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. 2020 Sep 14;16:335. doi: 10.1186/s12917-020-02550-w

In-gel digestion coupled with mass spectrometry (GeLC-MS/MS)-based salivary proteomic profiling of canine oral tumors

Sekkarin Ploypetch 1,2, Sittiruk Roytrakul 3, Narumon Phaonakrop 3, Suthathip Kittisenachai 3, Kantinan Leetanasaksakul 3, Sirinun Pisamai 4, Chanin Kalpravidh 4, Anudep Rungsipipat 2,5, Gunnaporn Suriyaphol 1,2,✉
PMCID: PMC7489029  PMID: 32928212

Abstract

Background

Various types of oral tumors, either benign or malignant, are commonly found in dogs. Since saliva directly contacts the tumors and saliva collection is non-invasive, easily accessible and cost effective, salivary biomarkers are practical to be used for the diagnosis and/or prognosis of these diseases. However, there is limited knowledge of protein expression in saliva for canine oral tumors. The present study aimed to investigate novel biomarkers from the salivary proteome of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP), using an in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). The relationships between protein candidates and chemotherapy drugs were explored and the expression of potential biomarkers in saliva and tissues was verified by western blot analysis.

Results

For saliva samples, increased expression of protein tyrosine phosphatase non-receptor type 5 (PTPN5) was shown in all tumor groups compared with the CP group. Marked expression of PTPN5 was also observed in LOM and OSCC compared with that in BN and EOM. In addition, tumor protein p53 (p53), which appeared in the PTPN5–drug interactions, was exhibited to be expressed in all tumor groups compared with that in the CP group. For tissue samples, increased expression of p53 was shown in LOM compared with the control group.

Conclusion

PTPN5 and p53 were proposed to be potential salivary biomarkers of canine oral tumors.

Keywords: Dog, In-gel digestion coupled with mass spectrometry (GeLC-MS/MS), Oral tumors, Tumor protein p53 (p53), Protein tyrosine phosphatase non-receptor type 5 (PTPN5)

Background

Head and neck tumors comprise approximately 7% of all tumors in dogs. Among these, oral melanoma (OM) and oral squamous cell carcinoma (OSCC) are most commonly found [1]. The tumor, node and metastasis (TNM) classification of tumors in the oral cavity are described. Stages I and II refer to tumors with ≤2 cm and 2 to < 4 cm, respectively, defined as early clinical stages with no metastasis, whereas stage III refers to a tumor with ≥4 cm and/or lymph node metastasis and stage IV refers to a tumor with distant metastasis. The latter two are defined as late clinical stages and are most frequently observed in the animal hospital owing to the difficulty in routinely examining tumors in dogs’ mouths [2–4]. After surgical resection, patients with late clinical stage are normally treated with chemotherapy drugs such as carboplatin, a derivative of the anticancer drug cisplatin, doxorubicin (or Adriamycin®), cyclophosphamide and piroxicam. With a high rate of metastasis and recurrence of oral cancer, novel biomarkers are important for early clinical diagnosis, screening and prognosis of the diseases [5]. Saliva proteins have high potential to be appropriate biomarkers because saliva makes direct contact with an oral mass, and saliva collection is non-invasive and not difficult to manipulate [6]. Novel salivary proteome biomarkers have been discovered in human oral tumors [7–10]. However, in dogs with oral diseases, the evidence of proteomics in saliva is still limited [6]. The present study aimed to search for novel suitable biomarkers in saliva of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), periodontitis (P) and healthy controls (C) (CP group), using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Associations of disease-related proteins with the chemotherapy drugs cisplatin, cyclophosphamide, piroxicam and doxorubicin were exhibited. The candidate protein expressions in saliva and tissues were affirmed by western blot analysis.

Results

GeLC-MS/MS results

A total of 3726 proteins were identified. The distribution of the individual and overlapped proteins in EOM, LOM, OSCC, BN and CP groups was illustrated by a Venn diagram (Fig. 1). In addition, the molecular function, biological process, cellular component and the relative expression levels of the proteins uniquely expressed in each group and commonly expressed in all cancerous groups was analysed using the PANTHER software tools (Tables 1 and 2 and Supplementary Table S1). For the networks of protein–protein and protein–chemotherapy drug interactions, analysed by the Stitch program, version 5.0, edge confidence scores demonstrated the strength of the interactions at the functional level. Pathways with high edge confidence scores (> 0.700) were presented as thick lines. The associations of protein tyrosine phosphatase non-receptor type 5 (PTPN5) and tumor protein p53 (p53) with cisplatin and doxorubicin drugs were shown. Additionally, the correlation of PTPN5 and cyclophosphamide was demonstrated (Fig. 2). In the present study, increased expression of another protein involved in the SUMOylation process, RanBP2, was noted in a cancerous group (Table 2). RanBP2 regulated translocation of p53, a well-known target of SUMOylation, to the cytoplasm, leading to poor prognosis and prostate cancer progression [11].

Fig. 1.

Fig. 1

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Venn diagram of proteins differentially expressed in early-stage OM (EOM), late-stage OM (LOM), oral squamous cell carcinoma (OSCC), benign oral tumors (BN) and normal and periodontitis (CP). Circles indicate overexpressed proteins uniquely found in each group and commonly found in all cancerous groups

Table 1.

