Intermolecular Associations in 2D and 3D 985 Protein–ligand and protein–protein interactions studied by electrospray ionization and mass spectrometry W.I. Burkitt*, P.J. Derrick* 1 , D. Lafitte† and I. Bronstein‡ *Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K., †Faculte de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedis 05, France, and ‡Institute of Animal Health, Protein Structure Group, Compton Laboratory, Compton, Newbury RG20 7NN, U.K. Abstract Electrospray ionization has made possible the transference of non-covalently bound complexes from solution phase to high vacuum. In the process, a complex acquires a net charge and becomes amenable to measurement by MS. FTICR (Fourier-transform ion cyclotron resonance) MS allows these ions to be measured with sufficiently high resolution for the isotopomers of complexes of small proteins to be resolved from each other (true for complexes up to about 100 kDa for the most powerful FTICR instruments), which is of crucial significance in the interpretation of spectra. Results are presented for members of the S100 family of proteins, demonstrating how non-covalently bound complexes can be distinguished unambiguously from covalently bound species. Consideration relevant both to determination of binding constants in solution from the gas-phase results and to the elucidation of protein folding and unfolding in solution are discussed. The caveats inherent to the basic approach of using electrospray and MS to characterize protein complexes are weighed and evaluated. Introduction Interest in MS among biochemists has risen sharply in recent years, reflecting progress in developing useful methods for applying this mature instrumental technique to biological macromolecules. The overriding challenge in seeking to apply MS to biomacromolecules has always been how to vaporize samples. Progress in meeting this challenge has been steady for over 30 years, and different methods have been used at different times. The methods currently used most widely are MALDI (matrix-assisted laser-desorption ionization) [1,2] and ESI (electrospray ionization) [3,4]. Before MALDI and ESI, there was fast-atom bombardment [5], and before that plasma desorption [6], with field desorption [7] before that. All of these methods work well for proteins, and there has always been a slant evident in development work in MS towards proteins rather than, say, nucleic acids and carbohydrates. The growing realization that characterization of genes yields insufficient information for understanding biological processes has brought greater attention to protein comple- ments of cells and tissues, and has been a major factor generating interest in MS. MALDI and ESI differ significantly in that the former typically affects sublimation of a crystalline solid, whereas the latter concerns the vaporization of a liquid solution. Thus, if the study is to probe by MS the properties of Key words: electrospray ionization (ESI), Fourier-transform ion cyclotron resonance (FTICR), mass spectrometry (MS), non-covalent interaction, S100. Abbreviations used: MALDI, matrix-assisted laser-desorption ionization; ESI, electrospray ionization; FTICR, Fourier-transform ion cyclotron resonance. 1 To whom correspondence should be addressed (e-mail p.j.derrick@warwick.ac.uk). a biomacromolecule that is dependent upon or involves solvent interactions, the method of choice today is ESI. This paper will focus on protein–ligand and protein– protein interactions studied by ESI. In principle, any mass- spectrometric technique can be used to measure gaseous protein ions formed by ESI, but this paper will concentrate on experiments using FTICR (Fourier-transform ion cyclotron resonance) [8]. The reason for this sharp focus is that FTICR affords resolution (in terms of mass) sufficiently high to resolve 13 C isotopic components of small proteins (molecular mass 100 kDa). FTICR offers mass resolutions higher than those of other mass-spectrometric techniques, although there is the caveat that resolution (more precisely resolution in terms of mass-to-charge ratio) is inversely proportional to mass with FTICR. The importance of resolving 13 C iso- topomers with ESI is that the spacing of adjacent 13 C peaks is related to 1/z (where z is the net charge of the ion in units of electronic charge), and knowing z allows the mass m of an ion to be determined from the measured mass-to- charge ratio m/z. If the 13 C isotopic peaks are not resolved, the conundrum that m/z is measured and both m and z are unknown must be resolved by some means in order to interpret the experimental results. ESI ESI involves nebulizing a liquid typically at atmospheric pressure with the aid of a high electric field. Barely visible droplets are created from protein solutions, and these droplets shrink through evaporation of solvent. The droplets become unstable and eventually isolated protein ions are obtained. C  2003 Biochemical Society