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Biological Sciences

Proteomics National Center for Research Resource

http://ncrr.pnl.gov

Sponsor: National Institutes of Health
Contact: David Camp

The long-term goal of the Research Resource is to make advanced proteomic technologies more accessible to and useful for the biomedical research community. The overall objectives of the Resource follow with selected current highlights.

Objective

Develop new analytical technologies

NCRR Technology Development

Develop new analytical technologies and associated computational and informatics tools for rapid, quantitative, and comprehensive proteomic measurements in support of biomedical research.

Broad characterization of the human blood plasma proteome

The increasing interest in characterizing the human blood plasma proteome—the complement of proteins in the soluble portion of blood—stems from the potential that it may contain signature proteins or "biomarkers" for essentially every disease state. Discovery of these biomarkers is particularly challenging, because human plasma contains not only "classic proteins" (such as serum albumin) that are present in high concentrations, but also "leakage" proteins (originating from virtually all cell types and tissues) present in much lower concentrations. These protein concentrations span a dynamic range >10 orders of magnitude.

Global categorization of detected human plasma proteins
Categorization of 1427 high-confidence plasma proteins that comprise the mass and time tag database and have passed the 95% confidence limit filtering criteria. This total is based on multiple analyses involving both depleted and non-depleted plasma samples. Full Image (png 43kb)

A new approach for global characterization of the human plasma proteome has been developed at the Proteomics National Center for Research Resources. The approach uses conventional methods to remove abundant plasma proteins, advanced separations techniques (also developed at the Center) to enable a large dynamic range of analysis, and mass spectrometric measurements.

After stepwise removal of the abundant albumin and immuglobulin proteins by affinity chromatography, analyses using ultra-high efficiency liquid chromatography coupled to tandem mass spectrometry revealed 2392 confidently identified proteins whose relative abundances spanned a dynamic concentration range of ~109 (from <30 pg/mL to 30 mg/mL). Tentative evidence indicated an additional 2198 proteins were identified with "moderate" confidence, for a total of 4590 distinct human blood plasma proteins. This extensive catalog of proteins represents an ~10-fold increase over previous global plasma proteome mapping efforts.

This new global characterization approach lays the foundation for future quantitative plasma studies designed to compare diseased with non-diseased samples for evidence of potential biomarkers, as well as for studies of protein-protein interactions.

Reference

Shen Y, J Kim, EF Strittmatter, JM Jacobs, DG Camp II, R Fang, N Tolic, RJ Moore, and RD Smith. 2005. "Characterization of the human blood plasma proteome." Proteomics (in press).

Meeting the proteomics throughput challenge

A robust, versatile gas-phase separation technology that is rapidly gaining acceptance as a versatile tool for post-ionization separations prior to mass spectrometric proteomic measurements is Field Asymmetric waveform ion mobility spectrometry (FAIMS). FAIMS uses electric fields to separate ions on the basis of their mobility through a gas. Advantages of this technology are its speed—separations require minutes instead of hours to tens of hours for condensed-phase alternatives—and its ion focusing effect that often improves sensitivity. However, FAIMS exhibits some limitations: low specificity, severe quantitation distortion, and inadequate resolution control. Until now, progress in overcoming these limitations has been impeded by the lack of a physical model that relates instrument parameters to operational performance.

The first comprehensive a priori computational model for FAIMS analyzers of any geometry was recently developed at the Research Resource. Following validation by experimental measurements, the model was successfully employed to optimize both design and operation of FAIMS devices. The fundamental limits on resolution, sensitivity, and throughput were identified, and paths for advancing the technology were discovered. Important among the discoveries is that signal discrimination based on scan direction can be used to increase the scan speed. Rapid scanning is critical for coupling liquid chromatography methods to FAIMS to further increase analytical sensitivity and with the throughput required for practical biomedical investigations involving large numbers of samples.

References

Shvartsburg AA, K Tang, and RD Smith. 2004. "Modeling the resolution and sensitivity of FAIMS analyses." J. Am. Soc. Mass Spectrom. 15:1487-1498.

Shvartsburg AA, K Tang, and RD Smith. 2004. "Understanding and designing field asymmetric waveform ion mobility spectrometry separations in gas mixtures." Anal. Chem. 76, 7366-7374.

Shvartsburg AA, K Tang, and RD Smith. 2005. "FAIMS operation for realistic gas flow and asymmetric waveform profiles including ripple." J.Am. Soc. Mass Spectrom. (in press).

Objective

Apply these technology developments within the construct of an integrated and iterative work path to challenging biomedical studies through a set of collaborative projects.

Global analysis of human epithelial cell response to epidermal growth factor stimulation

Biomedical Applications: Human Mammary Epithelial Cells (HMEC)
Biomedical Applications: Human Mammary Epithelial Cells (HMEC)

The first multi-parameter analysis of normal human mammary epithelial cell (HMEC) response to epidermal growth factor (EGF) stimulation has been completed by PNNL scientists at the Proteomics National Center for Research Resources. EGF stimulation initiates a transition from the characteristic cell growth interval phase to the DNA synthesis phase. Information about HMEC under normal growth conditions is important to studies investigating the transformations of these cells in human epithelial cancers, such as breast cancer.

An extensive inventory of HMEC proteins and genes was generated that encompasses over one million data points collected from eight different time points over a 24-h period. Following addition of EGF that caused dynamic changes in the expression of more than 700 genes, analyses were performed using microarray, Western blot, and liquid chromatography-mass spectrometry (LC-MS) techniques. Among the data are nearly 7000 intracellular and extracellular proteins identified from high-throughput LC-MS measurements and Western blot analysis of ~1000 antibodies (proteins produced by the immune system in response to foreign substances called antigens).

