Quantitative Proteomics Strategies To Explore The Saccharomyces cerevisiae Proteome

Woolerton, Yvonne Quantitative Proteomics Strategies To Explore The Saccharomyces cerevisiae Proteome. PhD thesis, University of Liverpool.

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Abstract

Quantitative proteomics aims at not just identifying, but accurately quantifying the cellular proteome, and while technological advances towards accurate and reliable quantification of proteins is advancing, this alone does not provide an accurate picture of a proteins role within a cell. There is a far greater level of functionality in the cellular environment than there are protein coding genes in the genome, owing partly to the organisation of individual proteins into larger assemblies. A single protein can form interactions with, potentially, a large number of other proteins, leading to a variety of different protein complexes, and subunits can move, break apart or combine depending on cellular conditions. This complex organisation is despite the normal proteomics strategies employing a destructive process, breaking protein structure down to the peptide level. Further difficulties in mapping the cellular proteome arise from the differential expression level of proteins, which in S.cerevisiae can span up to 5 orders of magnitude. This poses problems for the quantification of less abundant proteins in the cell, which can be masked by the more concentrated proteins. An attempt is made within this thesis to use quantitative proteomic techniques to build a picture of the S.cerevisiae cellular proteome. For the analysis of S.cerevisiae protein complexes ion exchange chromatography has been used to separate the cellular proteome into discrete fractions, each containing a different array of protein complexes. The aim here was to analyse the individual subunits of these complexes by LC-MS, with the use of label free quantification strategies. This enables the high throughput identification and quantification of 1800 proteins along with their potential interaction partners. However, for some of the complexes presented here the accuracy of the label free quantification is called into question, as complex subunits known to be equimolar are identified at different concentrations. In order to assess the accuracy of the label free data QconCATs were also designed to analyse the subunits of some complexes by label mediated quantification. In addition, an attempt is made to access proteins from the entire dynamic range of the cellular proteome using equaliser bead technology. This method uses a library of hexapeptide ligands bound to porous beads to bind, theoretically, every protein present in the sample to equal amounts. The beads are used here to bring up the less abundant proteins in the sample, while simultaneously reducing the amount of the abundant proteins. While this goal is achieved, it is also evident that certain proteins are able to bind the beads to a much larger extent than others, so rather than reducing the dynamic range of proteins identified, there is more of a shift in the dynamics, with previously mid-range proteins becoming highly abundant in the data presented here.

Item Type:	Thesis (PhD)
Additional Information:	Date: 2014-04 (completed)
Depositing User:	Symplectic Admin
Date Deposited:	11 Sep 2015 10:49
Last Modified:	17 Dec 2022 01:34
DOI:	10.17638/02025519
URI:	https://livrepository.liverpool.ac.uk/id/eprint/2025519