00 −0.01 0.00

2627.41 1778.99 1836.01 1350.77 1990.09 1504.85 1101.62 1738.95 3405.81 1465.85 1465.85

8.8238 2.2737 2.7853 1.3742 8.1009 2.6169 5.7772 1.156 8.1062 2.0128 4.8553

3.0757 1.3848 1.2446 0.8587 2.7944 2.4418 3.1118 0.8315 4.3326 0.6659 1.461

3.5905 1.6401 1.4193 0.5474 3.183 2.5038 3.5181 0.8515 5.8779 0.8756 1.7307

(continued on next page)

5161

99 APPQPFGPGFVPPPPPPPYGPGR 97 FVPPPPPPPYGPGR 97 GFVPPPPPPPYGPGR 95 GIFPPPPPQP 99 GPGFVPPPPPPPYGPGR 99 GPGIFPPPPPQP 80 GPYPPGPL 99 GPYPPGPLAPPQPF 99 GPYPPGPLAPPQPFGPGFVPPPPPPPYGPGR 95 GRIPPPPPAPY 99 GRIPPPPPAPY

−0.01 −0.02 0.00

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 51 4 0 –5 16 5

99 QSLNEDVSQEESPSVISGKPEGR

iTRAQ8plex@N-term iTRAQ8plex@N-term Gln->pyro-Glu@N-term; iTRAQ8plex(K)@19 iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term; iTRAQ8plex(K)@2 iTRAQ8plex@N-term; iTRAQ8plex(K)@8 iTRAQ8plex@N-term iTRAQ8plex@N-term Gln->pyro-Glu@N-term; Cation:K(E)@11; iTRAQ8plex(K)@19 Gln->pyro-Glu@N-term; Dehydrated(S)@8; iTRAQ8plex(K)@19 Gln->pyro-Glu@N-term; Phospho(S)@8; iTRAQ8plex(K)@19 iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term; iTRAQ8plex(K)@5 iTRAQ8plex@N-term iTRAQ8plex@N-term; iTRAQ8plex(K)@2 iTRAQ8plex@N-term; iTRAQ8plex(K)@8; Amidated@C-term Formyl@N-term; iTRAQ8plex(K)@7 iTRAQ8plex@N-term; iTRAQ8plex(K)@7 iTRAQ8plex@N-term; iTRAQ8plex(K)@5 iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term

5162

Protscore

64.41

% cov accession

95.8 P02810

Name

aPRP

Peptides (95%) conf

40

Sequence

Modifications

99 QPFGPGFVPPPPPPPYGPGR 97 RGPYPPGPLAPPQPF 99 RGPYPPGPLAPPQPFGPGFVPPPPPPPYGPGR 99 YPPGPLAPPQPFGPGFVPPPPPPPYGPGR 93 APPQPFGPGFVPPPPPPPYGPGR 99 GPLAPPQPFGPGFVPPPPPPPYGPGR 99 GPYPPGPLAPPQPFGPGFVPPPPPPPYGPGR 99 LAPPQPFGPGFVPPPPPPPYGPGR 99 PGPLAPPQPFGPGFVPPPPPPPYGPGR 99 RGPYPPGPLAPPQPFGPGFVPPPPPPPYGPGR 99 YPPGPLAPPQPFGPGFVPPPPPPPYGPGR 99

iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term DGGDSEQFIDEER

99 99

GGDSEQFIDEER GGDSEQFIDEER

99 97 97

GGHPPPPQGRPQ GGRPQGPPQGQSPQ GPPPPPPGKPQ

96 99 98 99 99 99 99 99 99 99

GPPPQGGRPQ GPPPQGGRPQGPPQGQSPQ GPPQGQSPQ GPPQQGGHP GPPQQGGHPPPPQGR GPPQQGGHPPPPQGRPQ GPPQQGGHPRPP GPPQQGGHPRPPR GPPQQGGHQQ GRPQGPPQQGGHQ

