Comparison of three different fluorescent visualization strategies for detecting Escherichia coli ATP synthase subunits after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The correlation between protein molecular weight and the number of lysine o r basic amino acid residues was found to be high for broad range molecular weight standards, subunits of Escherichia coli F1F0-ATP synthase and the tr anslated open reading frame of E. coli. A relatively poor correlation betwe en protein molecular weight and the number of cysteine residues was observe d in all cases. The ability of amine-reactive, thiol-reactive and basic ami no acid-binding fluorophores to detect the eight subunits of F1F0-ATP synth ase complex was assessed using 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) , monobromobimane (MBB) and SYPRO Ruby protein gel stain, respectively. Tho ugh 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, how ever, failed to detect subunits a, b and c of the hydrophobic F-0 complex, as well as subunit epsilon of the F-1 complex. All three fluorescent detect ion procedures permitted subsequent identification of representative subuni ts by peptide mass profiling using matrix-assisted laser desorption ionizat ion time-of-flight mass spectrometry (MALDI-TOF MS). The use of thiol-react ive fluorophores for the global analysis of protein expression profiles doe s not appear to be advisable as a significant number of proteins have few o r no cysteine residues, thus escaping detection.