Lee Kibeom, Pi Kyungbae, Lee Keeman
Department of Biotechnology, SongDo Techno Park, 7-50 Songdo-Dong, Yeonsu-Gu, Incheon, 406-840, South Korea.
Biotechnol Lett. 2009 Jan;31(1):31-7. doi: 10.1007/s10529-008-9837-8. Epub 2008 Sep 18.
A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea-urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes.
肺癌组织蛋白质组分析中的一个问题是存在具有不同组织学背景的复杂成分（鳞状细胞癌、小细胞肺癌和腺癌）。在二维电泳（2-DE）之前对蛋白质成分进行有效的溶解是非常关键的。溶解不佳与无法检测到蛋白质以及蛋白质斑点出现扩散、条纹和/或拖尾有关。在此，我们优化了人肺癌组织的溶解方法以提高蛋白质分辨率。含有硫脲-尿素混合物的等电聚焦（IEF）复水缓冲液提供了更好的分辨率，而不含硫脲的缓冲液始终产生较差的结果。此外，含有CHAPS和DTT的IEF复水缓冲液给出了更好的分辨率，而含有Nonidet P-40（NP-40）和/或Triton X-100的缓冲液则不然。含三丁基膦的缓冲液始终产生较差的结果。使用优化的条件，我们通过基质辅助激光解吸电离质谱（MALDI-MS）对人肺癌组织进行二维凝胶分析，以鉴定11个差异表达的蛋白质斑点。本研究提供了一种研究复杂哺乳动物蛋白质组的方法工具。