ΔMass

−0.13 −0.18 0.01 −0.08 −1.10 −0.36 −0.40 −0.33 −1.30 −0.35 −0.39 iTRAQ8plex@N-term; Phospho(S)@5 iTRAQ8plex@N-term iTRAQ8plex@N-term; Phospho(S)@4 iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term; iTRAQ8plex(K)@9 iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term

Theor m/z

CDS: TFA

CD: TFA

CS: TFA

2362.26 1895.05 3561.91 3251.73 2627.41 2894.57 3405.81 2740.49 2991.62 3561.91 3251.73 0.00

1.866 2.9055 5.7167 13.704 6.9453 10.7916 9.6094 2.7422 0.3593 5.6668 8.9695 1880.79

0.9118 0.9968 2.5686 4.9346 2.6073 5.8341 7.0175 1.5213 0.4315 1.8944 3.1086 3.3568

1.1063 1.2553 4.2356 8.7672 2.8396 5.7283 9.648 1.4156 0.343 2.896 5.6263 1.991

−0.01 −0.01

1685.79 1765.76

3.5056 5.6359

1.6872 2.4177

−0.01 −0.01 −0.01

1528.83 1694.89 1676.99

4.2839 13.6596 2.7131

2.1403 5.3195 0.784

0.00 0.00 0.00 0.00 −0.02 −0.01 0.00 0.00 0.00 −0.01

1294.72 2171.13 1199.63 1178.62 1810.96 2036.07 1528.83 1684.93 1337.69 1647.86

5.9031 7.6799 6.6635 2.8621 14.2761 5.6124 4.5761 11.8249 5.6506 9.2082

3.5311 3.5452 2.8578 2.1231 3.2827 1.5873 2.6965 3.7167 2.968 3.658

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 51 4 0 –51 6 5

Table 4 (continued)

99 99

97 98

FIDEER GPPPPPPGKPQ

99

98 95

GPPPPPPGKPQGPPPQGGRPQ GPPQGQSPQ GPPPPPPGKPQGPPPQGGRPQ GPPQGQSPQ GPPQGQSPQ GPPQGQSPQ

99 95 98

GPPQQGGHPPPPQGRPQ GPPQQGGHPPPPQGRPQ GRPQGPPQGQSP

97 97 99 97 99

GRPQGPPQQ GRPQGPPQQGGH GRPQGPPQQGGHPRPPR GRPQGPPQQGGHQQ DGGDSEQFIDEER

99 99

GGDSEQFIDEER GGDSEQFIDEER

99

99

iTRAQ8plex@N-term iTRAQ8plex@N-term; iTRAQ8plex(K)@23 iTRAQ8plex@N-term; iTRAQ8plex(K)@23

0.01 −0.02

1775.92 3129.69

4.6827 17.6073

3.5427 3.7939

−0.02

4101.19

35.5568

7.5486

iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term; Oxidation(D)@1; Phospho(S)@2 iTRAQ8plex@N-term iTRAQ8plex@N-term; iTRAQ8plex(K)@9 No iTRAQ8plex@N-term; iTRAQ8plex(K)@9 No iTRAQ8plex@N-term; iTRAQ8plex(K)@9 iTRAQ8plex@N-term iTRAQ8plex@N-term; Amidated@C-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term; iTRAQ8plex(S)@11; Oxidation(P)@12 iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term iTRAQ8plex@N-term; Phospho(S)@5 iTRAQ8plex@N-term iTRAQ8plex@N-term; Phospho(S)@4

