Award details

New approaches to quantitative proteomics: application to chemical communication

Reference	BB/I016554/1
Principal Investigator / Supervisor	Professor Robert Beynon
Co-Investigators / Co-Supervisors	Professor Jane Hurst, Dr James Langridge
Institution	University of Liverpool
Department	Veterinary Preclinical Science
Funding type	Skills
Value (£)	91,932
Status	Completed
Type	Training Grants
Start date	01/10/2011
End date	30/09/2015
Duration	48 months

Abstract

unavailable

Summary

This project addresses the application of high resolution and high sensitivity mass spectrometry to characterize the protein components in rodent scent marks. Recent research in rodent semiochemistry, in substantial part from the academic applicants laboratories, has revealed a depth and complexity to rodent chemical communication previously unanticipated. We are building a detailed picture of the receptor repertoire for these signals, and of the higher level processing that collates these signals into behavioural responses, but our understanding of the molecular composition of the scent marks is some way behind. Chemical communication is capable of conveying an incredibly subtle stream of information, especially between conspecifics. Scent marks, predominantly urinary, contain an astonishing array of information that transmits variable indicators of the state of the scent owner (e.g. health status, pregnancy, recent food ingested and time since deposition). This status information is primarily associated with proteins (lipocalins and ESPs) that provide information on genetically invariant parameters such as sex and individual identity. In this project, the student will bring to bear advanced mass spectrometric methodologies in the characterization of these proteins. The samples will be recovered from wild-caught rodents, and are sometimes vanishingly small (such as in tear secretions). The challenges in the study of these scent mark proteins are two fold. First, there is a pressing need for accurate quantification of the proteins in the scent mark. Second, it is clear that the highly polymorphic gene cluster that encodes these proteins is genetically unstable, leading to each wild animal being able to express a unique pattern of these proteins. Thus the challenges are both quantitative and qualitative. The student will address these challenges in collaboration with Waters, using a combination of intact mass profiling, making use of the high resolution QToF instruments and new algorithmic approaches to spectral deconvolution developed at Waters, label-free quantification using the Waters-developed MS^E analytical workflow, coupled with Hi3 peptide quantification, and through the use of selected reaction monitoring, using an artificial QconCAT concatenated standard peptide assembly (technology invented and patented by the academic partner). Finally, discovery of new polymorphic variants will be based on intact mass survey, followed by electron transfer dissociation (ETD, using a new front-end source designed at Waters but not yet widely available) to isolate and characterise the amino acid sequences of the variant proteins. The plan of the programme will follow the outline below, although we expect that from year 2 onwards, the student will take some responsibility for the direction of the research. Year 1: Induction, Design of QconCAT proteins, expression and validation, experience of wild rodent sample collection and diversity. Training in intact mass profiling, bioinformatics tools and peptide level label-fre and label-mediated quantification. Year 2: Development of ion mobility methodologies to improve resolution of complex mixtures of isoforms, and the use of gas phase cross-sectional area to assess the conformational stability and consequent degree of protonation on electrospray ionsation behaviour. Expression of recombinant lipocalins for model studies, appropriately engineered to alter electrostatic potential for charge state manipulation. Instruction on ETD fragmentation. Year 3: Application of ETD to discover amino acid sequence variation in new isoform variants, leading to quantification by surrogate peptides. Includes an exploration of the effect of sequence variation on ESI signal intensity and charge state for quantification. Year 4: Completion of thesis and papers, exploration of new areas of investigation.

Committee	Not funded via Committee
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	Training Grant - Industrial Case

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