X-tracker: a generic quantitation tool for MS-based proteomics:

Lead Research Organisation: Cranfield University
Department Name: Cranfield Health


The completion of the Human Genome Project, which set out to read the entire DNA 'blueprint' for a human being, was one of the greatest scientific achievements of recent times. However, there is a limit to what the genome itself can tell us about how a human being grows and functions, as it does not change according to time or tissue type, and varies little between individuals of the same species. To get a real insight into biology and disease mechanisms, we need to look at the proteins expressed from the genome, as these are the molecules that actually do the work in the body. As the protein complement of a cell varies tremendously over time, between tissue types, and in response to environmental conditions, developing methods to identify which proteins are present, and in what quantities, in a particular sample is crucial. Proteomics is the name given to the science of identifying all the proteins within a sample. This is typically achieved by separating the proteins, breaking them up into smaller peptides, and analysing them with mass spectrometry (MS) to generate a characteristic spectrum which can be matched to a sequence known to exist in the genome. Methods to quantify the amount of proteins in a sample, either in relation to other samples or absolutely, have been developed based around the same technology. In previous projects we have developed computational methods that help to identify proteins and to quantitate them. Respectively, these are the Genome Annotating Proteomic Pipeline (GAPP) and i-Tracker. There are however a number of quantitation methods available and more are being developed, but there is currently no easy way of both identifying and quantifying proteins within the same package for a number of different quantitation methods. Nor is there an easy way to modify or add methods to a system to suit techniques specific to one laboratory. The aim of this proposal is to provide a system that meets this need and thereby to facilitate performing quantitative proteomics experiments and also the dissemination of novel methods throughout the community.

Technical Summary

As part of a previous project we have developed a system to aid protein quantitation using the iTRAQ protocol marketed by Applied Biosystems. This software is called i-Tracker. Other packages have also recently been released that provide similar functionality, generally linking protein identifications from a separate commercial package, such as SEQUEST or Mascot, with algorithms to handle a small number of quantitation protocols. What is lacking however is a more generic tool that is simple to use, allows analysis based on many different quantitation methods, is easily extensible to cover novel variations or protocols and is entirely open-access. The purpose of this proposal is to request funding to develop such a tool, specifically to: 1. Link the open-source protein identification system X!Tandem to a suite of open-access quantitation modules, to be developed as part of this project. 2. Develop quantitation modules for existing laboratory methods and a framework within which other development groups may straightforwardly add their own quantitation modules to the system. 3. Provide an intuitive user interface to the system that allows for small variations in laboratory protocols to be processed. Examples include the use of more than one input file type, containing diverse information, or choosing to quantitate using both labelled and label-free methods. The choice of method(s) will guide the entry of user-specific information required. 4. Host a web-site that will provide access to these developments, all user documentation, and a place for requesting new modules to handle new protocols, to be provided by interested developers. 5. Allow output of the combined identifications and quantitative information in a format defined by the user to be suited to their needs.


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