Conformer selected top down sequencing - A novel approach to structural proteomics

Conformer selected top down sequencing – A novel approach to structural proteomics

Funded Value: £360,078
Funded Period: Feb 14 – Jan 17
Funder: BBSRC
Principal Investigator: Perdita Barran
Research Topic: analytical science; instrumentation eng. & dev.; protein folding / misfolding; proteomics; structural biology

The development of ‘soft ionisation’ techniques has 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.

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. This 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 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.”

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