"Is it not evident in these last hundred years…
that almost a new Nature has been revealed to us? … more noble secrets
in optics, medicine, anatomy discovered, than in all those credulous and
doting ages from Aristotle to us?"
Contents © 2004-2011 Massachusetts
The Proteomics Core serves several functions within the Large Scale Collaborative Research Program. The Core performs cellular and protein analyses that identify and characterize the cellular perturbations occurring as a consequence of traumatic and burn injury in the clinical studies. This Core provides an essential connection between a description of the pattern of gene expression (as determined by the Genomics Core) seen in patients with burns and trauma, and the functional outcome of that gene expression, and the correlation to subsequent post-injury pathology.
The Core has defined 3 primary objectives:
To achieve these goals, highly coordinated efforts with other cores and a multitude of developments within the Proteomics Core including high throughput sample processing, high throughput and high sensitivity instrumentation, and efficient data handling and processing are required.
The large-scale collaborative research program requires high-throughput comprehensive system-wide comparative quantitative analysis of gene products including mRNA and proteins and the detection of changes in gene expression and protein abundances as a result of severe trauma or burn injury. At the same time, these quantitative proteomic data are evaluated in the context of changes in the functional proteomics of these cell populations. We define ‘functional proteomics’ as a phenotypic description of cellular proteins focused on variables with known functional consequences. Characterization of the level of the MHC class II proteins and co-stimulatory molecules (e.g. CD86) on blood monocytes is one example of functional proteomics, as the expression of MHC class has functional consequences in severe injury. Although gene expression can be analyzed in blood leukocyte populations, changes in protein abundances do not necessarily correlate with changes in gene expression, thus demonstrating the need for these complementary measurements.
In addition, gene expression cannot be measured in human blood plasma. The Proteomics Core provides high throughput proteome-wide protein abundance measurement capabilities for analyzing large scale clinical samples including human blood plasma and enriched blood leukocyte populations and provide high quality quantitative proteomic data. The proteomics data integrated with flow cytometric (functional proteomics) data, genomics data and clinical data are anticipated to lead to the identification of signature proteins or genes that have predictive values for the identification of different functional phenotypes and clinical outcomes in patients with severe trauma or burn injury, and will eventually lead to a better treatment for such patients.
The generation of valuable clinical samples for proteomics analysis and the integration of the proteomics data with genomics data and clinical data require extensive interdisciplinary collaborative expertise that is beyond the capabilities of any individual investigator.
During the first five years of funding, the efforts in the Proteomics Core were primarily focused on the development of quantitative proteomics capability and the application of quantitative proteomics for characterizing proteomic changes in human blood plasma with the purpose of identifying novel proteins that are associated with specific clinical trajectories in patients with severe trauma or burn injury.
For the renewal Program, the Core will extend high throughput measurements to the comparative quantitative proteomic profiling of enriched blood leukocyte populations from trauma or burn patients. Since cellular proteins present in plasma are often at very low concentrations, it is challenging to identify these proteins of interest due to the overall dynamic range of plasma protein concentrations. Quantitative proteome profiling of specific blood leukocyte populations from patients with different outcomes or from normal subjects will allow the discovery of novel candidate protein markers from the specific cell types that are otherwise below the detection limit of current technologies in blood plasma. The Proteomics Core has demonstrated the achievement of extensive proteome coverage for several different mammalian cell types or tissues including human mammary epithelial cells, human hepatocyte (Huh)-7.5 cells and mouse brain tissue, where 4,000-8,000 proteins were consistently identified with high confidence for each sample type. This capability will be further optimized and extended to the blood leukocyte populations and protein abundances in these cells will be quantitatively analyzed using the dual-quantitation approach in combination with AMT tag strategy.
Simultaneous with the development of these high throughput comparative proteomic determinations, the analysis of functional and receptor markers in isolated leukocyte subsets requires standardization, evaluation, and testing of analyte panels that can more precisely identify the functional status of the cell populations being evaluated.
Major Functions of this Core
The responsibilities of the Proteomics Core include:
The Proteomics Core contains four main elements
The high throughput proteomics element in the Core performs the large-scale proteome sample analyses and provides quantitative proteomics data. The functional proteomics element in the Core performs the monocyte and T-cell phenotyping. The data integration and interpretation element involves the coordinated efforts from the Proteomics, Genomics, and Data Interpretation Cores for extracting the biological information from these multiplexed datasets. In particular, comparative quantitative proteomics data potentially provides orthogonal validation of genomics measurements (and vice versa).
The Proteomics Core is an integrated component of this large-scale interdisciplinary program using systems biology approaches for solving the life-threatening problem of inflammation following major trauma or burn injuries. High throughput quantitative proteomics and genomics have become two major system-wide tools for identifying essential genes or gene products involved in disease pathways. The capability of analyzing large scale samples and providing quantitative data is a significant contribution for this program to gain new knowledge into the inflammatory responses.
Pacific Northwest National Laboratory, Batelle under the leadership of Dr. Richard Smith with Dr. David Camp, III (high-throughput liquid chromatography and mass spectrometry analyses of peptides from human plasma and peptides from leukocyte lysates)
University of Rochester Department of Surgery under the leadership of Dr. Carol Miller-Graziano with Dr. Asit De (leukocyte isolation protocols and phenotyping of blood leukocyte populations) and Dr. Paul Bankey (sample collection from trauma and control subjects for the development and evaluation of new protocols for the Genomics and CSSP Cores)
An Example of Flow Cytometry
Flow cytometric techniques permit PACB investigators to look at intracellular proteins in several different cell populations simultaneously. A sample of whole blood was stimulated and then the sample incubated with several fluorescently-tagged antibodies that recognize specific cell surface or intracellular proteins. In the example shown the green colored dots represent the protein content from polymorphonuclear neutrophils, blue corresponds to monocytes and the red dots represent the signal from individual lymphocytes. The intensity of protein staining, along with calculations of the number and percentage of the total cell population showing the protein of interest can be calculated.