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.