Proteomics approaches for identification of tumor relevant protein targets in pulmonary squamous cell carcinoma by 2D-DIGE-MS.

Hao Lihong,Qi Xiaoyu,Guan Zhuzhu,Shao Shujuan,Song Yang,Zhou Xin,Yang Xiaohan,Gong Linlin,Guo Yiping,Xue Liyan,Dunfa Peng

doi:10.1371/journal.pone.0095121

Abstract

Potential markers for progression of pulmonary squamous cell carcinoma (SCC) were identified by examining samples of lung SCC and adjacent normal tissues using a combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF). The PANTHER System was used for gel image based quantification and statistical analysis. An analysis of proteomic data revealed that 323 protein spots showed significantly different levels of expression (P≤0.05) in lung SCC tissue compared to expression in normal lung tissue. A further analysis of these protein spots by MALDI-TOF-MS identified 81 different proteins. A systems biology approach was used to map these proteins to major pathways involved in numerous cellular processes, including localization, transport, cellular component organization, apoptosis, and reproduction. Additionally, the expression of several proteins in lung SCC and normal tissues was examined using immunohistochemistry and western blot. The functions of individual proteins are being further investigated and validated, and the results might provide new insights into the mechanism of lung SCC progression, potentially leading to the design of novel diagnostic and therapeutic strategies.

Highlights

Lung cancer is the leading cancer-related cause of death worldwide in both men and women
2-D differential in-gel electrophoresis (2D-DIGE) proteome map The proteomes of pulmonary squamous cell carcinoma (SCC) obtained from 7 cancer patients and paired normal tissues were analyzed by 2D-DIGE in a nonlinear pI range of 3.0–10.0 and a molecular weight range of 10 to 120 kDa
To systematically compare the performance of different staining methods used in differential protein profiling, a DIGE gel was run with an independently prepared proteome from a paired normal tissue stained with Cy3, from SCC tissue stained with Cy5, and from a mixture of all protein samples as the internal standard stained with Cy2

Summary

Introduction

Lung cancer is the leading cancer-related cause of death worldwide in both men and women. Despite advances in treatments such as surgery, chemotherapy and radiotherapy, the clinical prognosis for patients with lung cancer remains poor, and the overall 5-year survival rate is only 10-15% [1]. Twodimensional differential in-gel electrophoresis [2,3,4,5] (2D-DIGE), is an advanced quantitative proteomics technology which offers higher sensitivity, accuracy, and resolution compared with traditional two-dimensional polyacrylamide gel electrophoresis (2-DE). This method is used to pre-label different protein samples with fluorescent cyanine dyes (Cy2, Cy3, and Cy5) prior to separation by 2-DE. 2D-DIGE can be used to obtain an accurate and reproducible quantitation of differences between samples [4]

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