Stabilization of Proteins and Noncovalent Protein Complexes during Electrospray Ionization by Amino Acid Additives

Abstract

Ionization of proteins and noncovalent protein complexes with minimal disturbance to their native structure presents a great challenge for biological mass spectrometry (MS). In living organisms, the native structure of intracellular proteins is commonly stabilized by solute amino acids (AAs) accumulated in cells at very high concentrations. Inspired by nature, we hypothesized that AAs could also pose a stabilizing effect on the native structure of proteins and noncovalent protein complexes during ionization. To test this hypothesis, here we explored MS response for various protein complexes upon the addition of free AAs at mM concentrations into the electrospray ionization (ESI) solution. Thermal activation of ESI droplets in the MS inlet capillary was employed as a model destabilizing factor during ionization. Our results indicate that certain AAs, in particular proline (Pro), pose considerable positive effect on the stability of noncovalent protein complexes in ESI-MS without affecting the signal intensity of protein ions and original protein-ligand equilibrium, even when added at the 20 mM concentration. The data suggest that the degree of protein stabilization is primarily determined by the osmolytic and ampholytic characteristics of AA solutes. The highest stability and visibility of noncovalent protein complexes in ESI-MS are achieved using AA additives with neutral isoelectric point, moderate proton affinity, and unfavorable interaction with the native protein state. Overall, our results indicate that the simple addition of free amino acids into the working solution can notably improve the stability and accuracy of protein analysis by native ESI-MS.

Effects of Multidentate Metal Interactions on the Structure of Collisionally Activated Proteins: Insights from Ion Mobility Spectrometry and Molecular Dynamics Simulations

“…9, 30-32 4 Factors that influence the structural integrity of electrosprayed proteins are of great interest. [33][34][35][36][37][38][39] Protein binding to low molecular weight ions is of particular importance in this context. 35,37,40,41 These bound ions may originate from specific solution phase interactions.…”

“…Factors that influence the structural integrity of electrosprayed proteins are of great interest. − Protein binding to low molecular weight ions is of particular importance in this context. ,,, These bound ions may originate from specific solution phase interactions. ,,− In addition, native ESI is prone to forming nonspecific adducts. − Small cations and anions tend to associate with biomolecular analytes during the final stages of nanodroplet evaporation. For example, ESI of NaCl-containing protein solutions generates heterogeneous [M + z H + n (Na–H) + m (Cl+H)] z + assemblies. − …”

Atomistic Insights into the Formation of Nonspecific Protein Complexes during Electrospray Ionization

“…Our simulations relied on the premise that droplets containing more than one protein represent the prerequisite for nonspecific ESI clustering. ,,− Hence, the MD runs described below followed a strategy similar to earlier protein ESI simulations, , except that the initial droplets contained multiple protein molecules. The initial Rayleigh-charged droplets in our simulations had a 5.5 nm radius, consistent with the size in the ESI plume after several fission/evaporation cycles …”

“…This phenomenon can manifest itself as complex formation from monomeric proteins − or the assembly of complexes into higher-order oligomers. ,− There can also be a mix of specific solution binding and nonspecific clustering. , All of these clustering scenarios complicate the interpretation of native ESI-MS data. Various strategies have been proposed for mitigating this problem, ,,− but it is nonetheless challenging to distinguish specific from nonspecific complexes in a mass spectrum. Interestingly, there are also instances where nonspecific clustering is beneficial; for example, protein clusters can serve as model systems for benchmarking mass analyzer performance at high m / z and as a testbed for top-down dissociation experiments. ,,, …”

Quantitative Determination of Protein–Ligand Affinity by Size Exclusion Chromatography Directly Coupled to High-Resolution Native Mass Spectrometry

“…Moreover, nMS enables the measurement of binding stoichiometry and dissociation constants for a variety of biological complexes. Despite many positive features, nMS is limited to volatile buffers which are compatible with MS detection, whereas, in many cases, nonvolatile buffers or complex stabilizing additives are required for proper biomolecule folding and preservation of noncovalent complexes. ,− …”