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Biological Sciences

High-Throughput Proteomics and Metabolomics Measurement Capabilities and Integrated Proteomics R&D

Contacts: and

PNNL's Biological Separations & Mass Spectrometry group focuses on development of high-resolution separations and mass spectrometry and their applications in biological research. Our current research emphasis is in the area of proteomics—the study of the entire complement of proteins expressed in a cell under a specific set of conditions at a specific time.

LC-MS/MS laboratory
LC-MS/MS laboratory for generating peptide databases-one of several laboratories at PNNL for proteomics and mass spectrometry. Enlarged View

Our signature strengths in high-throughput proteomics measurement capabilities and integrated proteomics R&D are among the world's best, as they combine instrumentation and capabilities located within the Environmental Molecular Sciences Laboratory at PNNL with some of the world's leading researchers in proteomics, mass spectrometry, and biochemistry to allow increasingly challenging biological research applications. The EMSL is a U.S. Department of Energy national scientific user facility located on the PNNL campus.

These signature strengths are based on

Electrospray Ionization Mass Spectrometry
2009 R&D 100 Award Winner: The Ultrasensitive Electrospray Ionization Mass Spectrometry Source and Interface comprises a multi-nanoelectrospray ionization source and MS interface. This PNNL-developed technology provides ionization efficiencies that were before only achievable from very-low-flow-rate, specialized separations and enables it to be used with common, higher-flow-rate analyses. PNNL News release. Enlarged View
  • Extensive, prudent investments in advancing proteome measurement platforms and the supporting informatics infrastructure
  • Assembly of a large and expert team with diverse skill sets needed for development and application of capabilities for high-throughput proteomics
  • An operational framework for rapidly translating and efficiently implementing concurrent technological advances (including many originating at PNNL/EMSL)
  • Leveraging of all the above in multiple applications to advance human health, environmental, and energy research.

Research Areas

Our research areas support our signature strengths. These include

High-Throughput Proteomics Measurement Capabilities

Contact: , Director of Proteomics Research

Fundamental to PNNL's world-leading and unique high-throughput measurement capability are:

  • The accurate mass and time (AMT) tag high-throughput proteome measurement strategy
  • An extensive proteomics pipeline based on high resolution separations combined with high sensitivity MS and advanced informatics tools,
  • An infrastructure for processing and managing large volumes of data.
LC-MS Experiments
LC-MS features are identified by matching their mass and time coordinates to those associated with peptides identified previously using LC-MS/MS experiments. Enlarged View

High-throughput proteome measurement strategy—the AMT tag approach. Our ability to obtain high-resolution, high-sensitivity liquid chromatography-mass spectrometry (LC-MS) measurements enables the AMT approach—a high-throughput strategy developed by PNNL staff that is well suited to a wide range of proteomics applications.

In this two-stage strategy, digested proteins are analyzed using traditional LC-MS/MS measurements. To avoid repetitious, time-consuming measurements in subsequent analyses of the same organism, the processed data from these initial measurements are stored in a reference database in the form of accurate mass and normalized chromatographic elution time information for each peptide; that is, an "accurate mass and time (AMT) tag." This serves as a unique 2D marker for subsequent identifications of that peptide.

Once a reference database has been generated, future samples from the same organism need only be analyzed by high-resolution LC-MS, because numerous peptides can be identified from a single spectrum by matching mass and elution time characteristics (features) with those associated with AMT tags in the reference database (see figure)

LC-MS Experiments
High-throughput proteomics pipeline. Enlarged View

High-throughput proteomics pipeline. Proteomic analysis of biological samples produces large volumes of data from different mass spectrometry technologies. These datasets allow the identification of peptides and proteins as well as allowing quantitation of peptide and protein abundances.

These experiments include a liquid chromatographic (LC) separation step coupled with both MS and MS/MS experiments. Data analysis tools are used to perform database searches to

  • Identify peptides from each MS/MS dataset
  • Interpret and extract detected masses from MS datasets
  • Assign peptide identifications to features detected by MS.

Additionally, quality control analysis is performed at several stages of the processing to ensure instrument performance and sample preparation quality. The data processing pipeline used in the PNNL AMT tag-based proteomics approach is shown in this diagram.

AMT tag-based proteomics requires two basic analysis stages: creation of Mass Tag Databases containing Potential Mass and Time Tags (PMT tags) and peptide mass assignment from high mass measurement accuracy (MMA) instrumentation.

Integrated Proteomics R&D

Contact: , Director of Proteomics Research

Our unique and specialized high-throughput measurement capability enables both hypothesis and discovery driven studies across essentially every area of biological research. Results from these studies provide the foundation for greatly increasing our understanding of biological interactions and processes, and drive technology development and informatics and bioinformatics efforts to allow increasingly challenging biological research applications.

Biological Sciences

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