Currently, this inventory of data is being used to create a comprehensive map of signaling networks from which realistic models may be developed. These models will yield insight into how these pathways interact in a coordinated manner to dictate cellular response.

The first comprehensive quantitative analysis of viral and cellular proteins that compose infectious particles in human cytomegalovirus (HCMV)

These two panels represent electron micrographs of an infectious HCMV virion (left panel) and a noninfectious particle produced in infected cells called dense bodies (right panel).
These two panels represent electron micrographs of an infectious HCMV virion (left panel) and a noninfectious particle produced in infected cells called dense bodies (right panel).

Human cytomegalovirus (HCMV, one of the largest and most complex viruses) is a common and widespread pathogen that exploits the immune defenses in its host to initiate infection. Primary infection with HCMV in healthy individuals (after the first months of life) with normal immune response is typically benign, and the virus remains dormant in the host. However, for individuals whose immune response is comprised; for example, by diseases such as AIDS or through transplantation procedures, primary HCMV is nearly always followed by persistent and/or recurring infections. Treatments are limited because HCMV possesses a complicated and lively array of strategies to avoid host immune defenses. Understanding how HCMV induces and circumvents host immunity is critically important for designing therapeutics to combat this pathogen. This understanding can be gained by studying the proteins that compose the infectious particles of the virus.

Using state-of-the-art techniques at the Proteomics National Center for Research Resources, scientists have discovered and quantified a record set of viral and cellular proteins for HCMV. This collaborative research effort involved scientists from Oregon Health Sciences University, Princeton University, the University of Alabama, and PNNL. While previous studies suggested that a viron (a viral particle) contained ~30-40 proteins, this study revealed 71 proteins, of which 59 are viral structural genes. Additionally, a large number of host cellular genes were determined to be associated with the viron, which indicates the host cellular proteins play a greater role during the course of growth and development of the virus than previously thought.

This study provides the first comprehensive analysis of the viral and cellular proteins that compose infectious particles of a large complex virus. The results from this analysis will be used in future studies to determine the function of the newly identified viron proteins and their contribution to structure and infectivity.

Reference

"Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome." Susan M. Varnum SM, DN Streblow, ME Monroe, P Smith, KJ Auberry, L Paša-Tolic, D Wang, DG Camp II, KD Rodland, HS Wiley, W Britt, T Shenk, RD Smith, and JA Nelson, J. Virology. 78(20):10960-10966.

Proteome Characterization Sheds Light on Hepatitis C Virus

Hepatitis C virus (HCV) infects close to 2% of the U.S. population, which makes it the most common blood-borne infection in the United States. Approximately 85% of these infections progress to various degrees of liver disease, such as cirrhosis (characterized by irreversible scarring of the liver and impaired liver function) and primary liver cancer. Accumulation of fat in the liver that causes inflammation is often a precursor to scarring and/or liver deterioration. Most characterization efforts to elucidate the host response to infection have centered on identifying the potential gene (a unit of DNA that carries information for the biosynthesis of proteins in the cell) markers of HCV-associated liver disease. However, few studies have investigated HCV infection at the protein level in part because of the large sample volumes required for conventional protein analysis methods.

In a recent collaborative research effort, scientists from the University of Washington and PNNL completed the first large-scale investigation of HCV replication. Global characterization of the Huh-7.5 model liver cell line both in the presence (+) and absence (-) of HCV replicon (a genetic unit of replication) were carried out by powerful liquid chromatographic separations combined with mass spectrometric measurements at the Proteomics National Center for Research Resource at PNNL. The characterization revealed 4200 proteins, including HCV replicon proteins, which represents the most comprehensive protein database yet reported for a human cell line. Comparison of (+) and (-) HCV samples resulted in the discovery of altered abundances of several proteins of interest; for example, alternations of fat metabolism proteins consistent with the phenotype of HCV liver infection.

When analyses were extended to the highly complex proteome of small-sized liver biopsy samples from HCV-infected patients, a total number of proteins >1500 were still able to be detected from only 2 μg of digested liver biopsy proteins. These results, obtained by employing the Huh-7.5 protein database in conjunction with the high throughput mass and time tag analytical approach (routinely used at the Center for proteomics studies), demonstrate a significant advancement in clinical proteomics efforts and offer the unique opportunity to begin investigating the clinical significance of protein expression changes associated with HCV infection. Furthermore, these results lay the foundation for future studies to investigate HCV-induced alterations in fat metabolism. The apparent link between fat metabolism and progression of liver disease makes these cellular metabolic pathways attractive targets for discovering new drugs to prevent formation of scar-like tissues.

Reference

Jacobs JM, DL Diamond, EY Chan, MA Gritsenko, W Qian, M Stastna, T Baaas, DG Camp II, RL Carithers, Jr., RD Smith, and MG Katze, 2005. "Proteome analysis of liver cells expressing a full-length hepatitis C virus (HCV) Replicon and biopsies of post-translated liver from HCV-infected patients." J. Virol. 79: 7558-7569.

Objective

Provide access to the new technologies and disseminate any resources resulting from the technology development and collaborative research projects (e.g., sample preparation techniques, software tools, antibody library, AMT databases, etc.) among the biomedical research community. http://ncrr.pnl.gov/training/ and http://ncrr.pnl.gov/data/

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