Xh. Cheng et al., DIRECT MEASUREMENT OF OLIGONUCLEOTIDE BINDING STOICHIOMETRY OF GENE-VPROTEIN BY MASS-SPECTROMETRY, Proceedings of the National Academy of Sciences of the United Statesof America, 93(14), 1996, pp. 7022-7027
The binding stoichiometry of gene V protein from bacteriophage f1 to s
everal oligonucleotides was studied using electrospray ionization-mass
spectrometry (ESI-MS). Using mild mass spectrometer interface conditi
ons that preserve noncovalent associations in solution, gene V protein
was observed as dimer ions from a 10 mM NH4OAc solution. Addition of
oligonucleotides resulted in formation of protein-oligonucleotide comp
lexes with stoichiometry of approximately four nucleotides (nt) per pr
otein monomer. A 16-mer oligonucleotide gave predominantly: a 1:1 (pro
tein monomer: oligonucleotide) complex while oligonucleotides shorter
than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative
binding constants for a mixture of oligonucleotides were readily meas
ured using mass spectrometry; The binding stoichiometry of the protein
with the 16-mer oligonucleotide was measured independently using size
-exclusion chromatography and the results were consistent with the mas
s spectrometric data. These results demonstrate, for the first time, t
he observation and stoichiometric measurement of protein-oligonucleoti
de complexes using ESI-MS. The sensitivity and high resolution of ESI-
MS should make it a useful tool in the study of protein-DNA interactio
ns.