Overexpressed proteins uniquely found in normal controls and periodontitis, benign oral tumors, early-stage oral melanoma, late-stage oral melanoma and oral squamous cell carcinoma based on biological process involvement and protein score

Database Protein name Protein ID score Peptides Biological process Subcellular distribution
Normal controls and periodontitis
 XP_016007048.1 Semaphorin-4B isoform X1 13.9 QLVASYCPK

1. Negative chemotaxis

2. Semaphorin–plexin signalling pathway

1. Extracellular space

2. Integral component of plasma membrane
 XP_011988340.1 Visual system homoeobox 1 isoform X2 16.98 FPGRPLPSAARQK

1. Multicellular organism development

2. Regulation of transcription

1. Nucleus

2. Cytoskeleton
 XP_013973434.1 CDK5 regulatory subunit-associated protein 2 isoform X1 12.52 FTNQGKR Microtubule organizing center
 XP_002689199.3 Olfactory receptor 2 M5 26.19 MCWQVAAMSWAGGAR Olfaction Plasma membrane
 XP_008048855.1 Potassium voltage-gated channel subfamily Q member 1 34.67 LNIEDFR

1. Potassium ion export across plasma membrane

2. Cellular response to cAMP

1. Endoplasmic reticulum

2. Endosome

3. Plasma membrane
 XP_007125871.1 GLIPR1-like protein 1 14.03 AHNEAR Single fertilization Plasma membrane
 EHB15707.1 Transient receptor potential cation channel subfamily M member 5 26.14 TVAPKSLLFR Ion transmembrane transport Plasma membrane
Benign oral tumors
 KFO21119.1 Germ cell-less protein-like 1 7.86 KAVAAR Cell differentiation Nucleus
 XP_004629194.1 Poly [ADP-ribose] polymerase 12 21.09 KLGMSSELVHR Protein auto-ADP-ribosylation Nucleus
 XP_015289690.1 Lamin tail domain-containing protein 2 8.98 GLLPPMSSGK Cell population proliferation

1. Cytoskeleton

2. Nucleus
 XP_012868232.1 Telomeric repeat-binding factor 2-interacting protein 1 16.48 AEPDPEAAESVEPQTK

1. Negative regulation of DNA recombination at telomere

2. Positive regulation of NF-κB transcription factor activity

 Nucleus
 XP_012373519.1 Myb-related protein B 16.69 MLPGRYVPGGGVGAR

1. Mitotic cell cycle

2. Regulation of cell cycle

 Nucleus
 XP_012865682.1 Erythrocyte membrane protein band 4.2 12.59 QWSAVVEDR

1. Cell morphogenesis

2. Hemoglobin metabolic process

Cytoskeleton

1. Cytoplasm

2. Membrane
 XP_005371197.1 Long-chain-fatty-acid–CoA ligase ACSBG2 5.91 APGTGFLTEMLR cell differentiation
Early-stage oral melanoma
 XP_011760132.1 Putative protein SSX6 12.53 GGNMPGPTGCVR Regulation of transcription, DNA-templated Nucleus
 XP_004326275.1 Bromodomain testis-specific protein-like 14.28 DNAKPMNYDEKR Chromatin remodelling Nucleus
 XP_006868797.1 Zinc finger protein GLI2-like 16.61 GGSLENSSIPDLSR Nucleic acid binding Nucleus
Late-stage oral melanoma
 EPQ15807.1 Transformation/transcription domain-associated protein 9.28 AMAILTPAVPAR

1. DNA repair

2. Histone deubiquitination

1. Golgi apparatus

2. Nucleus
 XP_009240233.1 Glutathione S-transferase-like 20.93 ARISHILTINK Glutathione transferase activity Cytoplasm
 XP_011282224.1 Protein FAM186A 32.14 SVEQSFLELLIEEDR No data

1. Nucleus

2. Cytoplasm
 XP_004412391.1 Deleted in lung and oesophageal cancer protein 1 7.49 AGPPKNK Negative regulation of cell population proliferation Cytoplasm
Oral squamous cell carcinoma
 XP_007944568.1 Ankyrin repeat domain-containing protein 26-like 6.56 ADIKENMVIDMQANCMILXK Protein interaction Cytoplasm
 XP_012392091.1 Cytohesin-4 isoform X2 9.84 YPGELSSGEAEELQR Regulation of ARF protein signal transduction Nucleus
 XP_007532207.2 Probable C-mannosyltransferase DPY19L4 17.69 KPKSSGNK Protein C-linked glycosylation via 2′-alpha-mannosyl-L-tryptophan Membrane
 EHB17858.1 Dynein heavy chain 11, axonemal 3.80 ATSEMR Determination of left/right symmetry Cytoskeleton
 XP_004275614.1 Fanconi anaemia-associated protein of 100 kDa 7.99 XGMDDR Interstrand cross-link repair Nucleus
 OBS77059.1 Protein A6R68_16468 7.01 DQVSDDVSVQSSGPNCQR Regulation of transcription by RNA polymerase II Nucleus
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Table 2.

Overexpressed proteins commonly found in early-stage oral melanoma, late-stage oral melanoma and oral squamous cell carcinoma based on biological process involvement and protein score

