Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis.
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.
Lee
Kibeom
K
Department of Biotechnology, SongDo Techno Park, 7-50 Songdo-Dong, Yeonsu-Gu, Incheon, 406-840, South Korea. klee02@empal.com
Pi
Kyungbae
K
Lee
Keeman
K
D002021:Buffers; D003902:Detergents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007525:Isoelectric Focusing; D008175:Lung Neoplasms / Q000737:chemistry*; D009363:Neoplasm Proteins / Q000032:analysis*; D019163:Reducing Agents / Q000494:pharmacology; D012995:Solubility / Q000187:drug effects; D013890:Thiourea / Q000494:pharmacology
D002021:Buffers; D003902:Detergents; D009363:Neoplasm Proteins; D019163:Reducing Agents; D013890:Thiourea; D004229:Dithiothreitol