Utilizing MALDI-TOF MS and LC-MS/MS to access serum peptidome-based biomarkers in canine oral tumors

Tumors frequently found in dogs include canine oral tumors, either cancerous or noncancerous. The bloodstream is an important route for tumor metastasis, particularly for late-stage oral melanoma (LOM) and late-stage oral squamous cell carcinoma (LOSCC). The present study aimed to investigate serum peptidome-based biomarkers of dogs with early-stage oral melanoma, LOM, LOSCC, benign oral tumors, chronic periodontitis and healthy controls, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography tandem mass spectrometry. A principal component analysis plot showed distinct clusters among all groups. Four peptides were identified, including peptidyl-prolyl cis-trans isomerase FKBP4 isoform X2 (FKBP4), steroid hormone receptor ERR1 (ESRRA or ERRA), immunoglobulin superfamily member 10 (IGSF10) and ATP-binding cassette subfamily B member 5 (ABCB5). FKBP4, ESRRA and ABCB5 were found to be overexpressed in both LOM and LOSCC, whereas IGSF10 expression was markedly increased in LOSCC only. These four proteins also played a crucial role in numerous pathways of cancer metastasis and showed a strong relationship with chemotherapy drugs. In conclusion, this study showed rapid screening of canine oral tumors using serum and MALDI-TOF MS. In addition, potential serum peptidome-based biomarker candidates for LOM and LOSCC were identified.

chromatography tandem mass spectrometry (LC-MS/MS) [10][11][12][13][14][15] . In dogs, MALDI-TOF MS and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) has been applied to study peptidomic and proteomic profiles of canine oral tumor tissues and saliva 16,17 . However, a serum-based peptidomic biomarker of canine oral tumors has not yet been reported. Because of the highly metastatic behavior of canine oral cancers, novel circulating biomarkers can provide strong supporting evidence for diagnosis, prognosis, monitoring, and selecting treatment options. In addition, blood circulation supplies the organs and also tumors. Thus, blood sample is a widely useful source of circulating biomarkers. This study aimed to investigate the serum peptidome-based biomarkers of EOM, LOM, late-stage OSCC (LOSCC), benign oral tumors (BN), chronic periodontitis (PD) and healthy controls (CTRL), using MALDI-TOF MS and LC-MS/MS techniques. All 32 replicates in each pooled sample group demonstrated the homogeneity within the group. In the component analysis, ClinProTools revealed the different proteomic profiles from 1 to 20 kDa among 6 groups: CTRL, PD, BN, EOM, LOM and LOSCC. A 3-dimensional view of the principal component analysis (PCA) plot showed distinct clusters among these groups (Fig. 1). Therefore, there are differentially expressed protein and peptide profiles in each group. Peptide mass fingerprints (PMFs) from 1500 to 2500 Da were demonstrated (Fig. 2). In this mass range, significantly different peaks among the 6 groups could be detected.

MALDI-TOF MS results.
The MALDI-TOF MS results had an accurate outcome with the 95% confidence interval. The cross-validation and the recognition capability calculated by ANOVA and W/KW test in the EOM, LOM, LOSCC, BN, PD and CTRL groups was all 100%, indicating that the results were of high reliability. There were up to 19 different peaks identified among all analyzed groups, and four of them were significantly differentially overexpressed in canine oral cancer groups (EOM, LOM and LOSCC) with a mass ranging from m/z 1000 to 2500 Da, including m/z 1574.10, 1892.10, 2158.09 and 2182.14/2183.83 Da by ClinProTools software (Fig. 3). The peptide sequences of unique peaks were analyzed by LC-MS/MS.