Database Protein name Protein ID score Peptides Biological process Subcellular distribution
XP_005376885.1 ATP synthase subunit s, mitochondrial isoform X1 4.77 HQTMLFGK ATP biosynthetic process Mitochondria
XP_004411845.1 Carbonic anhydrase 12 isoform X1 33.40 SLHAAAVLLLLCFK Carbonate dehydratase activity Integral component of membrane
XP_015354861.1 Cell division cycle-associated protein 2 17.63 RSFCAPTLSSK Cell cyclecell division Nucleus
XP_004625867.1 dihydroorotate dehydrogenase (quinone), mitochondrial 17.17 IPIIGVGGVSSGQDAMDK ‘de novo’ UMP biosynthetic process Mitochondrion inner membrane
XP_014948096.1 Hermansky–Pudlak syndrome 3 protein isoform X1 9.93 ACPPISMDVCALR Organelle organization,pigmentation Cytosol
XP_004644982.1 KN motif and ankyrin repeat domain-containing protein 3 14.22 FALNQNLPDLGGSR Negative regulation of actin filament polymerization Cytoplasm
XP_008158631.1 Leucocyte immunoglobulin-like receptor subfamily A member 6 3.43 EPAEVEELK Adaptive immune response Membrane
XP_003787787.1 Negative elongation factor C/D 7.47 SNFIMMN Transcription by RNA polymerase II Nucleus
XP_011285357.1 Neurexin-2-β 13.66 VVVVLGGQGSSG Neuron cell–cell adhesion signal transduction Membrane
XP_005629058.1 Origin recognition complex subunit 1 isoform X1 6.66 SRPTPSHPATPRAK DNA replication,mitotic cell cycle Nucleus
XP_006896914.1 Phosphoenolpyruvate carboxykinase, cytosolic [GTP] isoform X1 18.32 ARVSQM Gluconeogenesis Cytosol
XP_004620060.1 Phospholipase B1, membrane-associated-like 11.55 RMENNSGINFNEDWK Phospholipase activity Integral component of membrane
XP_012626009.1 Progesterone receptor isoform X2 17.75 VLLLLNTTR DNA-binding transcription factor activity Nucleus
XP_008151988.1 Secernin-2 13.13 QGGITAEAMMDILRDK Exocytosis Extracellular exosome
XP_007489730.1 Sodium/iodide cotransporter 6.99 DSKEYPQEVK Cellular response to cAMP Membrane
XP_016811442.1 T-box transcription factor TBX18 isoform X2 12.54 MYSGELGPI DNA-binding transcription factor activity Nucleus
XP_004045865.1 Uncharacterized protein LOC101132572 12.64 RFTLSLDAPAPTQGVCK Unknown Unknown
XP_006190947.1 Zinc finger protein ZIC 3 8.6 THTGKGEGGR Cell differentiation Nucleus
XP_011744397.1 28S ribosomal protein S14, mitochondrial 16.97 KNTXLPK Mitochondrial translational elongation and translation Mitochondria
XP_007505382.1 3-hydroxyisobutyrate dehydrogenase, mitochondrial isoform X1 8.97 SMASKTPVGFVGLGNMGNPMAK 3-hydroxyisobutyrate dehydrogenase activity Mitochondria
XP_004448347.1 α-ketoglutarate-dependent dioxygenase alkB homolog 4 isoform X1 7.08 LVSLNLLSSTVLSMSR Demethylation Mitochondria
XP_005065718.1 Ankyrin repeat domain-containing protein 34B 20.75 QKALMTTNGPK Unknown Nucleus
NP_036833.1 β1 adrenergic receptor 13.02 QGFSSESK Adenylate cyclase-activating adrenergic receptor signalling pathway Endosome,plasma membrane
ELK12127.1 Cytochrome b-c1 complex subunit 2, mitochondrial 11.51 DNMAYTGEGLR Aerobic respiration Mitochondria
XP_006883886.1 E3 SUMO-protein ligase RanBP2 11.07 LSQSGHMLINLSRGK centrosome localization Nucleus
BAD96349.1 Heme oxygenase (decyclizing) 2 variant 11.2 KSSGALEK Heme oxygenase (decyclizing) 2 variant Endoplasmic reticulum
OBS70980.1 Pyrroline-5-carboxylate reductase 9.86 LTAFXPAPK L-proline biosynthetic process Mitochondria
XP_015976454.1 Laminin subunit α1 15.83 YXNGTWYK Cell adhesion Extracellular region or secreted
KFO28259.1 Mitochondrial import receptor subunit TOM20 like protein 10.02 LFSVQMPLAKLPTTGQR Protein import into mitochondrial matrix Mitochondria
EAW72809.1 Signal sequence receptor, delta (translocon-associated protein delta), isoform CRA_c 3.09 APTQAPMR Regulate the retention of ER resident proteins Endoplasmic reticulum
XP_006865897.1 Tyrosine-protein phosphatase non-receptor type 5 21.9 AEGLRGSHR Cellular response to cytokine stimulus Endoplasmic reticulum
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Fig. 2.

Fig. 2

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Involvement of tyrosine-protein phosphatase non-receptor type 5 (PTPN5) and tumor protein p53 (TP53) in networks of protein chemotherapy drug interactions, cisplatin and doxorubicin, analysed by Stitch, version 5.0; a Interactions of PTPN5 and TP53 with cisplatin; b Interactions of PTPN5 and TP53 with doxorubicin; c Interactions of PTPN5 and TP53 with cyclophosphamide. Red circles: PTPN5 and TP53. Abbreviations: ataxia telangiectasia mutated (ATM), breast cancer 4721, early onset (BRCA1), cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A), cyclin-. 34,473 dependent kinase inhibitor 2A (CDKN2A), CREB binding protein (CREBBP), E1A binding 474 protein p300 (EP300), K (lysine) acetyltransferase 2B (KAT2B), mitogen-activated protein kinase 4758 (MAPK8), Mdm2 (MDM2) and sirtuin 1 (SIRT1).

Western blot analysis results

Western blot analysis unveiled an enhanced expression of PTPN5 and p53 in saliva of tumor groups compared with that in the CP group (Figs. 3 and 4). In addition, the expression of PTPN5 in LOM and OSCC was augmented compared with that in BN and EOM (Fig. 3). For tissue samples, we did not detect PTPN5 antibody binding to the tissue proteins (Data not shown). For the p53 western blotting, increased expression of p53 was observed in LOM compared with the control group (Fig. 5). Peptide sequences of PTPN5 and p53 western blot analysis were verified by LC-MS/MS (Fig. 6).

Fig. 3.

