This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Immunoprecipitation of protein complexes from cell lysates and subsequent mass spectrometric analysis of gel separated proteins has become widely appreciated as powerful tool to study protein interactions in vivo. Unfortunately, the method does not reveal further structural information about the architecture of a particular protein complex. Other methods, like the yeast-two-hybrid assay can detect binary protein interactions but rarely disclose all protein interactions within a stable assembly. Here, we have investigated a new approach to map the structural connectivity of proteins in a complex. Protein complexes from Saccharomyces cerevisiae are isolated after genomic tagging of one of its components using immunoprecipitation technology. The complex components are electrophoretically separated and identified by MALDI-ion trap MS and MS/MS experiments with the additional aim to generate a high-quality set of MS/MS reference data. Strains with a deleted component of the same complex are chosen from a library and crossed with strains bearing the genetic tag used for immunopurification. Protein identification is carried out after digestion of gel separated complex components (immunopurified from these cells) using particular signatures from an MS/MS reference data set, derived from the initial identification of the complex components. These MS/MS signatures were carefully chosen and tested in order to meet two main quality criteria: 1) unambiguous sequence assignment and 2) highly sensitive detection. Intense fragmentation on the C-terminal side of aspartate or glutamate residues as well as frequent disruptions of peptide bonds at the N-termini of proline yield a low number of intense signals. Peptides that display such characteristic behavior in MALDI-ion trap experiments also account for high scores in searches with programs like Sonar MS/MS and are therefore preferred as signatures. Currently, our list of MS/MS signatures comprises 91 entries, containing the masses of selected unique digest peptides of a total of 32 Rpd3/Sin3-complex-members, each yielding up to three intense mass spectrometric fragments in hypothesis-driven MS/MS experiments. Protein A tagged Sin3, Rpd3 and Ume1 allow the purification of the same histone deacetylase complex which displays significant homology to the human HDAC-1 complex. When Rpd3 is deleted from strains containing tagged Ume1 a significant portion of the wild type protein complex is still recovered. However, gel electrophoresis and tandem MS analysis indicate that 4 particular proteins are no longer associated with the complex. A similar pattern is observed when Rpd3 is deleted from strains containing tagged Sin3. These initial results demonstrate that the deletion of one member of a complex might have a pruning effect on a branch of proteins formerly grouped or directly associated with the deleted partner. A more detailed study should give us clues about the detailed architecture of particular protein complexes. The use of MS/MS signatures obtained from MALDI-ion trap experiments permits sensitive protein identification of digested complexes, allowing us to reduce the required starting material from 10g cells per immunopurification to 1-2g of cellular material.
Showing the most recent 10 out of 67 publications
