Proteome coverage is often defined as the number of proteins identified in an experiment. However, most of these proteins are only partially covered by identified peptides. Even if the majority of proteins in a cell are identified, the majority of the proteome sequence is not. It may be difficult to identify and quantify regions of interest that are not observable with standard tryptic digestion.
Confetti is our map of HeLa proteome coverage that can be achieved using an extensive panel of digests, accessing more sequence than is possible in typical tryptic experiments. We performed 48 single, double and triple enzyme digests. Unfractionated and SAX fractionated MS analysis was performed on Orbitrap Elite and Q Exactive mass-spectrometers, and the results are detailed in our publication in Mol. Cell. Proteomics:
Guo X*, Trudgian DC*, Lemoff A, Yadavalli S, Mirzaei H Mol. Cell Proteomics 2014 Apr
* Equal Contribution
Our Confetti online tool allows users to search for a protein of interest, and identify coverage from the panel of digests. Confetti offers fucntionality to identify candidate peptides for targetted SRM assays, and to view and download spectra acquired.
Access Confetti at https://proteomics.swmed.edu/confetti
Proteomics groups and facilities often invest a great deal of time and effort in the optimization of mass-spectrometry acquisition methods. However, the standard reverse phase linear gradient of approx 0-30% ACN used to separate peptide mixtures for LC-MS is rarely adjusted. This standard gradient is 'good enough' for most gel-bands and complex mixtures, but becomes quite inefficient when analyzing samples that have been pre-fractionated at the peptide level. SCX, HILIC, OFFGEL, AEX, or high pH RP pre-fractionation are all only partially orthogonal to the on-line separation. Fractions can be heavily biased toward hydrophobic or hydrophilic peptides, meaning much of the standard gradient is wasted.
GOAT allows optimized gradients to be generated for LC-MS/MS runs quickly and easily. The only input required is a vendor raw format or MzML file, acquired during a run using a user-specified standard gradient. GOAT uses MS/MS scan information to evenly distributed peptides (or other compounds of interest) throughout your LC-MS/MS run.
Free for non-profit academic use at the GOAT website.
Trudgian DC*, Fischer R*, Guo X, Kessler BM, Mirzaei H. PROTEOMICS 2014 Apr
* Equal Contribution
In collaboration with the University of Oxford
CPFP is a web-based data analysis pipeline aimed at proteomics core facilities and large proteomics labs. Based on the ISB Trans-Proteomic Pipeline, it now incorporates a number of additional tools. The pipeline was initially developed at the University of Oxford, where it is used by the Oxford Central Proteomics Facilities. CPFP is now developed in the Mirzaei Lab and Proteomics Core at UTSW, where it is also the primary analysis platform.
Development at UTSW has concentrated on allowing CPFP to support the analysis of very large or computationally complex analyses via cloud and cluster computing. CPFP at UTSW uses the TACC Lonestar4 and Stampede HPC systems to run database searches. The pipeline is flexible, and can also be used in a totally cloud based, or hybrid configuration on the Amazon Web Service Cloud.
CPFP is an open-source project at cpfp.sourceforge.net.
Trudgian DC, Mirzaei H, Journal of proteome research, 2012 Oct;