Advantages and drawbacks of nanospray for studying noncovalent protein–DNA complexes by mass spectrometry

Abstract

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 MS/MS. Reproducibility problems in the nanospray single-stage MS 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 spectra of protein-DNA mixtures. Competition experiments 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 µL of sample. The MS/MS experiments on the dimers reveals 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.

Dimerization and DNA Binding Properties of theBacillus licheniformis 749/I BlaI Repressor

“…The absence of BlaI monomer-half-OP1 complex seems to exclude the possibility that the binding of BlaI occurs according to the monomer pathway. Previously, we obtained similar results by mass spectrometry that only the BlaI dimer could bind to the OP1 operator (26) and the semi-conserved OP1 operator. the binding reaction was performed in conditions where BlaI was predominantly present as a monomer ([BlaI] ϭ 0.016 M).…”

Estrogen receptor–ligand complexes measured by chip‐based nanoelectrospray mass spectrometry: An approach for the screening of endocrine disruptors

“…If it is assumed that there is a similar ionization and mass spectrometer transfer efficiencies for the different hERa LBD ions, the folded monomer was found to represent 15% of the total protein concentration based on the nanoESI mass spectra, which correspond to 10%-15% recovery for the purification step. As described by Gabelica et al (2002), when species have different m /z values, such for the different hERa LBD ions, discrimination can occur during the ES process, the transmission into the mass spectrometer, and in the detector. Considering the actual knowledge in this field, such discrimination is difficult to predict for this system.…”

Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues

“…Advantages of nanoESI include improved desolvation, greater salt tolerance, and higher ion yield [32,41,42]. However, nanoESI tends to be less robust in terms of signal stability and reproducibility than regular ESI [43,44]. It is often implied that nanoESI is better suited for preserving proteinligand interactions [32,45], although experimental evidence suggests that this is not necessarily the case [46][47][48].…”