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 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 epsilon 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.
Berggren
K N
KN
Proteomics Section, Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, Oregon 97402, USA.
Chernokalskaya
E
E
Lopez
M F
MF
Beechem
J M
JM
Patton
W F
WF
D000595:Amino Acid Sequence; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001643:Bridged Bicyclo Compounds; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005456:Fluorescent Dyes; D005663:Furans; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021122:Protein Subunits; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001643:Bridged Bicyclo Compounds; D005456:Fluorescent Dyes; D005663:Furans; D021122:Protein Subunits; C008394:2-methoxy-2,4-diphenyl-3(2H)-furanone; D025243:Bacterial Proton-Translocating ATPases; D003545:Cysteine; C028381:monobromobimane