−0.01 −0.02 −0.01 0.00 0.00

1910.04 1932.02 1718.90 1718.90 1667.71

4.0669 9.5097 15.0219 9.6835 3.6536

1.0998 1.8093 1.8392 1.4374 1.3368

0.00 0.00

1112.59 1676.99

6.4584 10.6486

1.7087 2.8207

0.00

3220.69

9.4287

4.2216

−0.06

3220.69

10.9522

5.4427

0.00 0.01

1199.63 1198.65

1.7041 8.9578

0.7278 4.1687

−0.31 −0.02 −0.19

2036.07 2036.07 1830.01

7.5491 4.5999 6.0046

3.3435 1.8051 3.1339

0.00 0.00 −0.32 −0.01 0.00

1268.70 1519.80 2123.16 1775.92 1880.79

5.3657 3.6905 13.6373 6.8084 3.3568

3.385 2.6306 4.2703 3.2573 1.991

−0.01 −0.01

1685.79 1765.76

3.5056 5.6359

1.6872 2.4177

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 51 4 0 –5 16 5

99 99 95 99 96

GRPQGPPQQGGHQQ GRPQGPPQQGGHQQGPPPP PPGKPQ GRPQGPPQQGGHQQGPPPPP PGKPQGPPP QGGRPQ PQGPPQQGGHPRPPR RGRPQGPPQQGGHQQ RPQGPPQQGGHQQ RPQGPPQQGGHQQ DSEQFIDEER

5163

5164

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 51 4 0 –51 6 5

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].

REFERENCES Fig. 8 – Box plot of Gravy score of identified peptides in Procedure F (TFA), Procedure G from A (CS), Procedure G from B (CD) and Procedure G from C (CDS).

peptides were considered for analysis. This resides in the fact that iTRAQ peptide derivatization increases the molecular weight allowing the detection and identification of small peptides usually excluded due to limitations in MALDI ionization step. For instance, the peptide sequences GPYPPGPL with m/z 796.41 (P–B peptide (SMR3B)) and GKPEGR with m/z 642.34 (bPRP4) excluded in routine LC-MALDI analysis were identified after iTRAQ labeling. Peptide semi-quantitation showed, in general, that Procedure G allowed the extraction of higher amounts of each peptide, being more pronounced in CDS saliva. The extraction of salivary peptides by Procedure G showed even better results in terms of number of different peptides, as well as distinct quantities of each peptide extracted when compared with the traditional TFA extraction. In addition, this procedure allowed the analysis of salivary peptides even after addition of urea, as well as detergents, in order to promote better protein solubilization. Thus, the high number of peptides and quantity found for CD and CDS treatment in extracts obtained from Procedure G emphasizes the importance of salivary complexes in trapping components which could lead to false conclusions. To the best of our knowledge, this is the first work using iTRAQ labeling devoted to the analysis of peptides extracted after salivary complex disruption.

5.

Conclusion

Overall, data suggest that a considerable amount of protein/ peptides is lost when a clearance step with centrifugation is performed to saliva samples. Besides all those expected proteins from epithelial cells and bacteria, many salivary proteins like amylase, mucins, cystatins and histatin were identified in the pellet fraction, either using in-gel and off-gel approaches. Considering the good reproducibility obtained in proteome analysis with iTRAQs, we suggest the use of this strategy for protein and peptide quantification in saliva. Based on our results, we strongly recommend the addition of urea to saliva immediately after its collection followed by a sonication step for the proteome/peptidome analysis.

