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The Proteomics Core continued to perform analyses for many members of the SWEHSC who are part of the SWEHSC Research Cores:
- The laboratory of Dr. Clark R. Lantz has used the Proteomics Technology to identify potential pulmonary protein biomarkers in the lung lining fluid of mice chronically exposed to low dose arsenic and to validate these protein changes in human populations exposed to arsenic.
- In the laboratory of Dr. Qin Chen, biomarkers of cardiomyocyte injury associated with oxidative stress in vitro and in vivo are sought following numerous lines of evidence that indicate a role of oxidative stress in initiation and progression of heart failure.
Dr. Clark R. Lantz
The laboratory of Dr. Clark R. Lantz has used the Proteomics Technology to identify potential pulmonary protein biomarkers in the lung lining fluid of mice chronically exposed to low dose arsenic and to validate these protein changes in human populations exposed to arsenic. In the design of Dr. Lantz’s experiments, mice were administered 10 or 50 ppb of arsenic (sodium arsenite) in their drinking water for 4 weeks and subsequent to that, the Proteomics Technology was crucial in identifying proteins in the lung lining fluid using 2-D gel electrophoresis (N=3) or multidimensional protein identification technology (MUDPIT or LC-LC-MS/MS) (N=2) coupled with mass spectrometry.
The proteomics results indicated proteins, whose expression in mouse lung lining fluid was consistently altered by arsenic and included glutathione-S-transferase omega-1 (GST-omega-1), contraspin, apolipoprotein A-I and A-IV, peroxiredoxin-6 and receptor for advanced glycation end products (RAGE).
Of special interest to Dr. Lantz’s team, was the identification of RAGE, a protein that is involved in chronic diseases, such as diabetes, atherosclerosis, coronary artery disease and lung cancer. As such, its identity was further validated in the Proteomics Technology by re-analysis of the raw mass spectrometric data my multiple protein identification algorithms.
Following these results, Dr. Lantz chose to determine whether arsenic-induced alterations in RAGE were also present in humans. Regression analysis demonstrated a significant negative correlation (p=0.016) between sputum levels of RAGE and total urinary inorganic arsenic, similar to results seen in the animal model.
It was concluded that combinations of proteomic analyses of animal models followed by specific analysis of human samples provides for an unbiased determination of important, previously unidentified biomarkers that may be related to human disease.
Dr. Qin Chen
In the laboratory of Dr. Qin Chen, biomarkers of cardiomyocyte injury associated with oxidative stress in vitro and in vivo are sought following numerous lines of evidence that indicate a role of oxidative stress in initiation and progression of heart failure. Among the main cell types within the heart, cardiomyocytes (CMCs) undergo hypertrophy or apoptosis, while cardiac fibroblasts (CFs) are responsible for fibrosis during the process of heart failure.
In an effort to identify cell type specific biomarkers of oxidative injury, Dr. Chen ’s lab isolated CMCs and CFs from rat hearts and treated cells with sublethal doses of H2O2. Crucial to this type of biomarker discovery, the Proteomics Technology used classical ESI-LC-MS/MS approaches for detection of secreted protein factors in the conditioned media. A comparison of the profiles of secreted proteins among the two cell types lead to the finding that H2O2 at sublethal doses caused an elevation in the level of Cystatin C protein in the conditioned medium from CMCs but not from CFs.
Validation of the proteomics data by RT-PCR analyses of Cystatin C mRNA and Western blot analyses of Cystatin C protein confirmed that H2O2 induces a dose dependent elevation in CMCs. Dr. Chen subsequently carried this discovery further by validating the potential of using Cystatin C as a cardiac injury biomarker in vivo (i.e.: by inducing cardiomyopathy in mice by chronic administration of doxorubicin, Dox).
Her findings indicated that while elevated Cystatin C protein was detected in the plasma, increased levels of Cystatin C protein or mRNA were found in cardiac tissue from Dox treated animals. In a mouse model of myocardial infarction induced by left descending coronary artery occlusion, an increase in the level of Cystatin C protein was detected in the plasma of myocardial infracted mice. These findings, made possible through initial exploratory proteomic approaches in the Proteomics Technology, suggest that Cystatin C can be useful as a biomarker of cardiomyocyte injury associated with oxidative stress in vitro and in vivo.