Multi-step conformational transitions in heat-treated protein therapeutics can be monitored in real time with temperature-controlled electrospray ionization mass spectrometry.
Guanbo WangPavel V BondarenkoIgor A KaltashovPublished in: The Analyst (2018)
Heat-induced conformational transitions are frequently used to probe the free energy landscapes of proteins. However, the extraction of information from thermal denaturation profiles pertaining to non-native protein conformations remains challenging due to their transient nature and significant conformational heterogeneity. Previously we developed a temperature-controlled electrospray ionization (ESI) source that allowed unfolding and association of biopolymers to be monitored by mass spectrometry (MS) in real time as a function of temperature. The scope of this technique is now extended to systems that undergo multi-step denaturation upon heat stress, as well as relatively small-scale conformational changes that are precursors to protein aggregation. The behavior of two therapeutic proteins (human antithrombin and an IgG1 monoclonal antibody) under heat-stress conditions is monitored in real time, providing evidence that relatively small-scale conformational changes in each system lead to protein oligomerization, followed by aggregation. Temperature-controlled ESI MS is particularly useful for the studies of heat-stressed multi-domain proteins such as IgG, where it allows distinct transitions to be observed. The ability of native temperature-controlled ESI MS to monitor both the conformational changes and oligomerization/degradation with high selectivity complements the classic calorimetric methods, lending itself as a powerful experimental tool for the thermostability studies of protein therapeutics.
Keyphrases
- heat stress
- mass spectrometry
- ms ms
- molecular dynamics
- molecular dynamics simulations
- single molecule
- protein protein
- multiple sclerosis
- heat shock
- monoclonal antibody
- liquid chromatography
- small molecule
- endothelial cells
- high resolution
- gas chromatography
- single cell
- living cells
- high glucose
- quantum dots
- subarachnoid hemorrhage
- brain injury
- simultaneous determination