Comparison of three different fluorescent visualization strategies for detecting Escherichia coli ATP synthase subunits after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Comparison of three different fluorescent visualization strategies for detecting Escherichia coli ATP synthase subunits after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
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十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后三种不同荧光可视化策略检测大肠杆菌 ATP 合酶亚基的比较
作者：
K. Berggren;E. Chernokalskaya;M. F. Lopez;J. Beechem;Wayne F. Patton
蛋白质分子量与赖氨酸或碱性氨基酸残基的数量之间的相关性被发现对于宽范围的分子量标准、大肠杆菌F1 F0-ATP合酶的亚基和大肠杆菌的翻译的开放阅读框架是高的。杆菌在所有情况下，观察到蛋白质分子量和半胱氨酸残基的数量之间的相关性相对较差。分别使用2-甲氧基-2，4-二苯基-3（2 H）-呋喃酮（MDPF）、单溴比曼（MBB）和SYPRO Ruby蛋白凝胶染色评估胺反应性、巯基反应性和碱性氨基酸结合荧光团检测F1 F0-ATP合酶复合物的8个亚基的能力。虽然实验上没有荧光团提供准确的估计亚基化学计量的这种复杂的，MDPF和SYPRO红宝石蛋白凝胶染色能够半定量检测每个亚基。然而，MBB未能检测到疏水性F0复合物的亚基a、B和c，以及F1复合物的亚基ε。所有三种荧光检测程序均允许随后使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）通过肽质量分析鉴定代表性亚基。使用巯基反应性荧光团进行蛋白质表达谱的全局分析似乎并不可取，因为大量蛋白质具有很少或没有半胱氨酸残基，从而逃避检测。
The correlation between protein molecular weight and the number of lysine or basic amino acid residues was found to be high for broad range molecular weight standards, subunits of Escherichia coli F1F0‐ATP synthase and the translated open reading frame of E. coli. A relatively poor correlation between protein molecular weight and the number of cysteine residues was observed in all cases. The ability of amine‐reactive, thiol‐reactive and basic amino acid‐binding fluorophores to detect the eight subunits of F1F0‐ATP synthase complex was assessed using 2‐methoxy‐2,4‐diphenyl‐3(2H)‐furanone (MDPF), monobromobimane (MBB) and SYPRO Ruby protein gel stain, respectively. Though experimentally none of the fluorophores provided accurate estimates of the subunit stoichiometry of this complex, MDPF and SYPRO Ruby protein gel stain were capable of semiquantitative detection of every subunit. MBB, however, failed to detect subunits a, b and c of the hydrophobic F0 complex, as well as subunit ε of the F1 complex. All three fluorescent detection procedures permitted subsequent identification of representative subunits by peptide mass profiling using matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). The use of thiol‐reactive fluorophores for the global analysis of protein expression profiles does not appear to be advisable as a significant number of proteins have few or no cysteine residues, thus escaping detection.