Characterization of the human proteome has become one of the newest interests in clinical discovery research. Because many proteins are known to be involved in inflammation and the body’s immune response, scientists have great interest in defining the human proteome like they did in defining the human genome and transcriptome. Human blood contains not only proteins released from potentially every cell type and tissue as a result of disease processes or damage, but also a diverse population of circulatory immune cells. The presence of specific proteins or protein parts known as peptides, as well as specific alterations of the cellular proteomes of immune cells, can provide a wealth of important information about the onset or presence of most disease states. Measurement of the circulatory and cellular proteins present in blood is now possible using new high-throughput proteomic technologies.
Read MoreProteomics at PNNL
The Proteomics Center at Pacific Northwest National Laboratory (PNNL) represents a large interdisciplinary team of researchers focused on the development of instrumentation, enabling technologies, methodologies, informatics tools, and the application of these tools to high throughput proteomics challenges. The Glue Grant program leveraged the extensive infrastructure developed at PNNL to provide a qualitative proteomics platform for the reproducible and reliable analysis of the proteins found in blood plasma, T-lymphocytes, monocytes, and neutrophils from trauma and burn patients.
The efforts of the Glue Grant proteomics investigators primarily focused on the development of quantitative proteomics and the application of quantitative proteomics to measure changes in the levels of proteins in human blood plasma and its enriched leukocyte populations with the purpose of identifying novel proteins associated with specific clinical recovery patterns of patients with severe trauma or burn injury.
The complicating feature of studying the plasma proteome is that some proteins are found in high amounts while others, called “low abundance” proteins (cytokines, for example) are present at very low levels. This tremendous range in protein concentrations presents serious challenges for technologies intended to measure either the altered presence or the relative abundances of proteins in the blood plasma.
PNNL proteomics approach
The PNNL proteomics strategy relied on the PNNL-developed accurate mass and time (AMT) tag approach that uses a dedicated mass spectrometry instrument coupled with an automated high-resolution nanoscale capillary liquid chromatography separation system (LC-MS/MS). To provide high throughput measurements for clinical samples, the PNNL investigators developed a novel dual-quantification approach where a reference sample is spiked into each clinical sample and the relative protein abundances are quantified, based on both reference to sample ratios and direct mass spectrometry peak intensities. Coupling this strategy with the dual quantification approach, the application of the AMT tag strategy provided a high dynamic range of coverage, high throughput, and high accuracy quantification.
First of its kind large-scale high throughput proteomics analyses of clinical leukocyte samples
About 530 gene/proteins displayed significant changes in both microarray gene expression (left) and mass spectrometry-based protein abundance (right)
The first clinical application of a high-throughput, large-scale, quantitative proteomics approach for biomarker discovery for the prediction of patient outcome following burn, trauma, or critical illness, analyzed patient blood plasma and demonstrated the following:
- Confident identification of more than 4,000 distinct plasma proteins
- Found differences in circulating levels of 43 proteins involved in the acute-phase response, hepatic signaling, the complement cascade, inflammation, and insulin resistance
- 32 of the proteins identified were not previously known to play a role in the response to injury
- Interleukin-4, interleukin-8, granulocyte macrophage colony-stimulating factor, monocyte chemotactic protein-1, and β2-microglobulin correlated well with survival and may serve as clinical biomarkers
Extending these initial findings in blood plasma, PNNL investigators optimized their methodologies to enable efficient analysis of very small proteomic samples of circulating blood leukocyte cell subpopulations (monocytes, neutrophils, and T-lymphocytes) in injured patients. As one might imagine, creation of the extensive plasma and cell-specific AMT tag (peptide/protein) quantitative databases from these small, non-conventionally-sized blood leukocyte samples represented significant challenges for proteomic sample processing and analysis to minimize contamination and achieve the necessary reproducibility and sensitivity.
The analysis of the proteomics of trauma in 486 monocyte samples from 100 patients using dual channel LC-MS/MS quantified approximately 3, 000 proteins. Glue Grant investigators applied a time series linear mixed model and a binary probit regression approach to analyze this large-scale proteomics data set. Combining the results from the two tests, 580 proteins were detected with differential expression over time at a False Discovery Rate < 0.01 and 530 proteins were mapped to gene expression data.
Pathway analysis revealed that these significant proteins are related to integrin signaling, ILK signaling, Fcy receptor-mediated phagocytosis, and virus entry via endocytic pathways. A comparison of these results with the gene expression data from the same samples showed that while more than 60% demonstrated a similar pattern across time, others had different trends between gene expression and protein expression, which warrants further investigation by our computational group. Based on our preliminary data analyses, a number of biological functions and canonical pathways in monocytes and T-cells were revealed to be significant in trauma patients. An extensive data set has been established that, for the first time, reveals the detailed proteome responses to injury for three different immune cell types.
The PNNL quantitative proteomics platform incorporating the “universal” reference represents the only global proteome discovery technology currently available for population-scale discovery. Glue Grant investigators are focused on statistical data analysis and biological interpretation of the large-scale, quantitative proteomics data with the goal of reaching a systems-level understanding of biological pathways in response to trauma and discovering novel candidates predictive of patient outcomes and clinical recovery paths.
