Differential Proteomics Analysis of Oligodendrogliomas and Astrocytomas Using iTRAQ Quantification

Background: Astrocytoma and oligodendroglioma are two histological subtypes of primary Central Nervous System (CNS) tumors. Because of the high cytoarchitectural variability and lacking accurate diagnosis biomarkers, distinguishing astrocytomas and oligodendrogliomas clinically remains challenging. Methods: The total protein lysates from of astrocytoma and oligodendroglioma clinical specimens were analyzed by 2DLC/MS/MS and quantified via Isobaric Tags Relative and Absolute Quantitation (iTRAQ). Differentially expressed proteins were further analyzed by Ingenuity Pathway Analysis (IPA) software. Lastly, potential bio-markers’ expression levels were validated by western blot. Results: A total 1856 proteins were identified. Among them, 83 proteins were increased and 82 proteins were decreased in astroglioma specimens compared to oligodendroglioma. Our bioinformatics study showed this protein profile change in astrocytoma more likely tend to enhance cell proliferation, migration and angiogenesis. Moreover, pathway-analysis showed that protein level of Rho Family GTPases pathway components was remarkably different between astrocytoma and oligodendroglioma. Lastly, two members of Rho family of GTPases, cell division control protein 42 homolog (CDC42) and transforming protein RhoA (RHOA) were found highly expressed in astrocytoma and oligodendroglioma, respectively. Discussion: Differential-proteomic analysis was validating to distinguish between astrocytomas and oligodendrogliomas. Two members of especially the Rho family of GTPases, CDC42 and RHOA, would be potential indicators to reflect the pathological characteristics of these two diseases. Citation: de Lin S, Li J, Wang P, Zang Y, Zhao Tb (2017) Differential Proteomics Analysis of Oligodendrogliomas and Astrocytomas Using iTRAQ Quantification. J Proteomics Bioinform 10: 128-134. doi: 10.4172/jpb.1000433 Volume 10(4) 128-134 (2017) 129 J Proteomics Bioinform, an open access journal ISSN: 0974-276X clinical investigation was conducted according to the principles which expressed in the Declaration of Helsinki. iTRAQ sample preparation Fifty-milligram samples from each of the twelve frozen tissue samples were used for proteomics analysis. Tumor samples were washed with PBS to remove the blood and then lyzed with lysis buffer (containing 8 M Urea, 2.5 M Thiourea, and 65 mM DTT). Cell debris was removed by centrifugation at 14,000 g at 4°C for 10 min. The protein concentration of each sample was estimated using the Bradford method. 1 mg proteins from each sample were pooled together and digested using Filter-Aided Sample Preparation (FASP) method [6]. Digested peptides from the astrocytomas and oligodendrogliomas were desalted with C18 columns (3 cc, 60 mg, Oasis). The desalted peptides were lyophilized and immediately stored at -80°C. 100 μg of the astrocytomas and oligodendrogliomas digested peptide were individually labeled with 115 and 116 iTRAQ regents respectively, according to the manufacturer’s protocol (ABsciex). After labeling, the labeled peptides were mixed and lyophilized by vacuum centrifugation.