Global Proteomics Analysis Laboratory
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Abstract
The proteomic and metabolomic laboratory at Purdue University's Bindley Bioscience Center performs global proteomics analysis for the CCE project. The facility uses proteomic and metabolomic tools to evaluate biological problems such as oxidative stress and apoptosis, cancer, aging and neurodegenerative diseases. Particular interests include using proteomics and metabolomics to study the impact of stimuli on cell cycle regulation and to tease out biological makers of disease. Several advanced platforms have been developed, as well as experimental strategies for biomarker and /or drug discovery identification.
Global proteomics analysis of plasma samples includes liquid chromatography - mass spectrometry (LC-MS) based proteomics followed by data mining that includes peak alignment and peak quantification. This approach will be used on groups of colorectal cancer and control patient samples (collected at IUSCC) to identify biomarkers which can predict susceptibility, treatment response and treatment outcome for colorectal cancer patients.
The global proteomics workflow uses Agilent's chip cube coupled the XCT PLUS ESI ion trap to deliver sensitive MS analysis of peptides for quantitation and MS/MS analysis of peptides for database searching.
See sample preparation protocol and instrument setup for detailed descriptions of the sample processing methodologies.
The XCT PLUS ESI ion trap in LC-MS mode generates ".D" files during instrument sample analysis. For peptide quantification, Bruker's CompassXport software is used to convert the XCT PLUS ".D" files to Level 1 LC-MS mzXML files. The mzXML file generated by the XCT PLUS in LC-MS mode has more detailed m/z information than the XCT PLUS run in LC-MS-MS mode. The LC-MS mzXML file can be used as input to the Proteomics Discovery Pipeline for data mining. [You must be logged in to access the Proteomics Discovery Pipeline link.]
The mzXML files will also be used by the Cancer Care Engineering statistical modeling group for integrative mathematical modeling.
For protein identification, the XCT PLUS LC-MS-MS generated ".D" files are converted to either
- mzXML Level 2 LC-MS-MS format file used for analysis by Sorcerer software
- PKL format file generated by DataExtractor, a component of Agilent's Spectrum Mill software
- MGF, the MASCOT generated format file used for MASCOT database searching.
These formats produce peak lists and fragmentation patterns which are matched against databases to identify proteins. Users can access the mzXML and MGF files converted from the D format, as use them as input to other identification software or databases. The PLK file is not accessible after conversion from the D format.
In general, the LC-MS and LC-MS-MS phases are separate runs of the XCT PLUS. The LC-MS phase includes hundreds of samples, and the LC-MS-MS phase includes only a handful of the original sample group.
Cite this work
Researchers should cite this work as follows:
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Ann Christine Catlin (2009), "Global Proteomics Analysis Laboratory," http://ccehub.org/resources/199.