These linear ion trap instruments are used for standard (single-dimensional) LC-MS/MS analyses as well as more specialized multi-dimensional LC-MS/MS (MudPIT) experiments. They and are equipped with microelectrospray sources, autosamplers and Eksigent LC pump systems to deliver nanoliter flow rates for high sensitivity analyses. High-throughput protein identification from MS/MS data is done with Sequest Cluster, a multiprocessor version of the widely used Sequest program, which runs on up to 100 nodes (Intel Xeon dual processors) on the Vanderbilt ACCRE Beowulf cluster, and datasets are visualized using software tools developed in-house (CHIPS and IDPicker).
The Orbitrap is a hybrid instrument consisting of a lower resolution LTQ linear ion trap front end coupled to a high resolution Orbitrap FT/MS analyzer. The Orbitrap mass analyzer is used for high resolution, accurate mass determination of precursor and/or product ion peptide masses, which can be particularly beneficial for the assignment of peptide identities and mapping sites of post-translational modification. MS data are processed and visualized as above for the LTQ systems.
Includes equipment for first dimension isoelectric focusing (Multiphor II and IPGphor) and second dimension SDS-PAGE (Ettan DALT 12) for large format (25 x 20 cm) gels. Protein samples are individually labeled with sensitive (sub picogram) cyanine fluorescent dyes (Cy2, Cy3 or Cy5) that exhibit a linear dynamic range of over four orders of magnitude. Using multiple 2D gels and a Cy2-labeled mixed-sample internal standard enables normalization of quantitative protein expression data across replicate samples from multiple experimental conditions in one concerted experiment. Additional instrumentation includes an Amersham Biosciences/GE Healthcare Typhoon 9400 Imaging system and Spot-Handling Workstation (SHW) which serves as a spot picker, digester and MALDI plate spotter, and permits high throughput, automated analyses of protein features selected from 2D gels for identification using the LTQ or MALDI-TOF/TOF MS systems.