Biological Sciences Division
Cutting-edge proteomics tool featured in two Analytical Chemistry articles
A cutting-edge proteomics analysis tool developed at Pacific Northwest National Laboratory was described in the May 15, 2005 issue of Analytical Chemistry, and approaches for using it coupled with another PNNL-developed technique were described in the October 15, 2005 issue. The automated-format 20-kpsi reversed-phase liquid chromatography (RPLC) system is an improvement on current widely practiced RPLC separations and analysis techniques.
In the May article, the authors reported that when coupled with tandem mass spectrometry (MS/MS), the RPLC system resulted in protein identification reproducibility approaching 90% between replicate experiments with a proteome coverage of >2000 proteins. The average protein MS/MS identification rate exceeded 10 proteins per minute, and 1207 proteins were identified in 120 minutes through assignment of 5944 different peptides. Additionally, a single run of the 20 kpsi RPLC system coupled to accurate mass Fourier transform ion cyclotron resonance MS detected >5000 different compounds from a metabolomics sample.
In the October article, the authors described using sub-micrometer separation media RPLC coupled to electrospray ionization-MS/MS to perform higher-throughput proteomics analyses—identifying greater than tenfold more peptides (4000)—than has been previously achieved with other methods. Their results provide a basis for optimizing the use of MS/MS-based proteomics approaches and suggest approaches for further improving throughput.
Development of the RPLC system was supported in part by the National Institutes of Health National Center for Research Resources and the U.S. Department of Energy's (DOE's) Office of Biological and Environmental Research. Work was performed in the Environmental Molecular Sciences Laboratory, a DOE national scientific user facility located at PNNL.
The May article was authored by PNNL staff Yufeng Shen, Rui Zhang, Ronald J. Moore, Jeongkwon Kim, Thomas O. Metz, Kim K. Hixson, Rui Zhao, Eric A. Livesay, Harold R. Udseth, and Richard D. Smith. Shen and Smith were the PNNL authors of the October article.
Shen Y, R Zhang, R.J Moore, J-K Kim, TO Metz, KK Hixson, R Zhao, EA Livesay, HR Udseth, and RD Smith. 2005. "Automated 20 kpsi RPLC-MS and MS/MS with Chromatographic Peak Capacities of 1,000-1,500 and Capabilities for Proteomics and Metabolomics." Analytical Chemistry 77(10):3090-3100. DOI:10.1021/ac0483062.
Shen Y, RD Smith, KK Unger, D Kumar, and D Lubda. 2005. "Ultrahigh-Throughput Proteomics Using Fast RPLC Separations with ESI-MS/MS." Analytical Chemistry 77(20):6692-6701. DOI:10.1021/ac050876u.