An in vivo control map for the eukaryotic mRNA translation machinery

Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non-intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non-stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNAi to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than-average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis

Multi-site rate control analysis identifies ribosomal scanning as the sole high-capacity/low-flux-control step in mRNA translation

Control of complex intracellular pathways such as protein synthesis is critical to organism survival, but is poorly understood. Translation of a reading frame in eukaryotic mRNA is preceded by a scanning process in which a subset of translation factors helps guide ribosomes to the start codon. Here, we perform comparative analysis of the control status of this scanning step that sits between recruitment of the small ribosomal subunit to the m7GpppG‐capped 5′end of mRNA and of the control exerted by downstream phases of polypeptide initiation, elongation and termination. We have utilized a detailed predictive model as guidance for designing quantitative experimental interrogation of control in the yeast translation initiation pathway. We have built a synthetic orthogonal copper‐responsive regulatory promoter (PCuR3) that is used here together with the tet07 regulatory system in a novel dual‐site in vivo rate control analysis strategy. Combining this two‐site strategy with calibrated mass spectrometry to determine translation factor abundance values, we have tested model‐based predictions of rate control properties of the in vivo system. We conclude from the results that the components of the translation machinery that promote scanning collectively function as a low‐flux‐control system with a capacity to transfer ribosomes into the core process of polypeptide production that exceeds the respective capacities of the steps of polypeptide initiation, elongation and termination. In contrast, the step immediately prior to scanning, that is, ribosome recruitment via the mRNA 5′ cap‐binding complex, is a high‐flux‐control step

Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards

Medical Biochemistr

QconCAT method development and applications in proteomics

Quantitative data is an excellent resource in any proteomics study but is essential in many. In recent years this area has expanded from relative to absolute quantification with a wide range of methods available for absolute quantitative proteomics. In general protein quantification is based on either label-mediated or label-free strategies. Common label-mediated approaches are isotope dilution strategies, such as AQUA, coupled with mass spectrometry, where analyte signal is compared to a stable isotope labelled standard added in known abundance. These methods are suited to small-scale studies but increasing demand for large-scale proteome quantification exposed the need for alternative quantification methodologies. The QconCAT technology, first published in 2005, is a label mediated approach which utilises the principle of surrogacy to quantify analyte proteins based on a signature peptide, or peptides, for each protein. QconCATs are concatenations of quantotypic peptides for a group of proteins, the QconCAT gene is designed in silico and expressed heterologously in E.coli with [13C6]arg and [13C6]lys to elicit a stable isotope labelled multiplexed absolute quantification standard. In this thesis I describe several developments to the QconCAT production protocol. These developments reduce the production time from ~19d, using the initial method, to less than 7d. Time gains have been made across the whole workflow in the areas of protein expression, cell lysis, and product purification. Moreover verification of the QconCAT is delayed until the final product is synthesised, made possible by evidence of high quality reproducible expression. I explain how these alterations allow for production of several QconCATs in parallel, giving added efficiency. The success of the method is demonstrated through the use of multiple QconCATs. As a result of this work it is now possible to make at least eight QconCATs per week and the rate-limiting step of the quantification workflow has migrated from standard preparation to data processing. The final study in this thesis discusses methods for accurate quantification of the QconCAT protein and additional applications of QconCATs for testing mass spectrometer performance

Oncoproteomic applications for detection of breast cancer. Proteomic profiling of breast cancer models and biopsies

The CD-ROM disc containing supplementary material is kept in the cardboard box in the Systems Office.The heterogeneity of breast cancer (disease stage and phenotype) makes it challenging to differentiate between each subtype; luminal A, luminal B, HER2, basal-like and claudin-low, on the basis of a single gene or protein. Therefore, a collection of markers is required that can serve as a signature for diagnosing different types of breast cancer. New developments in proteomics have provided the opportunity to look at phenotype-specific breast cancer cell lines and stage-specific liquid biopsies (nipple aspirate fluid [NAF], plasma samples) to identify disease and phenotype specific signature. An 8-plex iTRAQ quantification strategy was employed to compare proteomic profiles of a range of breast cancer and ‘normal-like’ cell lines with primary breast epithelial cells. From this, 2467 proteins were identified on Orbitrap Fusion and Ultraflex II, of which 1430 were common. Matched pairs of NAF samples from four patients with different stages of breast cancer, were analysed by SCX-LC-MS and a total of 1990 unique gene products were identified. More than double the number of proteins previously published data, were detected in NAF, including 300 not detected in plasma. The NAF from the diseased patients have 138 potential phenotype biomarkers that were significantly changed compared to the healthy volunteer (7 for luminal A, 9 for luminal B, 11 for HER2, 14 for basal-like and 52 for claudin-low type). The average coefficient of variation for triplicate analyses by multiple reaction monitoring mass spectrometry (MRM-MS), was 9% in cell lines, 17 % in tissue biopsies, 22% in serum samples and 24% in NAF samples. Overall, the results provide a strong paradigm to develop a clinical assay based on proteomic changes in NAF samples for the early detection of breast cancer supplementary to established mammography programmes.The supplementary material submitted with the thesis is not available online