Fig. 3

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Western blot analysis of salivary tyrosine-protein phosphatase non-receptor type 5 (PTPN5) of dogs with benign oral tumors (BN), early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC) and periodontitis and normal controls (CP); a Representative western blot for PTPN5 at 57–68 kDa; b bar graph of ratios of PTPN5 protein intensity to total blotted proteins in each lane in a membrane; a-b and c-d denote a significant difference at P < 0.05; e-f denote a significant difference at P < 0.001; g-h denote a significant difference at P < 0.0001

Fig. 4.

Fig. 4

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Western blot analysis of salivary tumor protein p53 (p53) of dogs with benign oral tumors (BN), early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC) and periodontitis and normal controls (CP); a representative western blot for P53 at 53 kDa; b bar graph of ratios of P53 protein intensity to total blotted proteins in each lane in a membrane; a-b denote a significant difference at P < 0.05; a-c denote a significant difference at P < 0.01; a-d denote a significant difference at P < 0.001

Fig. 5.

Fig. 5

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Western blot analysis of tumor protein p53 (p53) in tissues of dogs with benign oral tumors (BN), late-stage oral melanoma (LOM), oral squamous cell carcinoma (OSCC) and normal controls (C); a representative western blot for P53 at 53 kDa; b bar graph of ratios of P53 protein intensity to total blotted proteins in each lane in a membrane; a-b denote a significant difference at P < 0.05

Fig. 6.

Fig. 6

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Verification of expressed protein sequences by LC-MS/MS; a MS/MS fragmentations of LRVISLR found in salivary tyrosine-protein phosphatase non-receptor type 5 (PTPN5); b ALPPSTSSSPPQK found in salivary tumor protein p53 (p53)

Discussion

In the present study, GeLC-MS/MS was used to identify novel salivary biomarker candidates in canine oral tumors. PTPN5 and p53 were plausibly shown to be candidates in LOM and OSCC. PTP is a group of protein tyrosine phosphatases that have divergent functions, either promoting or suppressing cancer. Several oncogenic PTPs have been reported to be highly expressed in human breast cancer [12]. In contrast to receptor-type PTPs that localized to the plasma membranes, the non-receptor type PTPs, PTPNs, are located in the cytosol. PTPN5 is in the same non-receptor Cys-based classical PTPs as PTPN1 and PTPN11, which promoted tumorigenesis in ovarian cancer, gastric cancer, prostate cancer, breast cancer, leukaemia, colorectal cancer and uveal melanoma [13–19]. PTPN1 has been reported to be increased in canine oral cancer tissues by MALDI-TOF MS plus LC-MS/MS [20]. PTPN1 functioned via Src/Ras/Erk and PI3K/Akt pathways, whereas PTPN11 functioned via EGFR/Ras/MAPK pathways [15, 17, 21–23]. To the best of our knowledge, this study presented for the first time the association of salivary PTPN5 expression and canine oral cancers, particularly LOM and OSCC. Since most families of PTPs served as biomarker targets of several anticancer drugs, including PTPN11, PTPN6 and PTP1B, potential inhibitors of PTPN as candidate anticancer drugs for oral tumors should be investigated [24]. In the present study, we did not observe the expression of PTPN5 in any tissue proteins by western blotting. The plausible explanation included the expression of PTPN5 in saliva was not originated from the tumor tissues while proteins in saliva can be produced from salivary glands or can also be transferred from systemic circulation [25].

In the present study, we also exhibited the enhanced expression of p53, in tumor groups, particularly in saliva of LOM and OSCC and in tissues of LOM group. Likewise, p53 was found in the interaction networks of PTPN5 and the chemotherapy drugs cisplatin and doxorubicin. p53 is a tumor suppressor protein; however, mutant p53 protein has been shown to be a biomarker in several cancers, such as human breast cancer, colorectal cancer, ovarian cancer, oesophageal squamous cell carcinoma, non-small cell lung cancer, and a prognostic marker in breast cancer, oesophageal squamous cell carcinoma, colon cancer, non-small cell lung cancer and B cell lymphoma [26–33]. In human head and neck squamous cell carcinoma, p53 mutation played an important role in tumorigenesis and progression. It has been used not only as a risk and prognostic biomarker, but also as a predictive biomarker in the clinical response to chemotherapy treatments [34–38]. Several studies, aiming to treat cancer in humans, have investigated the promoting function of wild-type p53 and degradation of mutant p53 [29, 39, 40]. Further investigation of p53 in canine oral tumors for potential prognostic and therapeutic biomarkers should be performed.

In the present study, increased expression of another protein involved in the SUMOylation process, RanBP2, was noted in a cancerous group (Table 2). In our previous study of salivary proteomics of canine oral tumors using MALDI-TOF MS and LC-MS/MS, the expression of sentrin-specific protease 7 (SENP7) was found to be increased in saliva of dogs with BN, EOM, LOM and OSCC. And according to the western blot analysis to validate MS results in individual samples, the enhanced expression of SENP7 has been observed in LOM and OSCC, compared with that in CP and BN [6]. SENP7 functions to edit the poly-small ubiquitin-related modifier (SUMO) chains during SUMOylation, a post-translational modification of target proteins involving in several carcinogenic mechanisms [41]. In the present study using the same samples with the previous one, we found the expression of predicted SENP7 (Accession number: XP_008265236.1) in CP, BN, EOM and LOM groups but not in the OSCC group (Additional file 1). And this is probably due to different MS techniques and data analysis methods including different sample preparations, ionization approaches, and statistical analysis [20]. For MALDI-TOF MS coupled with LC-MS/MS, unique PMF peak spectra were previously selected by ClinProTools program before being sequenced by LC-MS/MS. For GeLC-MS/MS, all proteins were loaded into the SDS-PAGE, trypsinized and applied to LC-MS/MS. Proteins was quantitated using DeCyder MS Differential Analysis software, searched against the NCBI mammal database using MASCOT software and grouped by jvenn diagram. And that is the reason why we require traditional protein detection methods such as western blots to confirm the proteomic results.

