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Biological Sciences

Development of High-Throughput Proteomics Production Operations

Sponsor: DOE Office of Biological and Environmental Research
Contacts: Dick Smith, Matt Monroe

LC-Linear ion trap-FTICR (9.4 tesla magnet)

LC-Linear ion trap-FTICR (9.4 tesla magnet)

With recent advances in whole-genome sequencing for a variety of organisms, biological research is increasingly incorporating higher-level "systems" perspectives and approaches. Biology is transitioning from a largely qualitative descriptive science to a quantitative, ultimately predictive science. Key to supporting advances in microbial and other biological research at the heart of the U.S. Department of Energy's (DOE) Genomics: Genomes to Life (GTL) program is the ability to quantitatively measure the array of proteins (i.e., the proteome) in various biological systems under many different conditions. Ultimately, such measurements, and the resulting understandings of biochemical processes are expected to enable development of predictive computational models that could profoundly affect environmental clean-up and energy production by, for example, providing a more solid basis for mitigating the impacts of energy-production-related activities on the environment and human health.

Measurement capabilities with the desired comprehensive qualities are only now becoming feasible through new, advanced technologies. However, providing the capabilities needed to support the Genomics: GTL program goals will require further significant advances in measurement throughput and data quality, as well as additional types of data vital to understanding and modeling complex biological systems (e.g., information on protein modification states).

LC-FTICR (9.4 tesla magnet)

LC-FTICR (9.4 tesla magnet)

In FY 2001, a project was initiated at PNNL to develop a new approach for quantitative and high-throughput proteomics measurements using advanced instrumental approaches. This work resulted in a framework and technology platform for high-throughput, quantitative global proteomic measurements of microbial systems. The platform is based on a combination of advanced separations and mass spectrometric instrumentation and supporting computational infrastructure. Efforts have included the development of an ultra-high pressure capillary liquid chromatography (LC) platform combined with a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS) and supporting data analysis and management capabilities. These developments provided the first high-throughput mode "24/7" operation of such instrumentation, and resulted in its successful application to a set of microbial systems.

An important motivation for this project is the need for higher throughput proteomics measurements. The project aims to refine, evaluate, and implement technologies and protocols to provide much greater throughput. A "prototype high-throughput production" lab established at PNNL in FY 2002 was an early step in this direction.

With a paradigm established, our primary goals over the next 3 years are to greatly increase both data throughput and quality in conjunction with providing an infrastructure that supports the use of these global proteomics measurements in research at PNNL and elsewhere. The benefits of the increased data production will be evident from greatly improved data quality (e.g., the confidence of protein identifications, quantitative measurement of their abundances, and associated statistics for both), as well as the ability to manage, utilize and disseminate these data. Specific aims are to:

LC-Ion trap mass spectrometers

LC-Ion trap mass spectrometers

  • Significantly increase the overall data quality of global proteome measurements and provide data that are quantitative and have statistically sound measures of quality.
  • Increase overall data throughput by more than an order of magnitude in conjunction with the improved data quality.
  • Provide the informatics tools and infrastructure required to support improved data quality and increased throughput and to efficiently manage, use, and disseminate large quantities of data generated by GTL users.
  • Develop a foundation for the further expansion of proteomics measurements to enable more comprehensive coverage of protein modifications.

Among the expected outcomes are automated sample-handling and management processes, as well as established QA/QC measures to ensure high-quality results; redundant production lines; and configuration control of the pipeline.

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