| To realize the full potential of electrospray ionization-mass spectrometry (ES-MS) as a general tool for the determination of protein-ligand binding affinities, a robust experimental protocol is necessary. Using an antibody single-chain fragment (scFv) and its native ligand, alphaGal[alphaAbe]alphaMan, as a model system, the influence of experimental conditions on binding measurements performed with nanoflow electrospray (nanoES) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) were investigated. Mass spectra measured using short spray durations (< 10 min), which minimize pH changes, equimolar analyte concentrations, which minimize the formation of nonspecific complexes, and short accumulation times (< 2 s) in the hexapole of the ion source, which minimize dissociation of the gaseous complexes, accurately reflect the equilibrium distribution of bound and unbound protein in solution. Application of this methodology to the scFv and a series of carbohydrate ligands (alphaAbe(2-O-CH3-alphaMan), betaGlc[alphaAbe]alphaMan and alphaGlcalphaGlcalphaGal[alphaAbe]alphaMan) yields affinities which are in agreement with values obtained by microcalorimetry.; A potential strategy for minimizing nonspecific biological complexes in ES-MS experiments is to selectively dissociate them in the gas phase prior to detection. Blackbody infrared radiative dissociation (BIRD) was performed on the protonated (+10, +11) specific and corresponding nonspecific complexes of scFv with alphaGal[alphaAbe]alphaMan. This study revealed that the nonspecific complex was kinetically more stable at both charge states and, at the +10 charge state, energetically more stable than the specific complex, indicating that selective gas phase dissociation of the nonspecific complex is not feasible.; The mechanism of formation of nonspecific complexes of ubiquitin and carbonic anhydrase with carbohydrates ranging in size from mono- to tetrasaccharides and the nature of their stabilizing interactions were investigated using nanoES-FT-ICR/MS and BIRD. Nonspecific binding was favoured for small (mono- or disaccharide), hydrophilic carbohydrates over larger or hydrophobic carbohydrates, which tend to form gaseous monomer or cluster ions by nanoES; the efficiency was insensitive to the structure of protein and the charge state of the complex. Evidence that both ionic and neutral intermolecular hydrogen bonds stabilize the gaseous complexes was obtained. The use of multivalent metal ions was proposed as a strategy for minimizing the formation of nonspecific complexes of proteins and small molecules. |