With resources supported by the Glue Grant program, the technical expertise at PNNL provided the program with unique capabilities to obtain a throughput level that would be otherwise impractical, unaffordable, and unachievable. Interesting biological discoveries and candidate protein predictors for patient outcomes will be further revealed through the continuing exploration of the Glue Grant high throughput proteomics, genomics, and functional proteomics data.
Relevant publications
Shen Y, Jacobs JM, Camp DG 2nd, Fang R, Moore RJ, Smith RD, et al. Ultra-high-efficiency strong cation exchange LC/RPLC/MS/MS for high dynamic range characterization of the human plasma proteome. Anal Chem. 2004 Feb 15;76(4):1134-44. PubMed PMID: 14961748
Qian WJ, Jacobs JM, Camp DG 2nd, Monroe ME, Moore RJ, Gritsenko MA, et al. Comparative proteome analyses of human plasma following in vivo lipopolysaccharide administration using multidimensional separations coupled with tandem mass spectrometry. Proteomics. 2005 Feb;5(2):572-84. PubMed PMID: 15627965; PubMed Central PMCID: PMC1781926
Qian WJ, Monroe ME, Liu T, Jacobs JM, Anderson GA, Shen Y, Inflammation and the Host Response to Injury Large Scale Collaborative Research Program. Quantitative proteome analysis of human plasma following in vivo lipopolysaccharide administration using 16O/18O labeling and the accurate mass and time tag approach. Mol Cell Proteomics. 2005 May;4(5):700-9. PubMed PMID: 15753121; PubMed Central PMCID: PMC1829297
Shen Y, Kim J, Strittmatter EF, Jacobs JM, Camp DG 2nd, Fang R, et al. Characterization of the human blood plasma proteome. Proteomics. 2005 Oct;5(15):4034-45. PubMed PMID: 16152657
Liu T, Qian WJ, Gritsenko MA, Xiao W, Moldawer LL, Kaushal A, Inflammation and the Host Response to Injury Large Scale Collaborative Research Program. High dynamic range characterization of the trauma patient plasma proteome. Mol Cell Proteomics. 2006 Oct;5(10):1899-913. Epub 2006 May 9. PubMed PMID: 16684767; PubMed Central PMCID: PMC1783978
Qian WJ, Liu T, Petyuk VA, Gritsenko MA, Petritis BO, Polpitiya AD, Inflammation and the Host Response to Injury Large Scale Collaborative Research Program. Large-scale multiplexed quantitative discovery proteomics enabled by the use of an (18)O-labeled “universal” reference sample. J Proteome Res. 2009 Jan;8(1):290-9. PubMed PMID: 19053531; PubMed Central PMCID: PMC2752204
Zhou B, Xu W, Herndon D, Tompkins R, Davis R, Xiao W, Inflammation and the Host Response to Injury Large Scale Collaborative Research Program. Analysis of factorial time-course microarrays with application to a clinical study of burn injury. Proc Natl Acad Sci U S A. 2010 Jun 1;107(22):9923-8. PubMed PMID: 20479259; PubMed Central PMCID: PMC2890487
Kotz KT, Xiao W, Miller-Graziano C, Qian WJ, Russom A, Warner EA, Inflammation and the Host Response to Injury Collaborative Research Program. Clinical microfluidics for neutrophil genomics and proteomics. Nat Med. 2010 Sep;16(9):1042-7. PubMed PMID: 20802500; PubMed Central PMCID: PMC3136804
Qian WJ, Petritis BO, Kaushal A, Finnerty CC, Jeschke MG, Monroe ME, Inflammation and the Host Response to Injury Large Scale Collaborative Research Program. Plasma proteome response to severe burn injury revealed by 18O-labeled “universal” reference-based quantitative proteomics. J Proteome Res. 2010 Sep 3;9(9):4779-89. PubMed PMID: 20698492; PubMed Central PMCID: PMC2945297
Finnerty CC, Jeschke MG, Qian WJ, Kaushal A, Xiao W, Liu T, Investigators of the Inflammation and the Host Response Glue Grant. Determination of burn patient outcome by large-scale quantitative discovery proteomics. Crit Care Med. 2013 Jun;41(6):1421-34. PubMed PMID: 23507713; PubMed Central PMCID: PMC3660437
Zhou JY, Krovvidi RK, Gao Y, Gao H, Petritis BO, De AK, Inflammation and the Host Response to Injury Large Scale Collaborative Research Program. Trauma-associated human neutrophil alterations revealed by comparative proteomics profiling. Proteomics Clin Appl. 2013 Aug;7 (7-8):571-83. PubMed PMID: 23589343; PubMed Central PMCID: PMC3737403
Contact
| Richard Smith, PhD | David Camp II, PhD | Weijun Qian, PhD | Wenzhong Xiao, PhD |
| 509-371-6576 | 509-371-6586 | 509-371-6572 | 617-724-7261 |
Visit the Glue Grant program website
Visit the Pacific Northwest National Laboratory Integrative Omics website