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