LC-MS/MS results
The highest intensity of four peptide peaks (1574.10, 1892.10, 2158.09, and 2182.14/2183.83 Da) from cancerous groups appeared in the 18%, 24% and 26% ACN elution fractions ( Table 1). The four peptides were sequenced and identified as peptidyl-prolyl cis-trans isomerase FKBP4 isoform X2 (FKBP4), steroid hormone receptor ERR1 (ESRRA, also known as ERRA), immunoglobulin superfamily member 10 (IGSF10) and ATP-binding cassette subfamily B member 5 (ABCB5) by MS/MS fragmentation, respectively. The results from Mascot software analysis are shown in Fig. 4. FKBP4, ESRRA and ABCB5 were found to be overexpressed in both LOM and LOSCC, whereas IGSF10 expression was markedly increased in LOSCC only. Candidate protein biomarkers were evaluated for biological processes and molecular function using UniProtKB/Swiss-Prot 18 ( Table 1).
The protein interactions network of FKBP4, ESRRA, IGSF10 and ABCB5 showed a strong relationship with chemotherapy drugs, including doxorubicin, cisplatin, and cyclophosphamide and also with their predicted functional partners, including heat shock protein 90 alpha family class A member 1 (HSP90AA1), peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha (PPARGC1A), glomulin (GLMN), FKBP-associated protein, estrogen, rapamycin, tacrolimus, estrogen-related receptor gamma (ESRRG), heat shock protein 90 alpha family class B member 1 (HSP90AB1), estrogen receptor 1 (ESR1) and XCT790, using STITCH 5.0 (Figs. 5 and 6). These proteins have been reported to play a crucial role in numerous pathways of cancer metastasis as shown in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps, namely nucleotide-binding oligomerization

