HIGH THROUGHPUT PROTEIN IDENTIFICATION
High Throughput Proteomics at Roy J. Carver Biotechnology Center
The instrumentation for high throughput proteomics at the Protein Sciences Facility was recently acquired using funding provided by a Roy J. Carver Charitable Trust sponsored scientific instrumentation grant. The newly acquired instrumentation includes an Ettan 2-D gel electrophoresis apparatus, a Typhoon™ 9400 multilaser scanner for gel imaging/analysis, a robotic sample handling workstation, and an Ettan MALDI ToF Pro mass spectrometer (MS). A number of high throughput proteomic research projects are currently under way and more are planned.
The 2-D gel electrophoresis is with an Ettan IPGphor Isoelectrofocusing first dimension separation followed by a DALTsix electrophoresis second dimension separation. With the Ettan DIGE (Fluorescence Difference in Gel Electrophoresis) system samples are labeled with CyDyes™ (Cy2, Cy3 and Cy5) and multiplexed for co-separation during 2-D gel electrophoresis. The samples, usually a treatment and control, are each labeled with either Cy3 or Cy5; plus a Cy2 labeled internal standard for each treatment. The Cy2 labeled internal standard is an excellent way to remove gel-to-gel variation. Following 2-D electrophoresis, gels are scanned with a Molecular Dynamics Typhoon™ 9400 Imager with 3 direct excitable lasers and DeCyder™ Differential analysis software automatically detects, matches, and analyzes spots for very small differences in protein abundance between the two samples (Cy3/Cy5) on the same gel and between gels through Cy2 internal standards. After DeCyder™ analysis identifies gel spots for picking the gel is transferred to the Ettan Sample Handling Workstation (SHW). The SHW consists of a video camera and three robotic units working together to excise selected protein spots from gels for preparation for subsequent analysis and identification. The gel plugs are then destained, washed, trypsin digested, and spotted on a MALDI target. The SHW can be programmed to process up to 1152 samples per batch from a maximum of 12 large format 2-D gels in less than 36 hours. For mass spectrometry analysis we utilize an Ettan MALDI ToF Pro MS that can process 288 samples/batch and generate a protein identification list of the spots in about 6 hours.
All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.