Conformer selected Top Down Sequencing - A Novel Approach to Structural Proteomics

The development of 'soft ionisation' techniques have positioned mass spectrometry as the central go-to technique for proteomic investigations. Electrospray ionisation (ESI)-MS is used extensively in this post-genomic era to determine the primary structure of proteins and has really become the essential tool in so called 'bottom-up' proteomic analysis. Extensive effort and resource has gone into mass spectrometry based proteomics, the majority of which relies on so called 'bottom-up' characterization where proteins are enzymatically cleaved into peptides for MS analysis followed by database correlation to identify (and quantify) the proteins under study. This approach has many analytical advantages; critically it is high throughput and sensitive and clearly has succeeded in many studies however it has some drawbacks. Perhaps the most obvious is that "bottom up" approaches cannot provide direct information on the active fold and interactions of the proteins being analysed, this limits the functional data that could be obtained from these studies.
The analytical advantages of mass spectrometry also apply to its use to examine intact proteins and complexes and there is an emerging research field that identifies proteins this way - so called "top-down" methodologies where proteins are sequenced in the mass spectrometer. Most top down approaches destroy the functional form of the protein prior to sequencing, to enable higher throughput and to facilitate more productive analysis from higher charged parent ions. This is not quite the route we will take, rather we will probe intact proteins and complexes , with careful use of nano-electrospray ionisation (nESI) to retain solution structures.
This proposal will develop so called "top down" methods to examine conformations and dynamics of proteins and protein complexes. The research program will enhance the gamut of predominantly solution-phase based techniques which evaluate protein structure and interactions. Methodologies will assess conformational stability, and dynamics of proteins both in solution and in a solvent free environment. Funds and research time are requested to construct novel instrumentation with which to measure conformations, and unfolding and refolding dynamics over timescales ranging from microseconds to minutes. The proposed instrument will also be able to photo-dissociate mass and conformer selected ions, and detect the product ions. The program of work following technology development will focus on two areas:
1. Top down structural proteomics; combining photo-dissociation and ion mobility mass spectrometry (IM-MS) to map protein structure and interactions.
2. Performing FRET combined with IM-MS, to determine protein unfolding pathways and the effect of fluorescent makers on protein structure.
The equipment will comprise a novel duel ion guide mobility mass spectrometer, wherein IM-MS will be used to determine collision cross sections, and optical methods will interrogate structure and stability via FRET and/or photo dissociation. This combined IM-MS photo activation approach will be termed photo-IM-MS.
Ions will be externally generated via ESI and transferred into a customised ion mobility mass spectrometer. Once in the duel drift region of the IM-MS, the drift time of ions (under the influence of a weak electric field) is related to their rotationally averaged collision cross section with the buffer gas. This mobility measurement can be made, or alternatively the ions will be 'stepped' into a parallel stacked ring ion guide drift region (2SRIG), which has a laser beam passing through it as well as optical detection. Ions in this region will interact with light and either be optically detected, or be pushed back into the first mobility cell. On exiting the cell ions will be transferred to a time-of-flight mass spectrometer and thus will be detected as a function of both mass, charge state, and cross section and potentially following optical interaction

This research program will design, develop and implement an analytical platform which will allow us to determine collision cross sections on mass selected ions and also to characterise conformer selected ions using optical methods. We will be able to perform FRET (Fluorescence Resonant Energy Transfer) as well as ultraviolet and infra-red action spectroscopy and perhaps most widely applicable photo-dissociation. Our proposed approach is based on the technique termed ion mobility mass spectrometry (IM-MS) which determines the rotationally averaged collision cross sections of mass selected ions. The novel aspect will be the design of a parallel stacked ring ion guide mobility cell which will provide an off axis region to further optical interaction. We will develop "top down" sequencing/characterisation methods for intact and native proteins and protein complexes using photo-dissociation with ion mobility. Solution based methods will complement solvent free experiments. Preliminary work will use small peptides, model protein and protein complexes to demonstrate the technology. In later studies we will apply this instrumentation to study dynamic and more disordered systems.
The work will develop the commercially available mass spectrometer known as a Synapt HDS MS. Initial work will take place at our project partners Waters at the Waters Mass Spectrometry Technology Center in Manchester and subsequent development and implementation will take place in Edinburgh.
Molecular mechanics calculations will generate structures to compare with those determined experimentally and will be trained via experimental data. Studies will be supported by comparison to data obtained from conventional techniques, including, solution phase FRET, NMR, and AUC. We will also employ pulse labeling techniques with the mass spectrometer as the detector. This will allow us to evaluate the effects of solvation on protein fold and on conformation.

We do our research to make an impact. This goes beyond the fame or money rewards that many in other sectors might be content with. From taking good data, that we understand, we wish to develop new knowledge, and new ways of obtaining knowledge. We also want to share that knowledge and for it to be of wide use. In the Barran group it is a matter of great pride that 15 students have received PhD's in the past 5 years, and along with 4 postdoctoral researchers, have then gone on to work in many other areas, trained and educated by their experience. This is one of the most immediate impacts from our research portfolio, but there is more to what we do than that. We have an on-going aim to use evidence from the gas phase to understand biological function. As a consequence of this, we collaborate widely with life science researchers so that we can bring the accuracy and selectively of gas phase analysis to assist with their understanding of the complicated processes that go on in the messy environment of cells.

Optical methods are used widely by cell biologists and biophysical chemists and we will here use similar methods albeit in the reductionist environment of the gas phase, which will contrast and complement cell work. Here we will build a novel instrument capable of determining the structures and dynamics of protein and protein complexes, adding to the emerging field of top down protein characterization. This will allow for rapid characterisation of protein structure, using very small amounts of sample .

We will have impact in a number of areas with this technology development. An early outcome will be that we will enable spectroscopy to be performed on a high resolution mass spectrometer capable of analyzing large intact bioactive protein complexes. The fact that we can also examine conformer and mass selected species with optical methods will have relevance to the proteomics community and in particular to those that are interested in mapping protein structure.

Once constructed, we apply our new technology platform to examine disordered and dynamic proteins and their complexes where much is predicted but little has been experimentally verified. The competing technologies for understanding IDPs are NMR and ultra-analytical centrifugation, but both require more sample than electrospray and neither can access as short timescales as we will here. In addition the high throughput capabilities of mass spectrometry are well suited to providing a global understanding unfolding dynamics, an area that we will exploit once this technology is implemented.

The research will have the most immediate benefit to research groups interested in the chosen protein systems; this will of course be those working on transcription factors but also will be biophysics groups interested in the nature of the disordered state of proteins and in related amyloidgenic systems. We are making an instrument that could have wide application. The use of photo dissociation as applied to peptides and sugars is growing and the 2SRIG system could readily be applied to such species. There are other non-biological systems which may also be well interrogated with this technology and we will seek collaborations in synthetic chemistry to test this.


Waters are very keen to see the possibilities of this research, we will work closely with them and our business development executives to best exploit this technology; Waters are our preferred partner since the 2SRIG is a technology relation to existing Synapt instrumentation, and since we have had several years of successful collaboration with them.