Conclusion

The present study used GeLC-MS/MS and western blotting to reveal the potential salivary biomarkers of canine oral tumors, PTPN5 and p53. The network interactions between the candidate proteins and chemotherapy drugs were also demonstrated. For future work, signalling pathways and potential inhibitors of the target proteins should be investigated as potential anticancer drugs for canine oral tumors.

Methods

Animals

Saliva samples were recruited from dogs with EOM (n = 5), LOM (n = 24), OSCC (n = 10) and BN (n = 11) (age range 7–14 years) whereas tissue samples were taken from 11 LOM, 9 OSCC and 9 BN dogs. Patient characteristics were shown in Tables 3 and 4. Patients were scheduled for surgical operations at the Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University and private animal hospitals. They were diagnosed with no prior history of treatments with chemotherapy and/or radiotherapy. The TNM staging of OM and OSCC were determined according to the WHO, whereby EOM and LOM include stages 1–2 and 3–4, respectively [42, 43]. Regional lymph nodes were examined cytologically for metastasis. Tumor spreading to abdominal organs was checked by an ultrasound examination. Skull-to-abdomen radiography was performed by a Brivo DR-F digital X-ray system (GE Healthcare, Chicago, IL, USA) or an Optima CT660 64-slice CT scanner (GE Healthcare). Seven saliva samples and 10 normal gingival tissue samples were obtained from healthy dogs with no history or clinical signs of oral cavity or cancers (age range 7–8 years). A chronic periodontitis group contained 5 dogs showing gingivitis, dental tartar and/or periodontal attachment loss (age range 7–13 years). The sample collection protocol was approved by the Chulalongkorn University Animal Care and Use Committee (CU-ACUC), Thailand (Approval number 1631042) and written informed consents were obtained from all dog owners.

Table 3.

Patient characteristics of the saliva of canine samples

Sample no. Groupsa Histological examination Age (y) Sexb Breed
1 Control Normal gingiva 8 F Beagle
2 Control Normal gingiva 8 F Beagle
3 Control Normal gingiva 8 F Beagle
4 Control Normal gingiva 8 F Beagle
5 Control Normal gingiva 8 F Beagle
6 Control Normal gingiva 8 F Beagle
7 Control Normal gingiva 8 F Beagle
8 Periodontitis Gingival hyperplasia 10 Mc Mixed
9 Periodontitis Gingival hyperplasia 12 Fs Golden Retriever
10 Periodontitis Gingival hyperplasia 10 M Mixed
11 Periodontitis Gingival hyperplasia 9 M Pomeranian
12 Periodontitis Gingival hyperplasia 14 Fs Shi-tsu
13 Benigh oral tumor Peripheral odontogenic fibroma 7 Fs Poodle
14 Benigh oral tumor Acanthomatous ameloblastoma 10 F Shi-tsu
15 Benigh oral tumor Acanthomatous ameloblastoma 11 F Labrador retriever
16 Benigh oral tumor Peripheral odontogenic fibroma 10 Mc Mixed
17 Benigh oral tumor Peripheral odontogenic fibroma 10 M Poodle
18 Benigh oral tumor Peripheral odontogenic fibroma 8 Mc Siberian husky
19 Benigh oral tumor Peripheral odontogenic fibroma 10 Fs Siberian husky
20 Benigh oral tumor Peripheral odontogenic fibroma 9 Y M Shi-Tzu
21 Benigh oral tumor Peripheral odontogenic fibroma 14 Y M Golden Retriever
22 Benigh oral tumor Peripheral odontogenic fibroma 2 Y F Golden Retriever
23 Benigh oral tumor Acanthomatous ameloblastoma 11 Fs Golden Retriever
24 OSCC well differentiated 11 M Mixed
25 OSCC well differentiated 13 Fs Cocker spaniel
26 OSCC poorly differentiated 9 M Shi-tsu
27 OSCC well differentiated 14 Fs Pug
28 OSCC poorly differentiated 15 Mc Poodle
29 OSCC well differentiated 11 Fs Poodle
30 OSCC well differentiated 11 M Mixed
31 OSCC poorly differentiated 12 F Bangkeaw
32 OSCC well differentiated 12 F Mixed
33 OSCC poorly differentiated 11 M Mixed
34 Early-stage OM (I) Melanotic melanoma 10 M Poodle
35 Early-stage OM (I) Amelanotic melanoma 14 M Mixed
36 Early-stage OM (II) Melanotic melanoma 10 Fs Mixed
37 Early-stage OM (II) Melanotic melanoma 11 M chihuahua
38 Early-stage OM (II) Amelanotic melanoma 12 M Poodle
39 Late-stage OM (III) Melanotic melanoma 12 M Pug
40 Late-stage OM (III) Melanotic melanoma 12 M Labrador retriever
41 Late-stage OM (IV) Melanotic melanoma 14 M Cocker spaniel
42 Late-stage OM (III) Melanotic melanoma 8 M Schnauzer
43

Late-stage OM

(III)

 Amelanotic melanoma 11 M Poodle
44

Late-stage OM

(III)