[1] Amado FM, Vitorino RM, Domingues PM, Lobo MJ, Duarte JA. Analysis of the human saliva proteome. Expert Rev Proteomics 2005;2:521-39. [2] Kawas SA, Rahim ZH, Ferguson DB. Potential uses of human salivary protein and peptide analysis in the diagnosis of disease. Arch Oral Biol 2012;57:1-9. [3] Stone MD, Chen X, McGowan T, Bandhakavi S, Cheng B, Rhodus NL, et al. Large-scale phosphoproteomics analysis of whole saliva reveals a distinct phosphorylation pattern. J Proteome Res 2011;10:1728-36. [4] Messana I, Inzitari R, Fanali C, Cabras T, Castagnola M. Facts and artifacts in proteomics of body fluids. What proteomics of saliva is telling us? J Sep Sci 2008;31:1948-63. [5] Schipper R, Silletti E, Vingerhoeds M. Saliva as research material: biochemical, physicochemical and practical aspects. Arch Oral Biol 2007;52:1114-35. [6] Oppenheim FG, Salih E, Siqueira WL, Zhang W, Helmerhorst EJ. Salivary proteome and its genetic polymorphisms. Ann N Y Acad Sci 2007;1098:22-50. [7] Chevalier F, Hirtz C, Chay S, Cuisinier F, Sommerer N, Rossignol M, et al. Proteomic studies of saliva: a proposal for a standardized handling of clinical samples. Clin Proteomics 2007;3:13-21. [8] Yan W, Apweiler R, Balgley BM, Boontheung P, Bundy JL, Cargile BJ, et al. Systematic comparison of the human saliva and plasma proteomes. Proteomics Clin Appl 2009;3:116-34. [9] Xie H, Rhodus NL, Griffin RJ, Carlis JV, Griffin TJ. A catalogue of human saliva proteins identified by free flow electrophoresis-based peptide separation and tandem mass spectrometry. Mol Cell Proteomics 2005;4:1826-30. [10] Hu S, Xie Y, Ramachandran P, Ogorzalek Loo RR, Li Y, Loo JA, et al. Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry. Proteomics 2005;5:1714-28. [11] Wilmarth PA, Riviere MA, Rustvold DL, Lauten JD, Madden TE, David LL. Two-dimensional liquid chromatography study of the human whole saliva proteome. J Proteome Res 2004;3: 1017-23. [12] Messana I, Cabras T, Pisano E, Sanna MT, Olianas A, Manconi B, et al. Trafficking and postsecretory events responsible for the formation of secreted human salivary peptides: a proteomics approach. Mol Cell Proteomics 2008;7:911-26. [13] Vitorino R, Barros A, Caseiro A, Domingues P, Duarte J, Amado F. Towards defining the whole salivary peptidome. Proteom Clin Appl 2009;3:528-40. [14] Vitorino R, Lobo MJ, Duarte JA, Ferrer-Correia AJ, Domingues PM, Amado FM. Analysis of salivary peptides using HPLC-electrospray mass spectrometry. Biomed Chromatogr 2004;18:570-5. [15] de Jong EP, van Riper SK, Koopmeiners JS, Carlis JV, Griffin TJ. Sample collection and handling considerations for

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 51 4 0 –5 16 5

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

peptidomic studies in whole saliva; implications for biomarker discovery. Clin Chim Acta 2011;412:2284-8. Snyder SL, Sobocinski PZ. An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines. Anal Biochem 1975;64:284-8. Vitorino R, Lobo MJ, Ferrer-Correira AJ, Dubin JR, Tomer KB, Domingues PM, et al. Identification of human whole saliva protein components using proteomics. Proteomics 2004;4: 1109-15. Vitorino R, de Morais Guedes S, Ferreira R, Lobo MJ, Duarte J, Ferrer-Correia AJ, et al. Two-dimensional electrophoresis study of in vitro pellicle formation and dental caries susceptibility. Eur J Oral Sci 2006;114:147-53. Tastet C, Lescuyer P, Diemer H, Luche S, van Dorsselaer A, Rabilloud T. A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteins. Electrophoresis 2003;24:1787-94. Manadas B, English JA, Wynne KJ, Cotter DR, Dunn MJ. Comparative analysis of OFFGel, strong cation exchange with pH gradient, and RP at high pH for first-dimensional separation of peptides from a membrane-enriched protein fraction. Proteomics 2009;9:5194-8. Gan CS, Chong PK, Pham TK, Wright PC. Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ). J Proteome Res 2007;6:821-7. Topkas E, Keith P, Dimeski G, Cooper-White J, Punyadeera C. Evaluation of saliva collection devices for the analysis of proteins. Clin Chim Acta 2012. Rudney JD, Xie H, Rhodus NL, Ondrey FG, Griffin TJ. A metaproteomic analysis of the human salivary microbiota by three-dimensional peptide fractionation and tandem mass spectrometry. Mol Oral Microbiol 2010;25:38-49. White MR, Helmerhorst EJ, Ligtenberg A, Karpel M, Tecle T, Siqueira WL, et al. Multiple components contribute to ability of saliva to inhibit influenza viruses. Oral Microbiol Immunol 2009;24:18-24. Denny PC, Denny PA, Klauser DK, Hong SH, Navazesh M, Tabak LA. Age-related changes in mucins from human whole saliva. J Dent Res 1991;70:1320-7. Quintana M, Palicki O, Lucchi G, Ducoroy P, Chambon C, Salles C, et al. Inter-individual variability of protein patterns in saliva of healthy adults. J Proteomics 2009;72:822-30. Duan X, Young R, Straubinger RM, Page B, Cao J, Wang H, et al. A straightforward and highly efficient precipitation/on-pellet digestion procedure coupled with a long gradient nano-LC separation and Orbitrap mass spectrometry for label-free expression profiling of the swine heart mitochondrial proteome. J Proteome Res 2009;8:2838-50. Martins D, Menezes de Oliveira B, dos Santos Farias A, Oka Horiuchi RS, Crepaldi Domingues C, de Paula E, et al. The use of ASB-14 in combination with CHAPS is the best for solubilization of human brain proteins for two-dimensional gel electrophoresis. Briefings in Functional Genomics and Proteomics, 6; 2007. p. 70-5.

