Hedberg Jesper J, Bjerneld Erik J, Cetinkaya Siham, Goscinski Jan, Grigorescu Ioana, Haid Daniel, Laurin Ylva, Bjellqvist Bengt
GE Healthcare, Amersham Biosciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden.
Proteomics. 2005 Aug;5(12):3088-96. doi: 10.1002/pmic.200401126.
2-DE is still a relatively cumbersome and labor intensive method. Given the successful cysteinyl protection concept with hydroxyethyl disulfide (specific oxidation) during the first dimension separation, the possibility for a simplified equilibration procedure was investigated. This was achieved by maintaining the S-mercaptoethanol modified cysteinyls throughout the 2-D workflow including second dimension separation, spot handling, protein digestion, and protein identification. The traditional equilibration protocol encompassing thiol reduction and alkylation was compared with a one-step protocol employing continuous exposure to hydroxyethyl disulfide. Both equilibration protocols gave equally well-resolved spot maps with analytical protein loads regardless of IPG strip pH range. Using preparative protein loads, narrow range IPG strips gave comparable results for the two protocols while preparative load on wide range IPG strips was the only condition where classical reduction/alkylation outperformed hydroxyethyl disulfide equilibration. Moreover, with analytical protein loads, the hydroxyethyl disulfide equilibration time could be significantly reduced without apparent loss of spot map quality or quantitative protein transfer from the first- to the second dimension gel. MALDI-TOF mass spectrometric protein identification was successfully performed with either iodoacetamide or hydroxyethyl disulfide as the cysteine modifier, yielding comparable identification results with high confidence in protein assignment, sequence coverage, and detection of cysteine-containing peptides. The results provide a novel and simplified protocol for 2-DE where the concept of hydroxyethyl disulfide as the cysteinyl protecting agent is extended to cover the entire 2-D work flow.
双向电泳仍然是一种相对繁琐且劳动强度大的方法。鉴于在第一维分离过程中使用羟乙基二硫化物（特异性氧化）成功实现了半胱氨酸保护的概念，我们研究了简化平衡步骤的可能性。这是通过在包括二维分离、斑点处理、蛋白质消化和蛋白质鉴定在内的二维工作流程中保持S-巯基乙醇修饰的半胱氨酸来实现的。将包含硫醇还原和烷基化的传统平衡方案与采用连续暴露于羟乙基二硫化物的一步方案进行了比较。无论IPG胶条的pH范围如何，两种平衡方案在分析蛋白质上样量时都能给出分辨率同样良好的斑点图谱。对于制备性蛋白质上样量，窄范围IPG胶条在两种方案下给出了可比的结果，而宽范围IPG胶条上的制备性上样量是经典还原/烷基化优于羟乙基二硫化物平衡的唯一条件。此外，对于分析蛋白质上样量，羟乙基二硫化物平衡时间可以显著缩短，而不会明显损失斑点图谱质量或蛋白质从第一维凝胶到第二维凝胶的定量转移。使用碘乙酰胺或羟乙基二硫化物作为半胱氨酸修饰剂均成功进行了基质辅助激光解吸电离飞行时间质谱蛋白质鉴定，在蛋白质归属、序列覆盖率和含半胱氨酸肽段检测方面均给出了具有高可信度的可比鉴定结果。这些结果为双向电泳提供了一种新颖且简化的方案，其中羟乙基二硫化物作为半胱氨酸保护剂的概念被扩展到涵盖整个二维工作流程。