 Melanotic melanoma 15 M Shi-tsu
45 Late-stage OM (III) Melanotic melanoma 13 Fs Golden Retriever
46 Late-stage OM (III) Melanotic melanoma 14 M Mixed
47 Late-stage OM (III) Melanotic melanoma 13 F Poodle
48 Late-stage OM (III) Melanotic melanoma 12 M Pomeranian
49 Late-stage OM (IV) Melanotic melanoma 15 M Golden Retriever
50 Late-stage OM (III) Amelanotic melanoma 13 M Cocker spaniel
51 Late-stage OM (III) Melanotic melanoma 14 M Golden Retriever
52 Late-stage OM (III) Melanotic melanoma 12 M Mixed
53 Late-stage OM (III) Amelanotic melanoma 10 M Mixed
54 Late-stage OM (III) Melanotic melanoma 14 M Mixed
55 Late-stage OM (III) Melanotic melanoma 15 M Poodle
56 Late-stage OM (III) Melanotic melanoma 8 M Golden Retriever
57 Late-stage OM (III) Melanotic melanoma 10 Fs Beagle
58 Late-stage OM (III) Amelanotic melanoma 10 M Mixed
59 Late-stage OM (III) Amelanotic melanoma 8 M Mixed
60 Late-stage OM (III) Amelanotic melanoma 12 Fs Dachshund
61 Late-stage OM (IV) Melanotic melanoma 14 M Poodle
62 Late-stage OM (III) Melanotic melanoma 12 F Golden Retriever
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Clinical stages are in parentheses

aOM Oral melanoma, OSCC Oral squamous cell carcinoma

bM Male, Mc Male castration, F Female, Fs Female spray

Table 4.

Patient characteristics of the canine gingival tissues

Sample no. Groupsb Histological examination Age (year) Sexb Breed
1 Control Normal gingiva 8 F mixed
2 Control Normal gingiva 6 M mixed
3 Control Normal gingiva 7 M mixed
4 Control Normal gingiva 4 Mc Beagle
5 Control Normal gingiva 1 F mixed
6 Control Normal gingiva 8 F Beagle
7 Control Normal gingiva 8 F Beagle
8 Control Normal gingiva 8 F Beagle
9 Control Normal gingiva 8 F Beagle
10 Control Normal gingiva 8 F Beagle
11 benign oral tumor Acanthomatous ameloblastoma 8 Fs Rottweiler
12 benign oral tumor Acanthomatous ameloblastoma 9 Mc mixed
13 benign oral tumor Peripheral odontogenic fibroma 6 Mc Shi-Tzu
14 benign oral tumor Acanthomatous ameloblastoma 7 F Beagle
15 benign oral tumor Acanthomatous ameloblastoma 8 Fs Chi hua hua
16 benign oral tumor Peripheral odontogenic fibroma 9 M Shi-Tzu
17 benign oral tumor Peripheral odontogenic fibroma 14 M Golden Retriever
18 benign oral tumor Peripheral odontogenic fibroma 2 F Golden Retriever
19 benign oral tumor Acanthomatous ameloblastoma 6 M Mixed
20 OSCC poorly differentiated 10 F Mixed
21 OSCC well differentiated 17 Fs Shi-Tzu
22 OSCC poorly differentiated 10 M Mixed
23 OSCC well differentiated 3 M Shi-Tzu
24 OSCC well differentiated 11 M Schnauzer
25 OSCC well differentiated 10 M mixed
26 OSCC well differentiated 15 Fs Miniature pinscher
27 OSCC well differentiated 10 Mc mixed
28 OSCC well differentiated 10 M Shi-Tzu
29

Late-stage OM

(IV)

 Amelanotic melanoma 12 Fs Mixed
30

Late-stage OM

(IV)

 Melanotic melanoma 13 F English cocker spaniel
31

Late-stage OM

(III)

 Melanotic melanoma 10 Fs Mixed
32

Late-stage OM

(III)

 Amelanotic melanoma 10 M Labrador Retriever
33

Late-stage OM

(III)

 Melanotic melanoma 14 M Golden Retriever
34

Late-stage OM

(III)

 Amelanotic melanoma 11 M Mixed
35

Late-stage OM

(III)

 Melanotic melanoma 10 Fs Poodle
36

Late-stage OM

(III)

 Melanotic melanoma 9 Fs Rottweiler
37

Late-stage OM

(III)

 Melanotic melanoma 12 M Mixed
38

Late-stage OM

(III)

 Amelanotic melanoma 10 F Shi-Tzu
39

Late-stage OM

(III)

 Melanotic melanoma 12 Mc German Shepherd
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Clinical stages are in parentheses

aOM Oral melanoma, OSCC Oral squamous cell carcinoma

bM Male, Mc Male castration, F Female, Fs Female spray

Sample collection and preparation

Saliva was collected on the day of surgery without stimulation. Dogs were fasted for at least 1 h and their mouths were rinsed with 0.9% sterile saline solution [9]. Whole saliva (0.5–1.0 mL) was collected for 5–10 min using a sterile cotton swab. After centrifugation at 2600×g for 15 min at 4 °C [44], Halt protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) was added to 200 μL of supernatant and samples were kept at − 20 °C until analysis. Total protein concentrations were determined by the Lowry method, using bovine serum albumin as a protein standard [45]. According to our previous peptide profiles obtained from MALDI-TOF MS data, showing the control and chronic periodontitis in the same cluster, control and chronic periodontitis samples were consequently combined as a CP group [6]. For the tissues, samples were kept in RNALater solution at − 20 °C until use.

