Two dimensional gel electrophoresis (2DE) resolves a mixture of proteins based on both isoelectric point and molecular weight of the individual proteins. Even when we followed a standard protocol for 2DE we got lesser number of proteins focused especially in the basic region of the IPG strip. Since the common troubleshooting measures did not solve the problem we replaced the protease inhibitor cocktail in the lysis buffer with PMSF which resulted in an ideal protein map following 2DE. We also found that the presence of the cocktail results in skewing of the mass spectra of a purified protein which eventually resulted in incorrect identification of the protein by MASCOT search. Later we found that dimethyl sulfoxide, the solvent of protease inhibitor cocktail also resulted in focusing of lesser number of proteins. Addition of 1% dimethyl sulfoxide to bovine serum albumin resulted in lesser sequence coverage for the protein by LC-MS/MS. Also, dimethyl sulfoxide was found to decrease the intensity value of the dominant peptide in a fraction when MALDI-TOF-TOF was done. Even though we do not provide a reason behind our observations, we guess dimethyl sulfoxide might have changed the charge distribution of peptides or proteins resulting in the peculiar observations we had.

中文翻译：

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在裂解缓冲液中包含溶解于二甲基亚砜的蛋白酶抑制剂混合物，可减少二维凝胶电泳可分离的蛋白质数量

二维凝胶电泳（2DE）可根据各个蛋白质的等电点和分子量解析蛋白质混合物。即使当我们遵循2DE的标准协议时，我们得到的蛋白质数量也减少了，尤其是IPG条带的基本区域。由于常见的故障排除措施不能解决问题，因此我们用PMSF代替了裂解缓冲液中的蛋白酶抑制剂混合物，从而在2DE之后产生了理想的蛋白质图谱。我们还发现，鸡尾酒的存在会导致纯化蛋白质的质谱图发生倾斜，最终导致通过MASCOT搜索对蛋白质进行错误识别。后来我们发现，蛋白酶抑制剂混合物的溶剂二甲亚砜也导致了较少数量的蛋白质聚集。向牛血清白蛋白中添加1％二甲基亚砜会导致LC-MS / MS对蛋白质的序列覆盖率降低。另外，发现当进行MALDI-TOF-TOF时，二甲基亚砜降低了主要肽的强度值。即使我们没有提供观察结果背后的原因，我们猜测二甲基亚砜可能已经改变了肽或蛋白质的电荷分布，从而导致了我们所特有的观察结果。