Heat-induced conformational changes in proteins studied by electrospray ionization mass spectrometry

Abstract

A simple and effective device for investigating heat-induced denaturation of proteins by electrospray ionization mass spectrometry is described. Results are presented for the denaturation as a function of temperature and solution pH of bovine ubiquitin and bovine cytochrome c. These results are in concert with and extend the earlier results of LeBlanc et al. (Org. Mass Spectrom. 1991, 26, 831). The cooperative effects of pH and temperature on the denaturation of ubiquitin and cytochrome c were investigated. Electrospray ionization mass spectrometry is also shown to be a useful probe of the reversibility of heat-induced denaturation of proteins. Finally, it is demonstrated that heat-induced denaturation can be used to improve the mass spectrometric response of proteins that do not normally yield useful spectra when the solubilized protein is electrosprayed at ambient temperatures.

Mass spectrometry and the amyloid problem—How far can we go in the gas phase?

“…To date, the relationship between ESI-MS charge state distributions (i.e., protein conformation in the gas phase) has correlated well with the conformational properties of proteins in solution, thus supporting the notion that protein structure in the solution phase can be retained after ionization and, for some time, into the gas phase [23,24]. But what about a more realistic situation in which a protein could populate a number of conformational states simultaneously?…”

“…Early ESI-MS experiments illustrated that the m/z charge state distribution of a protein depended on its folded state, with a more folded protein having a narrower charge state distribution with fewer charges on average than the same protein analyzed under alternative solution conditions which favor a less folded conformation [23]. To date, the relationship between ESI-MS charge state distributions (i.e., protein conformation in the gas phase) has correlated well with the conformational properties of proteins in solution, thus supporting the notion that protein structure in the solution phase can be retained after ionization and, for some time, into the gas phase [23,24].…”

Time of flight versus ion trap MS coupled to CE to analyse intact proteins

“…It is accepted that the shape of the charged distribution does mainly depend on the availability of the various chargeable basic sites (Arg, Lys, His and the N-terminus) [8], and thus to the tertiary structure of the protein (i. e. in a highly folded protein few basic sites will be available, however, in a fully unfolded state all basic sites should be accessible) [9]. This effect on the charge state distribution of protein has been verified studying the influence of acids [10], temperature [11] or solvents [12]. However, recently it has also been shown that proteins themselves can have an important influence on the response of other proteins [13].…”

Investigation of intact protein complexes by mass spectrometry

“…Indeed, soon after the introduction of MALDI and ESI, these ionization methods were already used for the study of intact proteins and noncovalent protein complexes (Ganem, Li, & Henion, 1991;Miranker et al, 1993;Mirza, Cohen, & Chait, 1993;. In particular, ESI is well suited to detect and investigate non-covalent complexes by transferring whole intact assemblies into the vacuum inside the mass spectrometer.…”