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

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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