This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Kasetsart University of Veterinary Medicine Animal Care and Use Committee (protocol: ACKU60-VET-020). Owner consent was obtained prior to the collection of serum and tumor tissue.
Serum and tumor specimens from patients
Canine OSA plasma and tissue specimens were obtained from 10 dogs, according to the rules of the ethical committee at the Faculty of Veterinary Medicine, Kasetsart University that underwent surgical resection at the university’s Veterinary Teaching Hospital. The clinical information of these patients, including age, gender, breed, body weight, serum alkaline phosphatase (ALP), and lung metastasis, was obtained from medical records. Histological classification was made by the veterinary pathologist of the Veterinary Teaching Hospital, Kasetsart University. Pulmonary metastasis was confirmed on thoracic radiography or through histopathological examination after autopsy. The control group consisted of 15 non-OSA orthopedic patients, from which serum and bone tissue samples were taken. Serum was collected and stored at -80 °C until use. Proteins from bone tissues were extracted with 200 μl of 0.5% SDS, incubated at 37 °C for a few hours, and centrifuged at 10,000g for 15 min. The supernatant was transferred to a new tube, mixed well with 2 volumes of cold acetone, and incubated overnight at -20 °C. The mixture was centrifuged at 10,000g for 15 min and the supernatant was discarded. The pellet was dried in a Speedvac, re-suspended in 200 μl of 0.5% SDS, and stored at -80 °C prior to use. The pellets were re-suspended in 0.15% sodium deoxycholic acid, and protein concentration was determined using the Lowry method .
Prefractionation of proteins using SDS-PAGE
Proteins were fractionated on an SDS-PAGE mini slab gel (8 x 9 x 0.1 cm, Hoefer miniVE, Amersham Biosciences, UK). The polyacrylamide gel was prepared according to the standard method described by Laemmli. The separating gel used for the fractionation of soluble proteins from mammalian cells contained 12.5% acrylamide. An equal volume of protein samples was mixed with 5 ml of 5X sample buffer (0.125M Tris-HCl, pH 6.8, 20% glycerol, 5% SDS, 0.2M DTT, 0.02% bromophenol blue), boiled at 95 °C for 10 min before being loaded onto the 12.5% SDS-PAGE. To estimate the size of polypeptides, a low molecular weight protein standard marker (Amersham Biosciences, UK) was used. Electrophoresis was performed in SDS electrophoresis buffer (25mM Tris-HCl, pH 8.3, 192mM glycine, 0.1% SDS) until the tracking dye reached the bottom of the gel. After the electrophoresis finished, gels were silver stained.
After protein bands were excised, the gel plugs were dehydrated with 100% acetonitrile (ACN), reduced with 10mM DTT in 10mM ammonium bicarbonate at room temperature for 1 h, and alkylated at room temperature for 1 h in the dark in the presence of 100mM iodoacetamide in 10mM ammonium bicarbonate. After alkylation, the gel pieces were dehydrated twice with 100% ACN for 5 min. To perform in-gel digestion of proteins, 10 µl of trypsin solution (10 ng/µl trypsin in 50% ACN/10mM ammonium bicarbonate) was added to the gels, followed by incubation at room temperature for 20 min, and then 20 µl of 30% ACN was added to keep the gels immersed throughout digestion. The gels were incubated at 37 °C for a few hours or overnight. To extract peptide digestion products, 30 µl of 50% ACN in 0.1% formic acid was added to the gels, and then the gels were incubated at room temperature for 10 minutes in a shaker. Extracted peptides were collected and pooled together in the new tube. The pooled extracted peptides were dried by vacuum centrifuge and kept at -80 °C for further mass spectrometric analysis.
Peptide identification by HCTultra LC-MS analysis
Peptide solutions were analyzed using an HCTultra PTM Discovery System (Bruker Daltonics Ltd., UK) coupled to an UltiMate 3000 LC System (Dionex Ltd., UK). Peptides were separated on a nanocolumn (PepSwift monolithic column, 100 μm diameter x 50 mm length). Eluent A and eluent B solutions were prepared from 0.1% formic acid, and from 80% ACN in water containing 0.1% formic acid, respectively. Peptide separation was achieved with a linear gradient from 10% to 70% of eluent B for 13 minutes at a flow rate of 300 nl/minute. A regeneration step and an equilibration step were at 90% of eluent B and at 10% of eluent B, respectively, for 20 minutes/run. Peptide fragment mass spectra were acquired in the data-dependent AutoMS mode with a scan range of 300–1500 m/z, 3 averages, and up to 5 precursor ions selected from the MS scan 50–3000 m/z. Peptide peaks were detected and deconvoluted automatically using DataAnalysis version 4.0. Mass lists in the form of Mascot generic files were created automatically and used as the input for Mascot MS/MS ions searches of the National Center for Biotechnology Information (NCBI) mammal database (www.matrixscience.com) for protein identification. For protein quantitation, DeCyder MS Differential Analysis Software (DeCyderMS, GE Healthcare) was used. Acquired LC-MS raw data were converted, and the PepDetect module was used for automated peptide detection, charge state assignments, and quantitation based on the peptide ion signal intensities in MS mode. The analyzed MS/MS data from DeCyderMS were submitted to the database using the Mascot software (Matrix Science, London, UK). The data were searched against the NCBI mammal database for protein identification. Proteins considered as identified proteins had at least two peptides with an individual mascot score corresponding to p<0.05. The relationship of candidate proteins and chemotherapeutic drugs were performed by the Stitch program, version 5.0.