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

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.

Salivary proteomics in monitoring the therapeutic response of canine oral melanoma

“…MASCOT software, version 2.2 (Matrix Science, London, UK), was used to search the peptide sequences against the NCBI mammal database for protein identification. 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 were among the criteria used in the database search [ 14 ]. One or more peptides with an individual MASCOT score corresponding to p<0.05 were used to identify proteins, which were then annotated by UniProtKB/Swiss-Prot entries ( http://www.uniprot.org/ ).…”

“…ECL western blotting detection reagents (GE Healthcare) was utilized to visualized the target proteins. A ChemiDoc Touch Imaging System (Bio-Rad Laboratories) was used to image the chemiluminescent blots and Image Lab 6.0.1 software (Bio-Rad Laboratories) was used to analyse protein band intensities [ 14 ]. Total protein normalization was performed with the modification of Aldridge et al .…”

“…Briefly, blotting membranes were incubated with Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific) for 15 min prior to being rinsed 4 times with PBST. Excised protein bands were incubated with 10 mM dithiothreitol in 10 mM ammonium bicarbonate overnight, trypsinized at 37°C for 3 h and subjected to the LC–MS/MS as mentioned above [ 9 , 14 ].…”

Protein Profile of Human Saliva as a Predictive and Prognostic Tool for OSCC in Tamol Chewer’s Population in Assam

“…Several studies have investigated the use of salivary proteins as a potential diagnostic marker for oral cancer. The identification of proteins is either cleaved by gel electrophoresis or enzymatic digestion by the procedure to produce peptides (Hu et al, 2008;Ploypetch et al, 2020). Approximately, 3000 proteins have been identified in saliva by using various procedures (Hu et al, 2008;Jarai et al, 2012;Ploypetch et al, 2020).…”

“…The identification of proteins is either cleaved by gel electrophoresis or enzymatic digestion by the procedure to produce peptides (Hu et al, 2008;Ploypetch et al, 2020). Approximately, 3000 proteins have been identified in saliva by using various procedures (Hu et al, 2008;Jarai et al, 2012;Ploypetch et al, 2020). Similarly, various potential biomarkers identified from the saliva of OSCC such as cytokeratin 19 fragment (Cyfra21-1) (Rathore et al, 2020), albumin (Nguyen et al, 2020), telomerase (Sannam et al, 2016), transferrin (Nguyen et al, 2020), glutathione (Singh et al, 2020) were identified.…”