Application of SIMSTEX to oligomerization of insulin analogs and mutants

Abstract

The propensity of various insulins and their analogs to oligomerize was investigated by mass spectrometric methods including measurement of the relative abundances of oligomers in the gas phase and the kinetics of H/D amide exchange. The kinetics of deuterium uptake show a good fit when the exchanging amides are placed in three kinetic groups: fast, intermediate, and slow. r-Human insulin, of the insulins investigated, has fewer amides that exchange at intermediate rates and more that exchange at slow rates, in accord with its higher extent of association in solution. We adapted PLIMSTEX (protein ligand interactions by mass spectrometry, titration, and H/D exchange) to determine protein/ligand affinities in solution, to determine self-association equilibrium constants for proteins, and to apply them to various insulin analogs. We term this adaptation SIMSTEX (self-association interactions using mass spectrometry, self-titration and H/D exchange); it gives affinity constants that compare well with the literature results. The results from SIMSTEX show that some mutants (e.g., GlnB13) have an increased tendency to self-associate, possibly slowing down their action in vivo. Other mutants (e.g., lispro and AspB9) have lower propensities for self-association, thus providing potentially faster-acting analogs for use in controlling diabetes. (J Am Soc Mass Spectrom 2006, 17, 1526 -1534

Carboxyl Group Footprinting Mass Spectrometry and Molecular Dynamics Identify Key Interactions in the HER2-HER3 Receptor Tyrosine Kinase Interface

“…H/DX labels amide hydrogens, whereas hydroxyl radical labeling modifies aromatic, aliphatic, and sulfur-containing side chains. MS footprinting strategies require less protein material than traditional structural methods and have been successfully employed in the characterization of protein-protein interactions, oligomerization, and folding dynamics (15)(16)(17)(18). These techniques suffer some limitations when investigating membrane-associated proteins, such as extensive back-exchange (in the case of H/DX) or nonspecific oxidation (in the case of hydroxyl radical labeling) resulting from the extensive post-labeling sample cleanup required to remove lipids.…”

A mechanistic study of the H/D exchange reactions of protonated arginine and arginine-containing Di- and tripeptides

“…Although H/D exchange reagents can access all labile hydrogen atoms of small peptides, the accessibility of exchange reagents toward labile hydrogen sites of larger peptides and proteins is conformation dependent [1], thus the rate of H/D exchange reactions and the number of hydrogen atoms exchanged can be used as a structural probe [2]. H/D exchange and mass spectrometry can be combined to study both gas-phase and solutionphase H/D exchange reactions [3][4][5][6][7][8][9], and the kinetics of H/D exchange can be utilized to infer the presence of distinct conformations of both solution-phase [10 -12] and gas-phase peptide and protein ions [13]. For example, McLuckey et al studied the gas-phase H/D exchange of bradykinin [M ϩ H] ϩ ions (amino acid sequence RPPGFSPFR) with DI and D 2 O, and they interpreted the observed bimodal distributions of product ions as evidence for two non-interconverting ion conformations having different reactivities toward deuterating reagents [14].…”

Carboxyl-Group Footprinting Maps the Dimerization Interface and Phosphorylation-induced Conformational Changes of a Membrane-associated Tyrosine Kinase

“…Titration of Her4 Kinase-Domain-To gain a more quantitative view of Her4 kinase-domain dimerization, we modified the protocol of our previously reported H/D exchange method (SIMSTEX) (24) and used carboxyl-group modification instead of H/DX to determine the isolated Her4 kinase-domain dimer association constant. We started the titration experiment by equilibrating nickel liposomes with different concentrations of the isolated Her4 kinase-domain protein.…”

“…Notable approaches include acetylation (18), amide hydrogen/deuterium exchange (H/DX) (19), and hydroxyl radical footprinting (20). MS-based footprinting methods have been successful for monitoring protein folding and unfolding dynamics (21), characterizing protein-ligand interaction (22,23), and protein oligomerization (24). For probing complicated systems, such as membrane-associated proteins, H/DX suffers limitations because of significant back exchange accompanying the demanding postlabeling purification.…”

“…Through a novel modification of our SIMSTEX method (Self-association Interactions using Mass spectrometry, Self-Titration and H/D Exchange) (24), we are able to use a self-titration platform whereby the extent of carboxyl-group footprinting as a function of concentration can be modeled to give the dimerization affinity of the isolated Her4 kinase-domain dimer. This dimer association constant is an important gauge of the Her4 kinase-domain's contribution to the overall dimerization of the full-length Her4 protein.…”