Probing protein denaturation during size exclusion chromatography using native mass spectrometry
2020
Size-exclusion chromatography
employing aqueous mobile phases with
volatile salts at neutral pH combined with electrospray-ionization
mass spectrometry (SEC-ESI-MS) is a useful tool to study proteins
in their native state. However, whether the applied eluent conditions
actually prevent protein–stationary phase interactions, and/or
protein denaturation, often is not assessed. In this study, the effects
of volatile mobile phase additives on SEC retention and ESI of proteins
were thoroughly investigated. Myoglobin was used as the main model
protein, and eluents of varying ionic strength and pH were applied.
The degree of interaction between protein and stationary phase was
evaluated by calculating the SEC distribution coefficient. Protein-ion
charge state distributions obtained during offline and online native
ESI-MS were used to monitor alterations in protein structure. Interestingly,
most of the supposedly mild eluent compositions induced nonideal SEC
behavior and/or protein unfolding. SEC experiments revealed that the
nature, ionic strength, and pH of the eluent affected protein retention.
Protein–stationary phase interactions were effectively avoided
using ammonium acetate at ionic strengths above 0.1 M. Direct-infusion
ESI-MS showed that the tested volatile eluent salts seem to follow
the Hofmeister series: no denaturation was induced using ammonium
acetate (kosmotropic), whereas ammonium formate and bicarbonate (both
chaotropic) caused structural changes. Using a mobile phase of 0.2
M ammonium acetate (pH 6.9), several proteins (i.e., myoglobin, carbonic
anhydrase, and cytochrome c) could be analyzed by SEC-ESI-MS using
different column chemistries without compromising their native state.
Overall, with SEC-ESI-MS, the effect of nonspecific interactions between
protein and stationary phase on the protein structure can be studied,
even revealing gradual structural differences along a peak.
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