5165

[29] Chen EI, Cociorva D, Norris JL, Yates JR. Optimization of mass spectrometry-compatible surfactants for shotgun proteomics. J Proteome Res 2007;6:2529-38. [30] Gorg A, Weiss W, Dunn MJ. Current two-dimensional electrophoresis technology for proteomics. Proteomics 2004;4:3665-85. [31] Gundry RL, White MY, Murray CI, Kane LA, Fu Q, Stanley BA, et al. Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. In: Ausubel FM, et al, editor. Current Protocols in Molecular Biology; 2009. Chapter 10:Unit10 25. [32] Graham DRM, Garnham CP, Fu Q, Robbins J, Van Eyk JE. Improvements in two-dimensional gel electrophoresis by utilizing a low cost “in-house” neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteomics 2005;5:2309-14. [33] Amado F, Lobo MJ, Domingues P, Duarte JA, Vitorino R. Salivary peptidomics. Expert Rev Proteomics 2010;7:709-21. [34] Iontcheva I, Oppenheim FG, Troxler RF. Human salivary mucin MG1 selectively forms heterotypic complexes with amylase, proline-rich proteins, statherin, and histatins. J Dent Res 1997;76:734-43. [35] Fleissig Y, Deutsch O, Reichenberg E, Redlich M, Zaks B, Palmon A, et al. Different proteomic protein patterns in saliva of Sjogren's syndrome patients. Oral Dis 2009;15:61-8. [36] Neyraud E, Sayd T, Morzel M, Dransfield E. Proteomic analysis of human whole and parotid salivas following stimulation by different tastes. J Proteome Res 2006;5:2474-80. [37] Haigh BJ, Stewart KW, Whelan JR, Barnett MP, Smolenski GA, Wheeler TT. Alterations in the salivary proteome associated with periodontitis. J Clin Periodontol 2010;37:241-7. [38] Hu S, Loo JA, Wong DT. Human saliva proteome analysis. Ann N Y Acad Sci 2007;1098:323-9. [39] Sondej M, Denny P, Xie Y, Ramachandran P, Si Y, Takashima J, et al. Glycoprofiling of the human salivary proteome. Clin Proteomics 2009;5:52-68. [40] Hu S, Loo JA, Wong DT. Human body fluid proteome analysis. Proteomics 2006;6:6326-53. [41] Besson D, Pavageau AH, Valo I, Bourreau A, Belanger A, Eymerit-Morin C, et al. A quantitative proteomic approach of the different stages of colorectal cancer establishes OLFM4 as a new nonmetastatic tumor marker. Mol Cell Proteomics 2011;10 (M111 009712). [42] Levine M. Susceptibility to dental caries and the salivary proline-rich proteins. Int J dentistry 2011;2011:953412. [43] Vitorino R, Barros AS, Caseiro A, Ferreira R, Amado F. Evaluation of different extraction procedures for salivary peptide analysis. Talanta 2012 May 30;94:209-15. [44] Kawashima Y, Fukutomi T, Tomonaga T, Takahashi H, Nomura F, Maeda T, et al. High-yield peptide-extraction method for the discovery of subnanomolar biomarkers from small serum samples. J Proteome Res 2010;9:1694-705.