Discussion
In the present study, PMFs and peptidomic clusters were obtained from the serum of canine oral tumors. In addition, FKBP4, ESRRA and ABCB5 were found to be overexpressed in both LOM and LOSCC, whereas IGSF10 expression was markedly increased in LOSCC only. FKBPs are members of the immunophilin families. A high level of FKBP4 was demonstrated in several human cancers, including breast cancer, ovarian cancer and osteosarcoma, using proteomic techniques [20][21][22][23] . In addition, marked expression of FKBPs was noted in other human cancers, such as cutaneous melanoma, glioma, prostate cancer, lymphoma and hepatocellular carcinoma [24][25][26][27][28] . Recently, FKBP4 has been reported to be a novel PI3K/ PDK1/mTORC2 interacting protein and promoted Akt phosphorylation through PI3K/PDK1 and mTORC2. Since the PI3K/PDK1/Akt signaling pathway activates and regulates cell proliferation, cell cycle and survival, FKBP4 is directly related to cell growth promotion and proliferation 21 (Fig. 5). With regard to ESRRA, it is an orphan nuclear receptor that was shown to be activated by peroxisome proliferator activated receptor gamma coactivator-1 alpha and beta (PGC-1a and PGC-1b) 29 . Several enzymes in metabolic pathways of proliferating    30,31 . In previous studies, overexpression of ESRRA was exhibited to promote cancer cell growth and proliferation 30,32,33 . Moreover, the epidermal growth factor/Erb-B2 receptor tyrosine kinase 2 (EGF/ERBB2) signaling pathway in breast cancer cells modulated the transcription activity of ESRRA, which was associated with poor prognosis in breast and ovarian cancers 32,34,35 . Implanted xenografts with knockdown of ESRRA expression in MDA-MB-231 cells can reduce the tumor growth rate of breast cancers 36 . In the present study, we found prominent expression of FKBP4 and ESRRA in LOM and LOSCC, possibly being novel prognostic biomarkers and therapeutic target candidates. Hence, their role in cancer cell growth and proliferation in LOM and LOSCC should be further investigated. For ABCB5 protein, ABCB5 is an ABC transporter and P-glycoprotein family member 37 . This protein had been reported to be expressed in human cutaneous melanoma and be associated with chemoresistance 38,39 . Moreover, it was upregulated in circulating melanoma tumor cells and correlated with pulmonary metastasis formation in human-to-mouse subcutaneous tumor xenotransplantation models 40 . In our present study, increased expression of ABCB5 in LOM and OSCC might represent a biomarker associated with stages of metastasis, as per a previous report 40 . For IGSF10, IGSF is classified based on molecules combined with immunoglobulins. IGSF10, a member of the IGSF, is reported to control the early migration of gonadotropin-releasing hormone expressing neurons and maintenance of the osteochondroprogenitor cell pool 41 . Inhibition of IGSF10 expression by IGSF10small interfering RNA was demonstrated to inhibit cancer cell proliferation, enhance adhesion between cells and matrix, and increase the survival rate of patients with lung cancer by activating the integrin-β1/FAK pathway 42 . We demonstrated very high and unique expression of IGSF10 in LOSCC, which revealed a potential biomarker candidate of LOSCC. In addition, its role in carcinogenesis and cancer progression should be further investigated.
Because of the high rate of metastasis and aggressiveness of melanoma in dogs, systemic medication such as chemotherapy should be used as a treatment option 2 . In the present study, using Stitch EMBL, the interaction network analysis revealed that a coordination of candidate proteins (FKBP4, ESRRA, IGSF10 and ABCB5) was involved in KEGG pathways of many cancers (Fig. 5). In addition, the four proteins appeared to link with chemotherapy drugs (carboplatin, doxorubicin and cyclophosphamide), which could probably lead to the development of serum biomarker-based liquid biopsy for efficient diagnosis, prognosis, recurrence and metastasis predictions (Fig. 6). The networks of the candidate proteins and the chemotherapy drugs showed a strong relationship with DNA topoisomerase II alpha (TOP2A), a biomarker of oral cancers (Fig. 6) 43,44 . The association of target proteins with TOP2A, chemotherapy drugs, particularly carboplatin, doxorubicin and metronomic chemotherapy based www.nature.com/scientificreports/ on cyclophosphamide, and/or immunosuppression agents such as rapamycin and tacrolimus should be further investigated. This might be of great benefit for veterinarians and researchers to understand the pathogenesis and biological process of canine oral cancer. MALDI-TOF MS was used to discover several specific serum peptide patterns of human cancers as well as tissues and whole saliva of canine oral tumors 16,17,45,46 . Since cancer cells usually invade blood vessels to metastasize to other distant organs, serum samples are of interest for exploring novel metastatic biomarkers [47][48][49] . To the best of our knowledge, this is the first publication of serum proteomics in canine oral tumors. Using MALDI-TOF MS, we found distinct PMF patterns among CTRL, PD, BN, EOM, LOM and LOSCC groups. Therefore, this instrument could be considered as a high sensitivity diagnostic tool for rapid screening for canine oral tumors. Using LC-MS/MS, four peptide regions of FKBP4, ESRRA, ABCB5 and IGSF10 were identified in LOSCC and/ or LOM. For dogs with recurrence or metastatic stages following surgery, the diagnosis and treatment are usually too late because the current metastasis stage diagnosis relies on cytology of regional lymph nodes and imaging analysis (X-rays, ultrasound, and computerized tomography scan). Our studies could probably help researchers to better understand, and encourage future studies of, disease recurrence and spreading following treatment.

Conclusion
Our data suggested that MALDI-TOF MS used with ClinProTools software has potential to be used for rapid screening in serum of canine oral tumors. The peptide fragment of serum proteins was identified using MALDI-TOF MS and LC-MS/MS, combined with the C18 ZipTip technique. FKBP4, ESRRA, IGSF10 and ABCB5 might be potential biomarkers for diagnosis, prognosis and metastasis prediction. The interaction network showed the proteins and chemotherapy drugs that were involved in the biological process and pathogenesis of canine oral cancer. Further studies should be performed in a larger population to evaluate these potential candidate biomarkers. The total of 67 serum samples were collected from the following: 5 EOM, 28 LOM, 10 LOSCC, 12 BN (age range: 7-14 years) and 5 dogs with PD (age range: 7-13 years); and samples representing normal oral health were gathered from 7 healthy dogs (CTRL) (range: 7-8 years) ( Table 2). The PD group was diagnosed by oral examination with appearance of gingivitis, dental tartar and loss of periodontal attachment. Whole blood (~ 3-4 mL) was collected from the cephalic or the saphenous vein. The collected blood was centrifuged at 3000g at 4 °C for 15 min. Serum samples (~ 500 µL) were mixed with protease inhibitor cocktails (Thermo Fisher Scientific) and stored at − 20 °C until use. Using bovine serum albumin as a standard, total protein concentrations from serum samples were evaluated by Lowry's assay.

