Investigating protein structure by top-down native protein mass spectrometry
Determining protein structure is the central challenge of structural biology, owing to the integral role of proteins in biological systems along with the complexity and diversity of their structures. It is not only an important challenge, but a difficult one; the large size of proteins gives rise to a limitless range of potential structures, while the traditional methods of NMR and crystallography both carry specific limitations. Hence, there is a drive to develop new, orthogonal techniques that could provide additional insight into protein structure. Mass spectrometry (MS) is a ubiquitous analytical technique across all fields of chemistry, and the development of “soft” ionisation techniques has allowed noncovalent interactions to be retained in the gas phase and observed by MS. This in turn has led to the development of “native” MS in which proteins can be analysed and studied in the gas phase with their structures largely intact. Top-down MS of native proteins has been suggested as a potential avenue for studying protein structure. Here, the top-down technique of electron capture dissociation (ECD), which does not disrupt noncovalent interactions, is investigated for its potential to observe protein structure in native MS. Firstly, ECD was used to determine the sites at which a range of metal ions bind to the unstructured protein α-synuclein. These results were analysed through both a traditional top-down workflow and a novel mass defect based analysis. This mass defect analysis demonstrated how metal-containing fragments can detected without determination of their monoisotopic signal and shows potential to be applied in a range of top-down experiments. Secondly, ECD was investigated for its ability to probe the structure of native proteins. Carbonic anhydrase was used as a model protein, and it was observed that ECD can be used to monitor gas-phase unfolding pathways in much greater detail than otherwise available. However, studying multiple structural homologues showed that ECD is not exclusively directed by structure, but rather that availability of positive charge plays an equally important role in directing fragmentation. This was further investigated by producing K→A variants of carbonic anhydrase specifically designed to target ECD-sensitive regions of the protein. Our results demonstrate that minor differences to the primary sequence of a protein can significantly alter the observed ECD fragmentation patterns.