Remodelling of the cardiac caveolar domain in heart failure and its putative influence on beta adrenergic signalling

Over 500,000 people in the UK have heart failure (HF). After an initial insult to the heart, sympathetic drive increases which leads to detrimental remodelling of cardiac β-adrenergic receptors (βAR) and further cardiac dysfunction. The main βAR expressed in the heart are the β1AR and β2AR. In heart failure, remodelling is characterised by reduced β1AR density, desensitisation of the remaining β1AR and aberrations of normal βAR signal compartmentalisation. Caveolae, flask-shaped lipid rafts, are present in most cells including cardiac myocytes and are characterised by the presence of caveolin and cavin proteins. Caveolar proteins create distinct micro-domains within the membrane and play a key role in compartmentalisation of signalling from both the β1AR and β2AR. Isolated reports of changes in caveolar structure and proteins in HF have implications for β-AR signalling, however the full array of caveolar protein changes in HF has not previously been assessed. Here we establish how the expression and membrane location of β-AR cascade and caveolar proteins changes in rat models of right ventricular (RV) and left ventricular (LV) failure induced by monocrotaline and aortic banding, respectively. For the RV model, we examined changes in β-AR responsiveness, and tested the potential for reversing functional and caveolar remodelling using a common LV therapy (the β-blocker metoprolol). Quantitative analyses of caveolar protein expression in myocyte and myocardial samples was also carried out using custom-designed calibrating peptides (CavCATs). Both HF models showed a reduction in caveolar protein expression, with protein redistribution also found in the RV model. Decreased expression of β-AR signalling proteins (β1AR, adenylyl cyclase) accompanied by increased expression of inhibitory proteins (Gαi, GRK2) was also observed in both models, with some remodelling of membrane distribution. β-blocker treatment in RV failure partially recovered expression of caveolar and β-AR cascade proteins. Cardiac β1AR responsiveness was reduced in RV failure and again, this was partially recovered by β-blocker treatment. Quantitative work highlights the importance of studying non muscle-specific caveolar protein isoforms in the cardiac myocyte given e.g. similar expression of Cav 3 and Cav 1 in these cells. Caveolae are dynamic membrane compartments which change in HF. This work suggests that caveolar changes affect β-AR signalling protein membrane location, which contributes to aberrations of signalling which (in the case of RV failure) can be reversed by β blockers

