Elsevier

Journal of Proteomics

Volume 77, 21 December 2012, Pages 480-491
Journal of Proteomics

iTRAQ-based quantitative proteomic analysis for identification of oligodendroglioma biomarkers related with loss of heterozygosity on chromosomal arm 1p

https://doi.org/10.1016/j.jprot.2012.09.028Get rights and content

Abstract

The oligodendroglioma (OG) type of glial cell tumors accounts for 2–5% of primary brain neoplasms and 4–15% of gliomas diagnosed worldwide. Allelic losses on 1p, or on 1p and 19q, correlate with chemotherapy response and good prognosis in OG patients; however, the underlying mechanisms are not yet clearly defined. Therefore, we utilized a quantitative proteomics strategy that combined 8-plex isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC/MS/MS) to identify molecular signatures, reveal mechanisms, and develop predictive markers of OG patients with 1p loss of heterozygosity (LOH). An initial screening of four OG patients with 1p LOH and four without were identified, and 449 differentially expressed proteins were quantified, 13 of which were significantly different between the two groups. Analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway suggested that 1p LOH may affect the actin network in OG. The differential expression of four of the 13 candidates (UBA1, ubiquitin-like modifier activating enzyme 1; ATP6V1E1, ATPase, H + transporting, lysosomal 31 kDa, V1 subunit E1; MAP2, microtubule-associated protein 2; and HMGB1, high-mobility group protein B1) was validated in 39 additional OG samples using immunohistochemistry. Decision tree modeling indicated that MAP2 expression is a powerful predictor of 1p LOH. Our results not only demonstrate the utility of iTRAQ-based high-throughput quantitative proteomic analysis in glioma research, but also provide novel markers that may help to reveal the mechanisms of 1p LOH-associated chemosensitivity, and to design diagnostic and prognostic assays and therapeutics for OG.

Graphical abstract

Highlights

► We use iTRAQ proteomic analysis to identify specific markers in OG tissues with 1p LOH. ► We identified 449 proteins from 8 cases of OGs using proteomic analysis. ► Thirteen proteins were differentially expressed between the OGs with and without 1p LOH significantly. ► Western blot and IHC analyses validated the proteomic results.

Introduction

Glioma is the most frequently diagnosed primary brain tumor. Despite the fact that the global incidence of glioma is high, treatment options remain limited to radiation, chemotherapy, or surgical excision. These treatments do improve the prognosis of glioma patients, but the overall prognosis remains poor. Of the glioma types, oligodendroglioma (OG) is especially common, accounting for 2–5% of primary brain neoplasms and 4–15% of gliomas. These tumors respond particularly well to chemotherapy and have good prognosis compared to other glioma types. Clinical studies indicate that the chemosensitivity and prognosis of OG patients strongly correlate with the presence of allelic losses on 1p, or 1p and 19q, in the resected tumor tissue. Further research has revealed that the OGs with or without 1p loss of heterozygosity (LOH) have profoundly different features, including tumor location, radiotherapy response, and prognosis. Thus, the two types of OGs, which are undistinguishable under optical microscopy, may in fact have different molecular biological characteristics [1], [2], [3].

Identifying the molecular profile of OGs may provide insights into the processes that underlie different phenotypes and aid in developing accurate diagnostic and therapeutic strategies [4], [5]. Except for the microscope differences related to 1p/19q LOH, other possible distinguishing features, such as differential expression of genes and proteins, and molecular biological mechanisms, have not yet been clearly defined for the OGs. These data will be critical to understanding the mechanisms responsible for the chemosensitivity in OGs with 1p LOH. Clinical testing of 1p LOH is most often carried out by the florescence in situ hybridization (FISH) method or microsatellite sequencing analysis [6]. Both techniques are labor intensive, time consuming, and costly. In the case of microsatellite analysis, repeated blood draws are required from the patient, which impacts patient comfort, patient compliance, and risk of bruising or infection. Thus, further studies are needed to not only elucidate the molecular signatures and mechanisms of OG, but to also provide easily assayable markers of chemosensitivity.

Modern proteomic technologies allow for high-throughput and simultaneous detection to provide large quantitative datasets at decreased time and cost. These technologies have been successfully used in research areas of both basic sciences and clinical medicine. Although genomic and transcriptomic studies have revealed many possible oncogenes and tumor suppressors, proteomic analysis has the advantage of directly studying the translated proteins, which represent the functional molecule in most of the biological processes. While only a handful of proteins can be studied simultaneously with traditional methods such as Western blotting, a typical proteomics study can yield information on hundreds or thousands of proteins. Such an approach has already facilitated the molecular profiling of several complex diseases, such as glioma [7], [8].

Isobaric tags for relative and absolute quantification (iTRAQ) are a newly developed quantitative proteomics technology, demonstrated to be efficient, reproducible, and capable of performing quantitative protein analysis on eight samples simultaneously [9]. In this study, we used the 8-plex iTRAQ technology combined with two-dimensional liquid chromatography tandem mass spectrometry (2D-LC/MS/MS) to identify specific protein markers in OG tissues with 1p LOH. Bioinformatic analysis of the resultant dataset of potentially associated proteins was carried out to provide novel insights into the molecular mechanisms underlying this tumor phenotype.

Section snippets

Sample collection and histopathologic examination

A total of 47 OG samples were obtained from Huashan Hospital, along with the corresponding patients' clinical information. Each patient provided informed consent, and the study protocol was approved by the local institutional review board. Pathologic analysis of the tumors was performed by two pathologists (Y. Wang and H. Chen), who worked independently and were blinded to the molecular analysis results. The fresh tumor samples were collected as described by Grzendowski et al. [6], and

Quantitative proteomic analysis of OGs using the iTRAQ method

The 1p LOH status of frozen OG samples and formalin-fixed and paraffin-embedded OG samples was first defined by FISH analysis (Fig. 1). Four frozen tissues with 1p LOH and four without 1p LOH were then selected for proteomic analysis to identify differentially expressed proteins associated with the mutation. The 95% CI minimal set of non-redundant proteins identified from the eight OG tissues was composed of 449 proteins. Fig. 2A depicts the distribution of these 449 proteins according to their

Discussion

The primary objective of our proteomic study was to identify promising biological markers between OGs with and without 1p LOH, as the 1p LOH phenotype is associated with chemosensitivity and good prognosis. Many previous studies have attempted to unravel the molecular biological underpinnings of the differences between these two groups of OG patients, but a clear and absolute understanding has remained elusive [1], [6], [14], [15], [16], [17], [18], [19]. To the best of our knowledge, our study

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    This work was supported by grants from the ShuGuang Project (No. 05SG05), the Science and Technology Commission of Shanghai Municipality (No. 07dz19505), the NSFC (No. 30970653), and the State Key Research Project (Nos. 2008ZX10002-017 and 2009ZX09301-011).

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