Structural Characterization of a Thrombin-Aptamer Complex by High Resolution Native Top-Down Mass Spectrometry

Abstract

Native mass spectrometry (MS) with electrospray ionization (ESI) has evolved as an invaluable tool for the characterization of intact native proteins and non-covalently bound protein complexes. Here we report the structural characterization by high resolution native top-down MS of human thrombin and its complex with the Bock thrombin binding aptamer (TBA), a 15-nucleotide DNA with high specificity and affinity for thrombin. Accurate mass measurements revealed that the predominant form of native human α-thrombin contains a glycosylation mass of 2205 Da, corresponding to a sialylated symmetric biantennary oligosaccharide structure without fucosylation. Native MS showed that thrombin and TBA predominantly form a 1:1 complex under near physiological conditions (pH 6.8, 200 mM NH4OAc), but the binding stoichiometry is influenced by the solution ionic strength. In 20 mM ammonium acetate solution, up to two TBAs were bound to thrombin, whereas increasing the solution ionic strength destabilized the thrombin-TBA complex and 1 M NH4OAc nearly completely dissociated the complex. This observation is consistent with the mediation of thrombin-aptamer binding through electrostatic interactions and it is further consistent with the human thrombin structure that contains two anion binding sites on the surface. Electron capture dissociation (ECD) top-down MS of the thrombin-TBA complex performed with a high resolution 15 Tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer showed the primary binding site to be at exosite I located near the N-terminal sequence of the heavy chain, consistent with crystallographic data. High resolution native top-down MS is complementary to traditional structural biology methods for structurally characterizing native proteins and protein-DNA drug complexes.

Native Top-Down Mass Spectrometry and Ion Mobility Spectrometry of the Interaction of Tau Protein with a Molecular Tweezer Assembly Modulator

“…Native top-down MS can also reveal the specific site(s) of noncovalent ligand binding and the location of surface residues [31, 37–41]. ECD fragmentation dissociates covalent backbone bonds but preserves non-covalently bound ligands.…”

The application of ion-mobility mass spectrometry for structure/function investigation of protein complexes

“…Moreover, it is expected that in the future, the IM-MS method will be expanded to include high resolution instruments such as FTICR and Orbitrap platforms, paving the way toward defining mobility times of different post-transnationally modified species of a single protein assembly, alongside MS n analyses [62,63]. This task is not trivial, however, due to the slow acquisition rate of these instruments, in comparison to the IM separation time.…”

Multiple Zones Modification of Open Off-Stoichiometry Thiol-Ene Microchannel by Aptamers: A Methodological Study & A Proof of Concept

“…For this purpose, fluorescent anti-sulforhodamine B aptamer was used for the proof of concept. As the affinity between an aptamer and its target seems favored in a low ionic strength medium (below 200 mM) [39], aptamers were dissolved in sodium phosphate buffer at ISx = 50 mM + 5 mM of MgCl 2 . AuNPs-coated OSTE plates treated with the buffer only were considered as control.…”

“…It is known that the ionic strength of the buffer used for the affinity test can have an important influence on the non-covalent interactions between the aptamer and its target [39]. Thus, buffer solutions at two different ISx (50 or 300 mM) were used to solubilize the aptamer and sulforhodamine B. Open-microchannels were functionalized with the same protocol used in Section 3.3: AuNPs-2 and aptamers were immobilized by droplet (3 µL) deposition: seven spots in a row of c.a.…”