DOSCATs: Double Standards in Quantitative Proteomics

Since its inception, the field of proteomics has shifted from being a qualitative discipline, generating long lists of proteins within a sample, to a quantitative one, where how much of a protein is reported. With the advent of systems biology, the routine analysis of biomarker levels, and the requirement for robust, reliable data comparable between different laboratories, the importance of absolute quantification, where proteins are quantified in absolute titre, is becoming increasingly important. There are two commonly used techniques for absolute protein quantification, based on either mass spectrometry (MS) or immunochemical techniques such as western blotting (WB). MS is generally considered the gold standard technique for quantification, but WB can offer greater sensitivity and is much more accessible to researchers. Neither are intrinsically quantitative techniques and so rely on standards; either isotope labelled peptides or recombinant proteins bearing an epitope are used for MS or WB respectively. To improve the robustness and reproducibility of quantitative data it would be advantageous to apply both techniques for orthogonal quantification, but due to the very different calibration standards, workflows rarely overlap. DOSCATs (Double Standard conCATamers) are novel calibration standards that can unite MS and WB workflows, allowing for the quantification of direct comparison of quantitative data between the two platforms. DOSCATs, based on QconCAT technology, combine a series of epitope sequences concatenated with peptides in a single artificial protein. Stable isotope labelled peptide for MS analysis are released upon digestion with an enzyme such as trypsin, and intact DOSCATs act to bear multiple epitopes for WB. Also included were restricted proteolysis sites that allow for a mobility shift within WB, lending greater flexibility to the standard. The aim of this thesis was to develop and optimise the use of DOSCAT technology so that they could be used to quantify target proteins in both quantitative platforms. A DOSCAT protein was designed and constructed to quantify five proteins of the NF-κB pathway. The DOSCAT was expressed and purified and the 9/13 peptides and 3/5 epitopes included in the sequence were observed by MS and WB respectively, demonstrating the proof of concept. However, restricted proteases performed poorly and three antibodies were discontinued by the manufacturer, so a second iteration of the NF-κB DOSCAT was designed. This was used to calibrate quantification by selected reaction monitoring MS (SRM-MS) and automated capillary WB. For three target proteins, protein fold change and absolute copy per cell values measured by MS and WB were in excellent agreement. Building on this success, another DOSCAT was built for six proteins implicated to be indicative of paediatric Streptococcus pneumoniae meningitis infection. All six proteins were quantified by SRM-MS although QWB failed to quantify two targets as either DOSCAT or endogenous protein was not detected. SRM-MS data agreed very well with previous datasets generated for the same samples by label-free MS and QWB using full length standards, however, absolute values for DOSCAT calibrated QWB were inconsistent. This could be due to antibodies recognising DOSCAT and endogenous protein with different affinities. This work demonstrates that DOSCATs can be used as multiplexed, dual purpose standards to unite MS and WB workflows. The DOSCAT approach has the potential to generate reliable quantitative information particularly relevant for systems biology studies and contribute to the desired increase in reproducibility of biological research

Protein and transcriptome signatures of cartilage ageing and disease

It is hypothesised that there are distinct mechanisms involved in cartilage ageing and disease which can be determined using next-generation technologies including mass spectrometry and RNA-sequencing. The aims of this thesis were to firstly characterise molecular mechanisms associated with age-related and arthritis-related changes in cartilage gene and protein signatures. Secondly the thesis developed new techniques to identify novel cleavage sites in matrix proteins and to quantify some known proteolytic events in articular cartilage using mass spectrometry-based proteomics platforms. Finally the levels of key proteinases and their inhibitors involved in the pathogenesis of OA were measured using mass spectrometry. Osteoarthritis (OA) is an extremely common cause of morbidity in both man and animals. OA involves the biomechanical failure of articular cartilage, together with changes in the subchondral bone and inflammation of the joints and leads to a variety of symptoms including pain, stiffness and reduced mobility. Age is an important factor in the development of OA and represents a huge challenge for society as whilst life span increases, the quality of life faced by an ageing population is often poor. Articular cartilage is susceptible to age-related diseases such as OA, but it is not an inevitable result of ageing and is a consequence of a complex inter-relationship between age and further predisposing factors. There have been major advances in technologies used to interrogate proteins and genes due to genome sequencing enabling gene and protein sequences to be determined. These ‘next-generation technologies’ include mass spectrometry (MS) and next-generation sequencing. This thesis has used these technologies in an attempt to address important questions relating to cartilage ageing and disease. The use of an inflammatory model of early OA in equine and human cartilage enabled the discovery and quantification of important proteins and pathways involved, using relative and absolute mass spectrometry techniques. In the equine secretome pathway enrichment analyses confirmed the up-regulation of glycolytic proteins. The novel proteins clathrin and LIM and SH3 domain protein-1 were identified for the first time in cartilage proteomics. QconCAT technology and gene expression analysis enabled normal and OA cartilage extract to be interrogated. Absolute quantification values were demonstrated for the first time for aggrecan; first and third globular domains, biglycan, cartilage oligomeric matrix protein, decorin and fibromodulin. Whilst a novel MS based technique enabled previously identified and novel extracellular matrix cleavage sites derived from matrix metalloproteinase 3 and a disintegrin and metalloproteinase with thrombospondin motifs 4 digestion of cartilage to be determined. Some of these sites of degradation were also evident in OA but not normal cartilage using matrix assisted laser desorption ionization imaging MS (MALDI-IMS). Tentative markers of OA and ageing cartilage were also demonstrated. Finally an RNA sequencing study on ageing equine cartilage found an age-related failure of matrix, anabolic and catabolic cartilage factors together with a reduction in Wnt signalling. This thesis developed novel proteomic methodologies to identify and quantify distinct differences between cartilage ageing and disease. Several proteins not previously described in cartilage were identified. In addition many novel cartilage degradation products were identified and age-related peptides were visualised in cartilage for the first time