This is a unique proteomic service that provides antibody affinity purification of endogenous protein complexes and MS-based identification and analysis of the associated proteins. Suitable antibodies for immunoprecipitation of the desired protein complex and optimization of the cell line, culture conditions and methods for cell lysis and antibody affinity purification are provided as services. The complexes are isolated under conditions of cellular activation by appropriate signals as requested by the cancer center investigator.
Isolation and Identification of Protein Complexes
Conditions for cell culture and antibody affinity purification of complexes will be optimized on a small scale. If an epitope-tagged construct has been made by the investigator, a cell line expressing the tagged protein will be used with an antibody to the tag. Also, complexes will be isolated under conditions of cellular activation of appropriate signaling pathways as instructed by the Cancer Center investigator. Initial immuno-isolations are carried out with 5mg of cell extracts (in 0.5ml) and resulting immuno-isolated complexes are resolved on SDS-gels Coomassie blue stained gels. Visible bands are then in-gel digested with trypsin and peptides are analyzed by mass spectrometry which identifies proteins by MS/MS. The detection of antigen is taken as criteria for a good quality antibody capable of isolating protein complexes. After optimization, protein complexes on a large scale (10 liters of cells) are isolated from 30mg protein extracts in 3 ml volumes. This is typically done with both S100 (cytosolic extract) and nuclear extracts. One half of the immuno-complex is washed with a low stringent buffer (NETN) and the other half is washed with a high stringent buffer (RIPA), and resulting immuno-complexes are resolved on an SDS-PAGE Coomassie blue stained gels. All visible and invisible bands are in–gel digested with trypsin and AspN and proteins are identified by MS with the nano-HPLC-ESI-LTQ. Acquired MS spectra are processed by BioWorks software to convert data into peptide and protein composition information.
Identification of Post-Transcriptional Modifications
The process of PTM analysis is basically the same as for protein identification with some modifications. Cellular protein complexes are isolated under conditions that maintain PTMs during lysis and in vitro processing, and larger amounts of starting cells and protein complexes are typically used to increase sensitivity of the mass spectrometry analysis. Modified target proteins are separated on SDS-PAGE, digested in gel by the most suitable peptidases and subjected to nano-HPLC/ESI/MS/MS (LTQ). MS/MS spectra are searched against a modified NCBI protein reference database using the BioWorks database™ search engine. Mass change is incorporated for the appropriate amino acid according to the type of PTM of interest.
Each MS spectra are manually verified for protein ID and PTM mapping. The identified proteins in immuno-isolated complexes include specific, non-specific and antibody cross-reactive unrelated proteins and their associated binding partners. The Qin lab has built a custom database for IP/MS (containing 1500 IP/MS datasets) and developed an analysis software package to filter nonspecific binding proteins and cross-reacting proteins. Both raw and filtered data are reported for a best estimate of specific, non-specific and cross reactive proteins in each isolated protein complex.
CPRIT Cancer Proteomics and Metabolomics
Additional services are available through CPRIT Cancer Proteomics and Metabolomics - a group funded through a grant from the Cancer Prevention Research Institute of Texas.