The Proteomics Core has optimized methods to develop a standard global quantitative proteomics approach for cancer center core users which has been used by more than IU Simon Comprehensive Cancer Center affiliated laboratories.
Our standard method uses isobaric mass tagging to multiplex global proteome quantitation. When used for biological replicate analysis, this method has reproducibly quantified 6,000-10,000 proteins for core users.
We have also applied these approaches to the phosphoproteome resulting in quantitation of many thousand to tens of thousands of phosphosites across up to 18 biological samples per experiment.
These approaches can be applied to analysis of proteins in cancer cells, isolated tumors, and other biosamples to measure changes in kinase pathways in response to various treatments or to establish baseline signaling profiles. We follow guidelines established by the NCI CPTAC consortium for many of our experimental workflows.
With quantitative proteomics methods inching closer and closer to full proteome coverage, novel applications of global proteomics can facilitate biological assays with significant promise for analysis of small molecule or drug characterization including global target screening and activity analysis.
The Proteomics Core, in cooperation with the Mosley lab, has developed a systematic service pipeline (from sample preparation to data analysis) for cellular thermal shift assays (CETSA) and thermal proteome profiling (TPP) that is now available to all cancer center users.
The CETSA approach has been used to perform:
- Target engagement studies
- Off-target interactions
The TPP approach has been used to perform:
- Monitoring of changes in cellular signaling states
- Biophysical analysis of the DNA damage response
More than 60 percent of the recent proteomics core projects by cancer center members involve analysis of affinity-purification samples for either protein identification or post-translational modification analysis (Androphy, Balakrishnan, Clapp, Corson, Hollenhorst, Johnson, Xiongbin Lu, Mayo, Mosley, Nalepa, Pili, Xie, and Zhang).
We have also performed amino acid site mapping of covalent small molecule modifications (Turchi and Meroueh).
To increase the rigor of this type of experiment, the proteomics core provides educational training and one-on-one consultation with cancer center users to discuss quantitative approaches to identify significant interaction partners with isolated proteins of interest.
The application of statistical methods to affinity-purification MS data is an area of expertise in the Mosley lab that have had recent publications in using these approaches to study dynamic protein-protein interactions as well as post-translational modifications.
Since August 2016, the proteomics core has updated its instrumentation and many of the available approaches for core users.
To facilitate IUSCCC member research, we have and will continue to conduct workshop-style information sessions run by core staff and/or technical representatives from various companies to educate on the advances in current quantitative proteomics methods.
Within this framework, we will highlight our core's existing strengths in:
- Chromatographic separations
- Post-translational modification analysis
- Quantitative protein-protein interaction analysis