Electrospray Mass Spectrometry of Biomacromolecular Complexes with Noncovalent Interactions—New Analytical Perspectives for Supramolecular Chemistry and Molecular Recognition Processes

Abstract

The development of “soft” ionization methods in recent years has enabled substantial progress in the mass spectrometric characterization of macromolecules, in particular important biopolymers such as proteins and nucleic acids. In contrast to the still existing limitations for the determination of molecular weights by other ionization methods such as fast atom bombardment and plasma desorption, electrospray ionization (ESI) and matrix‐assisted laser desorption have provided a breakthrough to macromolecules larger than 100 kDa. Whereas these methods have been successfully applied to determine the molecular weight and primary structure of biopolymers, the recently discovered direct characterization by ESI‐MS of complexes containing noncovalent interactions (“noncovalent complexes”) opens new perspectives for supramolecular chemistry and analytical biochemistry. Unlike other ionization methods ESI‐MS can be performed in homogeneous solution and under nearly physiological conditions of pH, concentration, and temperature. ESI mass spectra of biopolymers, particularly proteins, exhibit series of multiply charged macromolecular ions with charge states and distributions (“charge structures”) characteristic of structural states in solution, which enable a differentiation between native and denatured tertiary structures. In the first part of this article, fundamental principles, the present knowledge about ion formation mechanism(s) of ESI‐MS, the relations between tertiary structures in solution and charge structures of macro‐ions in the gas phase, and experimental preconditions for the identification of noncovalent complexes are described. The hitherto successful applications to the identification of enzyme–substrate and –inhibitor complexes, supramolecular protein–and protein–nucleotide complexes, double‐stranded polynucleotides, as well as synthetic self‐assembled complexes demonstrate broad potential for the direct analysis of specific noncovalent interactions. The present results suggest new applications for the characterization of supramolecular structures and molecular recognition processes that previously have not been amenable to mass spectrometry; for example, the sequence‐specific oligomerization of polypeptides, antigen–antibody complexes, enzyme–and receptor–ligand interactions, and the evaluation of molecular specificity in combinatorial syntheses and self‐assembled systems.

Characterization of noncovalent complexes of antimalarial agents of the artemisinin-type and Fe(III)-heme by electrospray mass spectrometry and collisional activation tandem mass spectrometry

“…Although the ESI technique is widely used in studies of noncovalent complexes of biomolecules [21][22][23][24][25][26], the correspondence between the specific noncovalent complexes formed in solution and gas-phase cluster ions produced by the ESI technique may be questioned [31][32][33]. Concern has been expressed that single molecules present in the solute can be randomly distributed in the sprayed droplets and can form any possible type of associate upon desolvation of the droplets.…”

“…In order to evaluate the potential of the drugs to form noncovalent complexes with Fe(III)-heme and their relative binding strengths, we resorted to electrospray ionization mass spectrometry (ESI-MS) and collision-induced dissociation tandem mass spectrometry (ESI-MS/CID/MS). Electrospray ionization mass spectrometry provides a rapid, sensitive and highly selective tool for probing noncova- lent interactions [21][22][23][24]. Most studies that are based on this approach rely on the ability of ESI to transfer noncovalent solution-phase assemblies intact into the gas phase.…”

An investigation of the binding of protein proteinase inhibitors to trypsin by electrospray ionization mass spectrometry

“…This not only avoids the denaturing conditions that have been used previously, but also allows the study of proteins under conditions in which they can be detected as complexes with substrates, products or inhibitors. Initially, ESI MS was restricted to the study of complexes in which the ligand (substrate or product) was covalently bound [1], but increasingly non-covalently bound enzyme complexes are being studied [2][3][4][5]. However, these studies have principally been concerned with the binding of relatively small molecules to enzymes [6], or with protein-protein interactions in natural oligomers, e.g.…”

Mass spectrometric determination of association constants of adenylate kinase with two noncovalent inhibitors

“…Problems with concentration dependent ion yields are circumvented by a data evaluation method that is insensitive to the overall ionization efficiency. The K a values found were 9.0 ϫ 10 4 M Ϫ1 (Ap4A) and 4.0 ϫ S oft ionization mass spectrometry (MS), in particular matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), is well known for its ability to bring high molecular weight biomolecules into the gase phase and to even preserve noncovalent complexes [1][2][3][4][5][6][7][8][9][10]. Besides its established applications, a recurring question concerns the applicability of mass spectrometry to measuring noncovalent interaction strengths [11].…”