Enhancing in-gel protein detection to promote coupled proteomic-genomic analysis

Abstract

Proteins are a distinct class of biological molecules involved in all cellular processes. The protein complement of an organism, organ, tissue, biological fluid or even a single cell is a highly complex mixture with a dynamic range of concentrations up to nine orders of magnitude. One of the few methods capable of resolving this complexity is two-dimensional sodium dodecyl sulphate polyacrylamide electrophoresis (2DE). Following resolution by 2DE, protein detection is carried out using either a stain or reactive fluorophore. These protein detection techniques must satisfy certain criteria; including high sensitivity and quantitative capacity (i.e. have a low limit of detection, with signal intensity proportional to protein concentration). Other considerations include ease of use, cost, and compatibility with subsequent mass spectrometric (MS) characterisation. The current industry standard for post-electrophoretic protein detection, SYPRO Ruby (SR), is reportedly capable of detecting 1 - 2 ng of broad range protein standards with a linear dynamic range (LDR) of 3 - 4 orders of magnitude; it is easy to use and can be imaged with commonly available equipment, although it has been described as expensive and of moderate mass spectrometry compatibility compared to other stains. Pre-electrophoretic approaches involve a covalent reaction between proteins and fluorescent dyes and the most commonly utilised method currently is differential gel electrophoresis (DIGE). DIGE reportedly detects as little as 0.25 ng of broad range protein standards with a LDR of 3 - 4 orders of magnitude. As this method requires optimisation of the pre-labeling reaction to each different sample, it is somewhat less straightforward to use than SR. DIGE is also expensive, and MS analysis usually requires protein material from a separate post electrophoresis stained preparative gel. For these reasons, new methods of detection are always being sought in an attempt to address the complexity of the proteome. If the ease of SR protocol could be combined with the specificity of reactive fluorophores for protein moieties, high sensitivity protein detection could be possible. Thus, the hypothesis that the use of reactive fluorophores for in-gel staining would optimise protein detection in proteomic analyses was tested. In order to identify whether using a reactive dye to post-electrophoretically stain protein could optimise protein detection, a series of amine- and cysteine-reactive dyes were tested for sensitivity in both pre- and post-electrophoretic applications. The data show that post electrophoresis staining is possible, and not significantly different in terms of lowest limit of detection from pre-labeling with isothiocyanate, succinimidyl ester, and maleimide derivatives of fluorescein and rhodamine. However, none of these reactive fluorophores were able to exceed the sensitivity of current industry standards, colloidal Coomassie Brilliant Blue and SR. Post-electrophoretic protein detection may have been ffected by physical characteristics (i.e. molecular weight, hydrophobicity, etc.) of the reactive fluorophores that prevented interaction with the targeted protein moieties. Also, the conformation of proteins in-gel may have limited the accessibility of the reactive fluorophores to the target amino acids. This suggested that the reaction between fluorophore and protein may need to be optimised on a protein-by-protein basis, thus limiting its potential for native proteome detection. Furthermore, without a dual-labeling approach, it seems that a proportion of proteins will always be missed by a reactive labeling method based on the absence or inaccessibility of the target amino acid. These studies focused on quantitative analysis (i.e. methods development) facilitated training in proteomic techniques which were then applied to a real biological problem involving chemotherapeutics. Platinum drugs have demonstrated efficacy in the treatment of solid tumours. A particular example, cisplatin, has seen widespread use and success in the treatment of testicular, ovarian, and head and neck cancers. However, side effects and resistance are critical factors in the decrease of cisplatin effectiveness over time. The novel platinum compound, [(5,6-dimethyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)]2+ (56MESS), has demonstrated a higher level of cytotoxicity in both L1210 and cisplatin-resistant cell lines compared to cisplatin. Compound design and initial DNA binding studies indicate that 56MESS intercalates with DNA rather than binding coordinately as cisplatin does. The Saccharomyces cerevisiae gene deletion mutant library was interrogated to gain an initial understanding of how the differences between cisplatin and 56MESS might affect the molecular mechanisms involved in the eukaryotic response to 56MESS. Genes associated with protein processing were implicated by the increase in 56MESS- sensitivity brought about by their deletion. Cisplatin-sensitive DNA repair mutants were also tested for 56MESS sensitivity and demonstrated very little cross-reactivity. The protein complement of yeast cells exposed to a non-lethal concentration of 56MESS was examined using one-dimensional SDS-PAGE, tricine electrophoresis and 2DE. These studies indicated that there were increased levels of peptides in 56MESS-exposed cells relative to water- and cisplatin-exposed cells. This suggested that proteome degradation occurred in response to the activity of 56MESS. As a very different mechanism of action was suggested, this represents an opportunity to target cellular pathways that could potentially circumvent cisplatin-resistance in the treatment of recalcitrant tumours. As a whole then, this thesis also illustrates the strength of a systems biology approach to the analysis of cellular processes.

Date of Award	2011
Original language	English