Biomarker identification by LC-MS/MS for patients with brain injury.

• Main Author: Teena.
• Other Authors: Chen Wei Ning, William
• Format: Final Year Project
• Language: English
• Published: 2009
• Subjects: DRNTU::Engineering::Chemical engineering::Biotechnology
• Online Access: http://hdl.handle.net/10356/16610
• Institution: Nanyang Technological University

This report is a feasibility study on using Liquid Chromatography- Mass Spectrometry (LC-MS/MS) in identifying potential biomarkers in brain injury. The feasibility of performing biomarkers discovery using serum samples obtained from brain injury patients suffering from small to moderate sized blood clots was examined. Samples were obtained from Tan Tock Seng Hospital (TTSH). A total of 9 brain- injured patients were recruited. However, only serum samples from 7 patients were prepared and labeled using i-TRAQ reagents and then sent for LC-MS/ MS test. From the results, the ratio of the amount of proteins present in the blood serum was calculated, i.e.: ratio of proteins present in Day 3: Day 1 and ratio of proteins present in Day 5: Day 1. These ratios were calculated for 4 Patients’ serum sample only, as the remaining results were unsatisfactory. The results obtained were analyzed to detect for possible presence of biomarkers. A total of 17 differentially expressed proteins were identified (Refer to Table 4A and 4B). The two proteins that showed the greatest change were Hemoglobin subunit alpha (Hemoglobin alpha chain) (Alpha-globin) -Homo sapiens (Human), with a fold change value of 1.5488 (upregulated) and Fibronectin precursor (FN) (Cold-insoluble globulin) (CIG) -Homo sapiens (Human), with a fold change value of 0.7649 (downregulated). This analysis managed to identify serum biomarkers that were common with those detected in a similar LC-MS/MS analysis of brain- injured patients’ serum (Hergenroeder et al., 2007 [1]). They include Alpha-1-antitrypsin precursor, Ceruloplasmin precursor (EC 1.16.3.1) (Ferroxidase), lg alpha-1 chain C region and lg mu chain C region. The results obtained support the use of serum as a source for discovery of biomarkers for brain- injury. This project faced some limitations. The LC-MS/MS test may not be able to detect all the proteins present in the sample. Due to limited resources and time constraints, any selected candidate proteins were not verified using Western Blot analysis and only one round of LC-MS/MS test was conducted for this study. Also, there was some delay in obtaining the blood serum samples from normal and healthy individuals. As a result, no LC-MS/MS test was conducted on the normal samples. Hence, the analysis was done purely on the changes in the serum protein level across the brain-injured patients, without a control. Improvements can be made to this project, such as immunodepleting the abundance serum proteins using antibody columns. Western Blot analysis could be used to further characterize any changes in serum abundance of the potential biomarkers. Continued work will be required to assess the roles, if any, of these serum biomarkers in the progression of brain injury, and to identify other biomarkers that could be used to predict adverse secondary events that may occur as a result of brain injury.