Method for Stabilizing Protein−Ligand Complexes in Nanoelectrospray Ionization Mass Spectrometry

Abstract

The interaction between the bovine pancrease trypsin (Tryp) and its competitive inhibitor benzamidine (1), in solution and the gas phase, is investigated using nanoflow electrospray ionization (nanoES) and Fourier transform ion cyclotron resonance mass spectrometry. In a recent study (Clark, S.M.; Konermann L. Anal. Chem. 2004, 76, 7077-7083), it was reported that the (Tryp + 1) complex could not be detected by ES-MS. Here, it is shown that, with gentle sampling conditions, it is possible to detect gaseous protonated ions of the (Tryp + 1) complex with nanoES-MS. However, the relative abundance of the detected (Tryp + 1)n+ ions is lower than expected, based on solution composition, which suggests that dissociation of (Tryp + 1)n+ ions occurs during MS sampling. The dissociation pathways and corresponding Arrhenius parameters for the protonated (Tryp + 1)n+ ions, at n = 7-9, are determined from time-resolved thermal dissociation experiments, implemented with the blackbody infrared radiative dissociation technique. The gaseous (Tryp + 1)n+ ions are found to have short lifetimes, e.g., <0.6 s, at temperatures of >100 degrees C. The use of solution additives, including polyols, carbohydrates, amino acids, and small organic molecules, to stabilize the (Tryp + 1)n+ ions during nanoES-MS analysis is investigated. Notably, the addition of imidazole to the nanoES solution is shown to preserve the (Tryp + 1)n+ ions. The Kassoc value, (1.9 +/- 0.2) x 104 M-1, determined for the (Tryp + 1) complex by the direct ES-MS method is in agreement with values determined by other analytical methods. The stabilizing effect of imidazole in nanoES-MS is further demonstrated for the interaction between carbonic anhydrase II and 5-(dimethylamino)naphthalene-1-sulfonamide. The stabilizing effect of imidazole may be due to enhanced evaporative cooling achieved by the dissociation of molecules of imidazole, bound nonspecifically, from the protein-ligand complex in the ion source.

Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

“…Notably, the K a value, 1.2×10 5 M −1 , which was calculated from the measured abundance ratio (R) of ligand-bound to unbound Lg ions, is roughly 5-fold smaller than the reported affinity [21]. As described previously, collisional heating of gaseous ions can occur during accumulation in the rf hexapole in the FTICR mass spectrometer used for the present measurements [17]. Indeed, it was found that R (and K a ) increased as the accumulation time decreased.…”

“…Recently, it was shown that the presence of imidazole, at high concentration (~10 mM), can protect protein-ligand complexes from in-source dissociation during ESI-MS analysis [17]. It was proposed that the stabilizing effect is due, at least in part, to enhanced evaporative cooling, which arises from the loss of multiple nonspecifically bound imidazole molecules from the gaseous protein-ligand complexes in the ion source [17].…”

“…Protein-ligand complexes stabilized predominantly by nonpolar interactions also tend to have low kinetic stabilities in the gas phase and are prone to dissociate in the ion source (i.e., in-source dissociation) [11,12]. In-source dissociation reduces the relative abundance of the ligand-bound protein ions and, consequently, leads to artificially low binding constants [11,[15][16][17]. In extreme cases, in-source dissociation results in false negatives, wherein no ligand-bound protein ions are detected by ESI-MS [15].…”

“…In extreme cases, in-source dissociation results in false negatives, wherein no ligand-bound protein ions are detected by ESI-MS [15]. However, using gentle sampling conditions, along with stabilizing solution or gas phase additives, it is sometimes possible to preserve the labile protein-ligand complexes during ESI-MS analysis and to quantify their association constants [17,18].…”

Characterization of acid-induced protein conformational changes and noncovalent complexes in solution by using coldspray ionization mass spectrometry

“…Nanospray [34 -36] is often a gentle ionization method of allowing the use of less harsh conditions including a lower heated capillary temperature, the absence of heater gas, and a lower spray voltage while also providing high ionization efficiency for small-sample consumption. These advantages favor the application of nanospray to study protein conformation and noncovalent complexes [37][38][39].…”