From a prospective observational study

From a prospective observational study, 30 subjects were chosen for serum proteome analysis. In the study group, 20 subjects had (newly diagnosed) type-2 diabetes (DM) on specific therapy, and 10 subjects were control. The diagnosis of DM and newly diagnosed was based on American Diabetes Association criteria. Newly diagnosed diabetes was diagnosed if the HbA1C level was less than 7.1%, having the age range of 18-40 years old. Inclusion criteria for the sample subjects is only oral hypoglycemic agents and exclusion criteria includes chronic inflammatory diseases, Class ??? and ?V congestive heart failure, Blood pressure > 180mm Hg (systolic) or >110mm Hg, (Diastolic) and pregnancy. The control (CTRL) group consisted of 10 clinically healthy individuals between 20 and 40 years of age. Exclusion criteria for the control group included pregnancy, alcohol consumption, tobacco products (former or current), chronic medical illness, and history of any drug treatment within the previous 3 months.
Ethical Statement
Informed consent was obtained from the subjects following the institutional review board guidelines for human subjects (Ethical review committee, BADAS, Bangladesh Institute of Research and Rehabilitation in Diabetic, Endocrine and Metabolic Disorder, Dhaka, Bangladesh). A special questionnaire concerning family history and health-related information was filled for all participants by direct interviews with the researchers.
Sample Collection and processing
10 mL venous blood were collected from 20 diabetic patients with 10 control subjects from Out Patients Department (OPD) of BIRDEM hospital. Before collection of blood informed written consent form were taken from each of the patients. Blood were collected from an antecubital vein in heparinized and Vacutainer tubes for serum. Blood were then allowed to clot at room temperature for 30 min. Samples were centrifuged at 2000Xg for 10 min after which serum were collected. Serum were stored at -800C until analyzed (Qin Fu et al., 2005).
Biochemical analysis
3 ml of blood and/or serum were analyzed from individual sample for different biochemical parameter. Fasting blood glucose (Barham, Trinder, 1972) and total lipid profile (TC, TG and HDL) (Trinder. 1988, Trinder 1969 and Lopes-verila M.E. et al., 1977) were measured enzymatically using Auto Lab Analyzer (Analyzer Medical system, Rome, Italy). LDL- cholesterol (C) was calculated from the equation of Friedewald equation (LDL–C = TC – HDL–C + (TG/5)), (Friedewald WT. 1972). HbA1c was measured using 3 mL EDTA blood by ion-exchange HPLC using Bio-Rad VARIANT. The separation of HbA1c is performed rapidly and precisely, without interference from labile A1c lipemia, or temperature fluctuations. HbA1c assay was standardized to the Diabetes Control and Complications Trial (DCCT) assay method. While measuring total renal profile, blood urea was measured using Enzymatic urea assay (Fossati P. Enzymatic urea assay. US patent 4,608,335, 1986). Pdf 21. Fasting Serum Creatinine level were estimated by alkaline-picrate methods using standard reagents (Randox Laboratories, UK). (pdf 22)
Proteomic analysis
Serum Delipidation and depletion
20µL serum was diluted with nanopure water (18.2 M?) and sonicated with acetonitrile for 10 min in an ultrasonic water bath (U300). The protein precipitate was pelleted by centrifugation at 12000 x for 10min at room temperature. The supernatant was transferred to a clean centrifuge tube and evaporated to dryness in centrifugal evaporator with no heating. (Rechard kay, 2008).
Protein separation: SDS-PAGE gel
Delipidated serum was run for protein separation by SDS-PAGE (12% resolving gel, Tris–HCl, pH 8.8 and 5% stacking gel). At first, protein samples were diluted with SDS-PAGE sample buffer (Tris–HCl, pH 6.8) at 1:2 and heated at 950C for 1–3 min (Julie L. Brunelle and Rachel Green, 2014). Molecular weight marker and prepared protein samples were loaded in the wells using a micro-pipettor with gel loading tips. A constant 85V for 35 min was supplied. When the bromophenol blue had run to the bottom of the gel, the power supply was off and disconnect the leads. The running buffer (Tris base 25 mM) was discarded and rinse the gel sandwich with water. The glass plates to access the gel was carefully separated.
Staining of SDS PAGE gel with coomassie blue R250
Gels were prefixed in 50% Methanol, 10% Acetic acid and 40% H2O for overnight. Than gels were stained in the above solution, with 0.25% Coomassie Blue R-250, for 4 hours, until the gels were in a uniform blue color. Staining was complete when the gels were no longer visible in the dye solution. Prior to complete staining, the gel will appear as a lighter area against the dark staining solution. The gels were de-stained for 24 hours in 5% Methanol, 7.5% Acetic acid and 87.5% H2O solution until background were clear. Then gels were stored in 7% Acetic acid solution for mass spectrum analysis.
Excision and in-gel digestion
The intended gels were washed with water (2x) and excised into 1 mm-cubes. A gel piece of roughly the same size from a non-protein containing region of the gel for use as a control. The gel particles were reduced by dithiothreitol in NH4HCO3 and incubated for 45 min at 56°C. After cooling down to room temperature, it was alkylated by iodoacetamide in NH4HCO3 for 30 min at room temperature in the dark. The gel particles were then washed with NH4HCO3 and acetonitrile followed by vacuum centrifuge to dry down. Finally, gels were digested by sequencing grade, modified trypsin enzyme solution (in 25 mM NH4HCO3) dissolved in acetic acid at 37°C for 30 minutes.
Identification of protein by ESI-MS
For mass spectroscopic experiment, each sample was mixed 1:1 (v/v) with solvent A (0.1% formic acid, 0.005% trifluoroacetic acid, and 3% acetonitrile in H2O) and solvent B (0.1% formic acid and 0.005% trifluoroacetic acid in 80% acetonitrile). A Symmetry 300 C18 trap from Dionex (Sunnyvale, CA) and a 15-cm long, 75-µm inner diameter PicoFrit column from New Objective (Woburn, MA), packed in-house with Jupiter C5 from Phenomenex (Torrance, CA), were used for the ESI experiments. The LC program consisted of a gradient from 3% to 35% B over 60 min, to 70% B at 65 min, and finally 95% B from 70 to 80 min. A Waters CapLC system with a flow rate estimated to be 300 nL/min was used to deliver solvent. The mass spectrometer used for ESI tandem mass spectrometry (ESI-MS/MS) was a quadrupole–time-of- flight (Q-TOF) operated with a spray voltage of 3.5 kV. Data-dependent MS/MS was generated using a 0.5-sec MS survey scan and 2.5-sec MS/MS scans on the three most abundant peaks found in the survey scan. The collision-induced dissociation (CID) energy was between 25 and 65 eV depending on the mass and charge state of the precursor ion. Mass spectra were calibrated using fragment ions generated from MS/MS of Glu-fibrinopeptide B (MW 1570.68).
Statistical and database analysis
Data from the ampherometric and biochemical experiments were analyzed using the Statistical Package for Social Science (SPSS) software for windows version 16.0 (SPSS Inc., Chicago, Illinois, USA). All the data were expressed as Mean ± Standard Deviation (SD) as appropriate. Statistical analysis of the results was performed by using the Dunnet t-test. The limit of significance was set at p