Advantages and drawbacks of nanospray for studying noncovalent protein-DNA complexes by mass spectrometry.
The noncovalent complexes between the BlaI protein dimer (wild-type and GM2 mutant) and its double-stranded DNA operator were studied by nanospray mass spectrometry and tandem mass spectrometry (MS/MS). Reproducibility problems in the nanospray single-stage mass spectra are emphasized. The relative intensities depend greatly on the shape of the capillary tip and on the capillary-cone distance. This results in difficulties in assessing the relative stabilities of the complexes simply from MS(1) spectra of protein-DNA mixtures. Competition experiments using MS/MS are a better approach to determine relative binding affinities. A competition between histidine-tagged BlaIWT (BlaIWTHis) and the GM2 mutant revealed that the two proteins have similar affinities for the DNA operator, and that they co-dimerize to form heterocomplexes. The low sample consumption of nanospray allows MS/MS spectra to be recorded at different collision energies for different charge states with 1 microL of sample. The MS/MS experiments on the dimers reveal that the GM2 dimer is more kinetically stable in the gas phase than the wild-type dimer. The MS/MS experiments on the complexes shows that the two proteins require the same collision energy to dissociate from the complex. This indicates that the rate-limiting step in the monomer loss from the protein-DNA complex arises from the breaking of the protein-DNA interface rather than the protein-protein interface. The dissociation of the protein-DNA complex proceeds by the loss of a highly charged monomer (carrying about two-thirds of the total charge and one-third of the total mass). MS/MS experiments on a heterocomplex also show that the two proteins BlaIWTHis and BlaIGM2 have slightly different charge distributions in the fragments. This emphasizes the need for better understanding the dissociation mechanisms of biomolecular complexes.
Gabelica
Valérie
V
Laboratoire de Spectrométrie de Masse, Département de Chimie, Bat B6c, Université de Liège, Belgium. v.gabelica@ulg.ac.be
Vreuls
Christelle
C
Filée
Patrice
P
Duval
Valérie
V
Joris
Bernard
B
Pauw
Edwin De
ED
D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization
D011506:Proteins; D004247:DNA