Analysis of salivary peptides by GeLC-MS/MS

Salivary peptides were analysed by GeLC-MS/MS as previously described with some modifications [20]. Briefly, 50 μg of pooled samples in each group (CP, BN, EOM, LOM and OSCC) were mixed with loading buffer [0.5 M dithiothreitol (DTT), 10% w/v SDS, 0.4 M Tris-HCl pH 6.8, 50% v/v glycerol, 0.1 mg/ml Bromophenol Blue] and boiled at 90 °C for 5 min prior to separating on 12.5% SDS-PAGE (Atto, Tokyo, Japan). Gels were fixed using 50% methanol, acetic acid and 37% formaldehyde and stained with silver nitrate solution, before being scanned using a GS-710 scanner (Bio-Rad Laboratories, Benicia, CA, USA) and stored in 0.1% acetic acid. After that in-gel tryptic digestion was performed where protein bands in each lane were divided into 17 segments and chopped into 1 mm3 pieces. Gel pieces were dehydrated using 100% acetonitrile (ACN) and dried. Cysteines were reduced and alkylated by 10 mM DTT in 10 mM ammonium bicarbonate and 100 mM iodoacetamide in 10 mM ammonium bicarbonate, respectively, prior to dehydrating twice in 100% ACN. After trypsin digestion in 50 mM NH4HCO3 (pH 7.8) overnight at 37 °C, peptides were extracted from the gels using 50% ACN in 0.1% formic acid (FA). Pooled samples were submitted to a reversed-phase high performance liquid chromatography (HPLC). The gradient-eluted peptides were analysed using an Ultimate 3000 LC System coupled to an HCTUltra PTM Discovery System (Bruker Daltonics, Bremen, Germany). Peptides were separated on a PepSwift monolithic column (100 μm internal diameter × 50 mm) (Thermo Fisher Scientific). Peptide separation was achieved with a linear gradient at a flow rate of 1000 nL/min from 4% ACN, 0.1% FA to 70% ACN, 0.1% FA for 7.5 min with a regeneration step at 90% ACN, 0.1% FA and an equilibration step at 4% ACN, 0.1% FA. The entire process took 20 min. Peptide fragment mass spectra were acquired in a data-dependent Auto MS mode with a scan range 400–1500 m/z. However, in the case of having more than 5 precursor fragments, peptides would be selected from the MS scan at 200–2800 m/z. CompassXport software (Bruker Daltonics) was used to convert data from LC-MS/MS into the mzXML format. Protein quantitation was performed using DeCyder MS Differential Analysis software (DeCyderMS, GE Healthcare) [46, 47]. The peptide sequences were searched against the NCBI mammal database for protein identification using MASCOT software, version 2.2 (Matrix Science, London, UK) [48]. Database query included taxonomy (mammals), enzyme (trypsin), variable modifications (oxidation of methionine residues), mass values (monoisotopic), protein mass (unrestricted), peptide mass tolerance (1.2 Da), fragment mass tolerance (±0.6 Da), peptide charge state (1+, 2+ and 3+) and maximum number of missed cleavages. Proteins were identified from one or more peptides with an individual MASCOT score corresponding to P < 0.05. Proteins were annotated by UniProtKB/Swiss-Prot entries (http://www.uniprot.org/) and classified according to their molecular function, biological process and cellular component using the PANTHER classification system, version 8.1 (www.pantherdb.org/) [49]. Protein list comparison among different sample groups was displayed using jvenn diagram (http://bioinfo.genotoul.fr/jvenn/example.html) [50]. The interaction network of candidate proteins and chemotherapy drugs was explored using the Stitch program, version 5.0 (http://stitch.embl.de/) [51].

Validation of MS results by western blot analysis

Protein concentrations of pooled saliva and tissue samples were determined by Lowry assay, SDS-PAGE and western blotting as described previously [6, 52]. Briefly, samples (10 μg) were mixed with loading dye, heated and applied to a pre-cast NuPAGE 4–12% (w/v) Bis-Tris gel (Thermo Fisher Scientific) using RunBlue MES Run Buffer (Expedeon, Heidelberg, Germany) at 200 V for 90 min. Protein standard marker was PageRuler prestained protein ladder (molecular weight range 10–180 kDa) (Thermo Fisher Scientific). After that, the proteins were transferred to TranBlot Turbo nitrocellulose membranes (Bio-Rad Laboratories) at 25 V for 14 min using Trans-Blot Turbo 5× transfer buffer (Bio-Rad Laboratories). Detection of total protein band intensities in each lane was performed by a Pierce Reversible Protein Stain Kit for Nitrocellulose Membranes (Thermo Fisher Scientific) according to the manufacturer’s instructions. Blocking non-specific protein binding was achieved by 5% bovine serum albumin (BSA) (GoldBio, St Louis, MO, USA) in Tris-buffered saline containing 0.1% Tween 20 (TBST) at 25 °C overnight. After washing with TBST, primary antibodies diluted at 1:1000 were incubated with a membrane at 4 °C overnight, including mouse monoclonal anti-human PTPN5 or STEP (F-9) (Cat. No. sc-514,678, Santa Cruz Biotechnology, Dallas, TX, USA) and mouse monoclonal anti-human p53 (DO-1) (Cat. No. sc-126, Santa Cruz Biotechnology, Dallas, TX, USA). Membranes were washed with TBST and then incubated with 1:10000 horseradish peroxidase conjugated-rabbit anti-mouse IgG secondary antibody (Abcam, Cambridge, UK) for 1 h at 25 °C. The proteins of interest were visualized with ECL western blotting detection reagents (GE Healthcare). Western blot imaging was performed using a ChemiDoc Touch Imaging System (Bio-Rad Laboratories). Protein bands intensities were analysed by Image Lab 6.0.1 software (Bio-Rad Laboratories). Total protein normalization was performed with the modification of Aldridge et al. (2008) [6, 53]. The ratios of target band intensities to the total proteins in each lane were calculated as previously described [6]. The western blotting was performed in triplicate.

Verification of expressed protein sequences by LC-MS/MS

LC-MS/MS was utilized to confirm PTPN5 and p53 (or TP53) protein identities as described previously [6]. Briefly, blotting membranes were incubated with Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific) for 15 min and washed 4 times with TBST. Protein bands were excised and stored in 10 mM DTT in 10 mM ammonium bicarbonate overnight. Samples were then trypsinized at 37 °C for 3 h and applied to the LC-MS/MS as mentioned above.