Peptide analysis by MALDI-TOF MS.
Sixty-seven samples were prepared with 0.1% trifluoroacetic acid (TFA) for analysis to a final concentration of 0.1 µg/µL. One microliter of a peptide, mixed with a matrix solution (5 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) in 100% acetonitrile (ACN) containing 5% TFA), was directly spotted onto the MTP384 target plate (Bruker Daltonics, Billerica, Massachusetts, USA) in eight replicates to prevent sample preparation bias. Target samples were tested using an Ultraflex III TOF/TOF system (Bruker Daltonics), using a linear positive mode with a mass range 1000-5000 Da. Each spectrum was obtained from 500 laser shots, with a 50 Hz laser. A ProteoMass peptide and protein MALDI MS calibration kit (Sigma Aldrich, St. Louis, Missouri, USA) was used for external calibration. MS spectra and peptide patterns were analyzed by flexAnalysis version 3.2 and ClinProTool version 3.0 software (Bruker Daltonics), respectively. The results were processed by a standard workflow that comprised fingerprint spectra, pseudo-gel view, dendrogram and PCA. Four from eight replicates of each sample were selected and pooled, together with other samples in each group. Next, 32 replicates of pooled samples were applied twice to MALDI-TOF MS to prevent sample preparation bias in each group. After the analysis by flexAnalysis and ClinProTool software was performed, the www.nature.com/scientificreports/ data were processed for peak selection and peak calculation. The recognition capability and cross-validation values of more than 90% exhibited the reliability of the peak selection 50 . ClinProTools v. 3.0 was used to analyze intensity values. Results with P < 0.05 were considered significant. Subsequently, the selected peptides from pooled samples in each group were purified, using C18 ZipTip (Merck Millipore, Burlington, MA, USA). Each sample was eluted in a 2% series of ACN in the range 0-100%. The second MALDI-TOF MS was performed for eluted fractions as mentioned above. The intensity of MS spectra was calculated by flexAnalysis (Bruker Daltonics). The fractions of selected peptides with the highest intensity were collected for further LC-MS/MS analysis.
Peptide identification by LC-MS/MS. The selected peptide fractions were sequenced by reversed-phase high performance liquid chromatography and a PTM Discovery System (Bruker Daltonics) coupled to an Ulti-Mate 3000 LC System (Thermo Fisher Scientific, Waltham, MA). A quadrapole ion-trap MS instrument (Bruker Daltonics) was used to analyze the serum peptides. Before analysis, a PepSwift monolithic nanocolumn (100 μm diameter × 50 mm length) was used to separate serum peptides based on differential polarity and molecular weight. The column was connected to electrospray ionization in the positive ion mode. Mobile phase A and B were prepared from 0.1% formic acid dilution and from 50% ACN in water containing 0.1% formic acid, respectively. A flow rate of 1000 nL/min was set to separate peptides with a 4-70% linear gradient of mobile phase B. Next, the peptides were regenerated and equilibrated using 90% and 4% eluent B, respectively, for 20 min per run. Peptide fragment mass spectra were selected from the MS scan at 200-2800 m/z. AutoMS mode was used to analyze peptide spectra with a scan range of 400-1500 m/z, 3 averages, and up to 5 precursor ions. The results of LC-MS/MS were converted into an mzXML file by CompassXport software (Bruker Daltonics). Protein was quantified using DeCyder MS Differential Analysis software (Amersham Biosciences, Little Chalfont, UK) 51,52 . The PepDetect module was used in MS mode for automated peptide detection, charge state assignments, and peptide ion signal intensities. The peptides were identified from MS/MS peptide mass values using Mascot software (Matrix Science, London, UK) 53 . The information about detailed analysis of the protein sequences and biological process was used in the annotation of UniProtKB/Swiss-Prot entries 18