Programmed Temperature Electrospray Ionization (ptESI) for Thermal Cycling of Proteins

Abstract

Temperature-controlled electrospray ionization (tcESI) coupled to mass spectrometry (MS) gives detailed structural information of proteins and has been used to study their thermal denaturation. In this work, our programmed-temperature electrospray ionization (ptESI) source is used to rapidly heat and cool a nanoESI capillary containing proteins in solution. The structural changes of acidified ubiquitin and lysozyme are investigated during both unfolding and refolding. ptESI uses fast scan rates, allowing for short experiment times, and results in data dense experiments. As mass spectra are acquired continuously while temperature is scanned. Both ubiquitin and lysozyme exhibited highly reversible unfolding and refolding at different scan rates, but only lysozyme displayed a scan rate dependence; the midpoint temperatures (Tm) shift to higher temperatures with increasing scan rates. This scan-rate dependence indicates the unfolding and refolding transitions are not at equilibrium and there is a partial kinetic component. The refolding of lysozyme appears to be frustrated as there is a large difference between the unfolding and refolding curves and midpoint temperatures (∆Tm). ΔTm also displays a scan-rate dependence, as it increases with increasing scan rate. This research demonstrates that ptESI can be used to determine Tm values, probe protein unfolding and refolding, and reveal kinetic dependencies for reversible transitions. Based on these results, I propose that ptESI can be used to investigate protein stability and scan-rate dependence in protein folding studies, in binding or thermostability assays, and can be a valuable tool for studying biomolecules.