The facility consists of a Applied Biosystems/MDS Sciex API 4000 QTrap (triple quadrupole linear ion trap tandem mass spectrometer (MS)) and a Thermo Finnigan LCQ Deca ion trap tandem MS. The MS’s are equipped with nano-electrospray ionization sources, nano-litre flow Agilent 1100 high pressure liquid chromatography systems, and 96/384 well auto-samplers.
Protein Interaction Mapping
To investigate the function of a protein we identify its protein interaction partners, these interactions generally define the function of a protein. We employ a co-immunoprecipitation-mass spectrometry (IP-MS) strategy in which complexes containing the protein of interest (“bait” protein) are immunoprecipitated and any interacting proteins (“preys”) identified by MS analysis. We isolate bait complexes by immunoprecipitation (IP) of ectopically expressed epitope-tagged bait proteins using antibodies (Abs) to the epitope tag, we also IP the endogenous bait protein when Abs to the bait protein are available. To facilitate our proteomic identification and validation efforts we have developed a vector system using in vitro recombination that allows us to fuse our open reading frames (ORFs) to a variety of epitope tags, fluorescent proteins, and transcription promoters. This allows us to rapidly shuttle the ORFs into other vectors for validation and functional experiments. Peptides in isolated multi-protein complexes are analyzed by LC-MS/MS and proteins identified by searching against protein and translated genome databases.