Background

Protein folding or unfolding reactions can vary from being highly cooperative to being non-cooperative in nature. A cooperative transition does not involve the formation of any intermediate states [1,2], whereas multiple intermediates are populated during a non-cooperative transition [3–7]. If the partially unfolded intermediate states are not populated to detectable extents, then the folding or unfolding reaction might appear to be a cooperative process [8–11]. Conventional ensemble-averaging probes such as fluorescence or circular dichroism (CD), which are commonly used to study protein folding and unfolding reactions, are unable to detect sparsely populated intermediate species. High-resolution methodologies are required that can not only detect co-existing conformations but also quantify them. Methodologies such as hydrogen exchange (HX) [12–16], sulfhydryl exchange (SX) [17,18], time-resolved fluorescence resonance energy transfer (trFRET) [19,20], and single-molecule FRET (smFRET) [21] have the potential to quantitatively measure the populations of co-existing conformations, and hence have been used to delineate the sequence of structural changes that occur during folding and unfolding. These methodologies have also enabled the structural characterization of transiently formed intermediate states very efficiently [22–25].

However, fluorescence studies require labeling of the protein with a high quantum yield dye, which is not very straightforward. Incorporation of the dye might also have some implications for the secondary and tertiary structure of the protein. On the other hand, HX studies typically rely on enzymatic digestion by pepsin for fragmenting the protein in order to determine where the protein exchange occurred. While pepsin digestion is easy to carry out [12,23,26,27], some proteins are inhibitors of pepsin, disallowing its use [16,28]. As electron transfer dissociation (ETD) involves the fragmentation of the protein inside the mass spectrometer, it has the advantage that it can be used for all proteins. This method was discovered in 2004 [29]. Briefly, a small chemical molecule having a lower electron affinity is allowed to transfer its electron to the protein backbone under a set of conditions. This energizes the protein molecule, leading to its fragmentation at the N-Cα bond, generating c and z ions. ETD has been used in the past to study posttranslational modifications of protein [30,31] and protein–protein interactions [32]. Coupling ETD with HX allows the study of the structural changes occurring in a protein during folding, and the structural characterization of the intermediate states [7,16,28]. The disadvantage of this method is the very low fragmentation efficiency of the protein. The intensities of the peptides are often very low, and many peptides cannot be detected reliably.

HX-MS is not only used to study protein folding but has also been used to study protein–protein interactions, protein–drug interactions, etc. Moreover, mass spectrometry alone has a broad application in proteomics studies. For larger proteins, enzymatic digestion does not give full coverage of the sequence; hence, enzymatic digestion can be coupled with ETD to increase the sequence coverage. This will enable the reliable detection and identification of the proteins and their interacting partners [33].