Statistical analysis

ANOVA statistical analysis, incorporated into the DeCyder MS differential analysis software, and MASCOT software, version 2.2 were used to analyse significantly different peptide peak intensities and MASCOT LC-MS/MS scores, respectively. Western blot analysis was performed by ordinary one-way ANOVA with Tukey’s multiple comparisons for PTPN5 and p53. Statistical analyses of protein expression data were conducted using GraphPad Prism, version 8.0.1 (GraphPad Software, La Jolla, CA, USA). Significance was accepted at the P < 0.05 level.

Supplementary information

12917_2020_2550_MOESM1_ESM.xlsx (491.8KB, xlsx)

Additional file 1. The relative expression levels of proteins found in normal controls and periodontitis (CP), benign tumors (BN), early-stage oral melanoma (EOM), late-stage oral melanoma (LOM) and oral squamous cell carcinoma (OSCC) as log2 intensities.

Acknowledgements

We sincerely thank Dr. Worapan Tadadoltip for sample collection. Special thanks to Ms. Janthima Jaresitthikunchai and Mr. Sucheewin Krobthong for technical assistance.

Abbreviations

ACN

Acetonitrile

Akt

Protein Kinase B

BN

Benign oral tumors

BSA

Bovine serum albumin

CU-ACUC

The Chulalongkorn University Animal Care and Use Committee

CP

Periodontitis and healthy controls

CT

Computer tomography

CPC

Chromosomal passenger complex

DTT

Dithiothreitol

EGFR

Epidermal growth factor receptor

EOM

Early-stage oral melanoma

Erk

Extracellular-signal-regulated-kinase

FA

Formic acid

GeLC-MS/MS

In-gel digestion coupled with mass spectrometry

HCTUltra

High-capacity ion trap mass spectrometry

HPLC

High performance liquid chromatography

i.d.

Inside diameter

IAA

Iodoacetamide

IgG

Immunoglobulin G

LC

Liquid chromatography

LOM

Late-stage oral melanoma

MALDI-TOF MS

Matrix-assisted laser desorption ionization mass spectrometry

MAPK

Mitogen-activated protein kinase

MES buffer

2-(N-morpholino) ethanesulfonic acid buffer

MS

Mass spectrometry

m/z

Mass per charge ratio

NCBI

National Center for Biotechnology Information

NH4HCO3

Ammonium bicarbonate

OSCC

Oral squamous cell carcinoma

p53

Tumor protein p53

PI3K

Phosphoinositide-3 kinase

PTM

Post-Translation Modification

PTPN1

Protein tyrosine phosphatase non-receptor type 1

PTPN5

Protein tyrosine phosphatase non-receptor type 5

PTPN6

Protein tyrosine phosphatase non-receptor type 6

PTPN11

Protein tyrosine phosphatase non-receptor type 11

PTP1B

Protein tyrosine phosphatase 1B

RanB2

E3 SUMO-protein ligase RanBP2

Ras

Ras protein

SDS

Sodium dodecyl sulfate

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SENP3

SUMO Specific-isopeptidase

SENP7

Sentrin-specific protease 7

Src

Proto-oncogene tyrosine-protein kinase

SUMO

Small ubiquitin-like modifier

TBST

Tris buffered saline buffer containing 0.1% Tween 20

TNM Stage

stages according to their primary sizes and metastatic profile, the tumor, node and metastasis

Topoll

Targeting DNA topoisomerase II

Tris-HCl

Tris hydrochloride

WHO

World Health Organization

Authors’ contributions

GS and SR designed the study. SP1, SP2, CK and AR collected samples. SP1, NP, SK and KL performed the experiments and analyses. GS and SP1 drafted the manuscript. GS and SR finalized the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the TRF Research Career Development Grant (RSA grant) (grant number RSA5980053) (to G.S.); the 100th Anniversary of Chulalongkorn University for a Doctoral Scholarship (to S.P.); the 90th Anniversary of Chulalongkorn University Scholarship (to S.P.); and an Overseas Research Experience Scholarship for Graduate Student (to S.P.). The funders had no role in the design of the study and collection, analysis, and interpretation of data, or in writing the manuscript.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval

All experimental protocols were approved by the Chulalongkorn University Animal Care and Use Committee (CU-ACUC), Faculty of Veterinary Science, Chulalongkorn University (Approval number 1631042). All procedures were performed in accordance with the relevant guidelines and regulations. Written informed consents were obtained from all dog owners.

Consent for publication

Not applicable.

Competing interests

The corresponding author, Dr. Gunnaporn Suriyaphol, is an Associate Editor of this journal. Other authors declare that they have no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Sekkarin Ploypetch, Email: sekkarin.ploy@gmail.com.

Sittiruk Roytrakul, Email: sittiruk@biotec.or.th.

Narumon Phaonakrop, Email: narumon.pha@gmail.com.

Suthathip Kittisenachai, Email: skittisenachai@gmail.com.

Kantinan Leetanasaksakul, Email: Kantinan.lee@biotec.or.th.

Sirinun Pisamai, Email: Sirinun.P@chula.ac.th.

Chanin Kalpravidh, Email: Chanin.K@chula.ac.th.

Anudep Rungsipipat, Email: Anudep.R@chula.ac.th.

Gunnaporn Suriyaphol, Email: Gunnaporn.V@chula.ac.th.

Supplementary information

Supplementary information accompanies this paper at 10.1186/s12917-020-02550-w.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12917_2020_2550_MOESM1_ESM.xlsx (491.8KB, xlsx)

Additional file 1. The relative expression levels of proteins found in normal controls and periodontitis (CP), benign tumors (BN), early-stage oral melanoma (EOM), late-stage oral melanoma (LOM) and oral squamous cell carcinoma (OSCC) as log2 intensities.

Data Availability Statement

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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