Verification Stage
Figure captions for software pipelines: verification
While many protein biomarker candidates have been discovered, few have translated into biomarkers of clinical utility. The Clinical Proteomic Technologies for Cancer initiative (CPTC) has developed a time-efficient, cost-effective method of triaging candidate lists of proteins into well-credentialed, high-priority biomarker candidates. A major effort of the CPTC has been to create the software for this so-called “verification” method. Briefly, verification assays are built for proteins received from discovery proteomics technologies. Then, a multiple-reaction monitoring (MRM) experiment is conducted. A number of CPTC software tools now exist to ease both the experimental setup of this experiment, as well as the analysis of the data. See below for further descriptions of these tools.
MRM Experiment Design
- ESP: The first step in developing effective MRM assays is to identify the appropriate peptides. Identification of the most effective signature peptides, particularly in the absence of experimental data, remains a major resource constraint in developing targeted MS–based assays. ESP is a computational method that uses protein physicochemical properties to select high-responding peptides and demonstrate its utility in identifying signature peptides in plasma, a complex proteome with a wide-range of protein concentrations. This method, which employs a Random Forest classifier, facilitates the development of targeted MS–based assays for biomarker verification or any application where protein levels need to be measured. The ESP predictor is freely available as a module in the GenePattern integrative genomics software package (http://www.genepattern.org/) under the category 'proteomics'.
- Skyline: Skyline is a Windows client application for building Selected Reaction Monitoring (SRM) methods and analyzing the resulting mass spectrometer data. It aims to employ cutting-edge technologies for creating and iteratively refining SRM methods for large-scale proteomics studies. The core focus of v0.5 is analysis of result data, building on the successful method creation features of v0.2. The library spectrum can then be used to identify fragment ion peaks ranked by intensity, and enable the user to define how many product ions (n = 3, 4, 5, and 6 in this comparison) are required to provide a specific and selective measurement given the target sample.
- MaRiMba: The MaRiMba software tool automates the creation of explicitly defined MRM transition lists required to program triple quadrapole mass spectrometers in such analyses. MaRiMba creates the MRM transition lists from pre-existing or custom spectral libraries, restricts output to specified proteins or peptides, and filters based on precursor peptide and product ion properties. MaRiMba can also create MRM lists containing corresponding transitions for isotopically heavy peptides, for which the precursor and product ions are adjusted according to user specifications. This open source application is operated through a graphical user interface incorportated into the Trans-Proteomic Pipeline (TPP), and it outputs the final MRM list to a text file for upload to MS instruments.
MRM Data Analysis
- Myrmidon: Myrmidon streamlines the automated analysis of large-scale SRM experiments. The software is compatible with files from several manufacturers of triple quadrupole instruments and can make use of SRM methods defined in Skyline software. Scaling across multiple replicates in multiple cohorts, the software assists in the recognition and integration of chromatographic peaks, producing reports suitable for statistical interpretation.
