Rajeswari Lakshmanan, Affiliation 1, Y. Jimmy Ytterberg, Affiliation 1, Thomas A. Drake, Affiliation 2, Paul M. Vespa, Affiliation 3, Joseph A. Loo, Affiliation 1, James F. Leblanc, Affiliation 2
Departments of 1Chemistry and Biochemistry, 2Pathology and Laboratory Medicine, and 3Neurosurgery, David Geffen School of Medicine,
University of California-Los Angeles, Los Angeles, CA 90095 USA |
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Traumatic Brain Injury (TBI) is a direct physical impact to the head from an external mechanical force. Accurate diagnosis is crucial, but currently there are no sensitive biomarkers for TBI. We have used the brain extracellular fluid collected from the injured patients by cerebral microdialysate (MD) to identify putative protein/peptide biomarker candidates. These biomarkers would be useful for diagnostic and prognostic purposes, and would also help to elucidate the mechanisms that underlie TBI. We have compared MD samples from two groups of TBI patients: those who had severe brain injury (abnormal samples) and from patients who had mild brain injury but had a better clinical outcome (control samples). We employed two different routes of proteomic techniques categorized as diagnostic and biomarker identification approaches. The diagnostic approach was aimed at finding a signature of peaks which can differentiate these two groups. We did this by enriching for intact peptides using C18 magnetic bead based capture followed by MALDI-TOF MS. For the biomarker identification approach, we applied classical proteomic top-down and bottom-up (trypsin digestion followed by LC-MS/MS) methodologies. Since phosphorylation regulates neuronal signaling in the brain, we also added phosphopeptide enrichment techniques.
We found several differences between these two sample groups. Both approaches show peptides/proteins that are present only in abnormal samples when compared to control. The LC-MS/MS data from the abnormal samples showed cytoskeletal proteins, proteins from blood and other proteins like ubiquitin and microtubule-associated protein 1B. Also, we were able to identify two phosphopeptides predominantly in the abnormal samples, both of which originated from fibrinopeptide A.
The peptides that are different could be used as a marker for the severity of injury and the proteins identified shed light into the mechanisms that are triggered after the trauma. We have demonstrated the applicability of MD fluid as a medium to find proteomic biomarkers in TBI. It has an inherent advantage of being less complex, by which, we can minimize the protein dynamic range problems that are often encountered when working with blood and CSF.
Acknowledgement
Thanks to W. M. Keck Foundation for the LC-MS/MS system. |
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