Introduction

Mass spectrometry (MS) owes its remarkable rise as a bioanalytical tool to the development of two “soft” ionization techniques: matrix-assisted laser desorption/ionization (MALDI) (1,2) and electrospray ionization (ESI) (3). The salient feature of soft ionization is that a wide range of analytes, from small metabolites to very large biomolecular systems, can be transferred intact into the gas phase, thus making them amenable to mass spectrometric analysis. For MALDI-MS, the sample is embedded into a crystalline matrix prior to analysis. For ESI, in contrast, ionization is initiated directly from the liquid phase by spraying analyte solution from the tip of an electrically charged capillary. The small droplets formed by the ESI emitter undergo rapid evaporation and fission, ultimately releasing the analyte as multiply protonated (or deprotonated) species (4).

Due to the seamless coupling to the liquid phase, ESI-MS is a powerful tool for studying chemical and biochemical processes in online experiments. The composition of a reaction mixture can be monitored continuously by direct infusion into the mass spectrometer, while the process of interest occurs in solution. The high selectivity of ESI-MS offers the unique opportunity of tracking a multitude of reactive species simultaneously. The result is a global view that provides both accurate kinetic parameters and detailed insights into reaction mechanisms (5).

The ESI charge state distribution is a highly sensitive probe for detecting changes in the solution-phase conformation of proteins. In the commonly used positive ion mode, unfolded polypeptide chains acquire a larger number of protons and will thus appear at lower mass-to-charge ratios (m/z) than their folded counterparts. Presumably, this effect is related to the changes in overall compactness upon unfolding, the larger surface area of an unfolded protein, and the fact that in the native state some protonation sites may be buried inside the interior of the protein (6). Also, possible charge compensation mechanisms are being discussed (7). In addition to providing information on the protein conformation, ESI-MS allows tracking the occurrence of protein-ligand and protein-protein interactions. This is due to the gentle nature of the ionization process that often preserves the composition of noncovalent complexes, such that the ligand binding state of a protein can be deduced from the mass of the corresponding ions ((Figure 1)).

Figure 1.

For many proteins, folding and unfolding are reversible processes that occur spontaneously upon exposure to a suitable solvent environment, typically within a time range of milliseconds to seconds. The mechanisms of these conformational transitions continue to be a hotly debated research topic (8). Many proteins have been characterized as apparent two-state folders, while others fold through transient intermediates. Studies on folding intermediates are complicated by their short lifetimes and by the fact that they often do not accumulate to a significant extent. Nonetheless, the detection and structural characterization of these elusive species represents a very important approach for deciphering the mechanisms of folding and unfolding. While deviations from two-state behavior can sometimes be inferred from equilibrium experiments, direct information on transient folding intermediates can be gained only through kinetic investigations. Optical stopped-flow spectroscopy is one of the classical approaches for studies of this kind. Additionally, pulsed hydrogen exchange quench-flow methods, combined with off-line analysis by nuclear magnetic resonance (NMR) or MS have proven to be extremely useful (9). Limitations of these well-established methods include the poor selectivity of optical detection and the very laborious nature of quench-flow experiments, where kinetic data must be pieced together from numerous single time point measurements. Online ESI-MS methods represent an interesting alternative for mechanistic investigations on protein folding and unfolding, especially in cases where structural transitions involve changes in the ligand binding state or quaternary structure. Following a brief summary of some technical aspects, we will highlight two examples that demonstrate the use of ESI-MS for kinetic and mechanistic studies on protein conformational changes.