Structure and energetics of peptides and small proteins

EPSRC Advanced Research Fellowship

This money allowed Dr. Barran to establish an independent research career.
It was mainly used to fund her salary for 5 years and to provide a small stipend towards project costs.

Until the mid 1980s, mass spectrometry (MS) struggled to analyse large molecules. This changed however with the advent of new, more powerful MS instruments and critically through the development of two new ionisation techniques: Matrix Assisted Laser Desorption Ionisation (MALDI) and Electrospray Ionisation (ESI) These soft ionisation techniques allow the transfer of extremely large, intact biomolecules into the gas phase with relative ease. ESI is particularly informative, since it generates highly charged ions which appear in a mass spectrum at low m/z and can be analysed on an MS with a relatively small mass range. The application of ESI to biomolecules was first shown by Fenn who received the 2002 Nobel Prize in chemistry along with Tanaka - who pioneered soft desorption ionisation a precursor of MALDI – and Wuthrich, an NMR spectroscopist.
While it is clear that MS can be used to study the mass of a protein, it is not necessarily clear how MS can be used to study protein conformation. One, quite indirect method is to use charge state analysis whereby the degree of ‘foldedness’ of a protein can be estimated by studying the distributions of charge states6; more highly charged protein ions tend to be more unfolded than lower charged protein ions. By analysing the abundances of higher and lower charged ions it is often possible to characterise gas phase unfolding transitions. Another, more direct method of analysing conformation by MS is hydrogen/deuterium exchange mass spectrometry, a technique which probes the solvent accessibility and hence structure of proteins in both the solution phase and the gas phase. These measurements however require some prior knowledge of the structure if they are to be useful. Therefore, they are most insightful when used to confirm the retention/loss of higher order structure in the gas phase.
Dr Barran and her group utlised all of these methods
A more direct method to study gas phase conformation is ion mobility spectrometry (IMS). The technique was developed by Cohen and Karasek in 1970 as a sensor. It has since been used to detect illegal drugs, chemical warfare agents, explosives and environmental pollutants. Ion mobility is a measure of how quickly a gas phase ion moves through a buffer gas under the influence of an electric field, this depends on two things: the rotationally averaged collision cross section of the ion and the charge present on it. By measuring the drift time of an ion through a known distance it is possible to determine its collision cross section which can then be used, in conjunction with computational studies, to obtain conformational information.
The use of IMS to elucidate structural information was pioneered by the groups of Bowers and Jarrold who used it to study conformations of gas phase atomic clusters. These methods have since been developed to provide structural information on biomolecules. This grant coupled with her first grant from EPSRC enabled the development of a new ion mobility mass spectrometer – the MoQTOF with which to analyse gas phase conformations of biomolecules. The timing of this proposal coincided with an rapid increase in the use of Ion Mobility to examine conformations of biomolecular molecules due principally to the availability of commercial ion mobility devices and in particular the ‘T-Wave Synapt’ which was launched by Waters/Micromass in 2006. These commercial available devices have already been used to good effect, but rely on the work from instruments such as that developed by Barran to calibrate data.
Barran has produced over 40 papers from the developmental work undertaken in this fellowship, and is now regconised as a world expert in IMS.