Use of FTICR Mass Spectrometric Proteomics Analysis for the identification of novel targets in pain research
EMSL Project ID
15897
Abstract
Treatment of chronic pain is one of the greatest challenges facing contemporary medical practice. Approximately one percent of the population (3 million people) in the United States suffers from moderate to severe neuropathic pain associated with injury to the peripheral nervous system. In recent years, neuropathic pain has received particular attention due to the severity and duration of the pain and its relative resistance to conventional analgesic therapies. Importantly, many analgesic drugs, from non-steroidal anti-inflammatory drugs to strong opiates, have proven to be either completely or partially ineffective. In fact, few pharmacological therapies have been approved specifically for the treatment of neuropathic pain. Unfortunately for many patients, pain continues to produce severe distress, dominating and disrupting the quality of their lives. The unmet clinical need, the personal suffering and societal economic costs of pain are therefore substantial. To bridge the gap between the ever-advancing understanding of the neurobiology of pain and the lack of success in clinical pain therapy, a greater and more sophisticated effort needs to be directed to the discovery of targets for new analgesics. In order to understand the molecular mechanisms of chronic pain, we have adopted a taxol-induced neuropathic pain model in rats, a well-known animal model with clinical relevance. Taxol is an antineoplastic agent used to treat patients with metastatic breast cancer, ovarian cancer, uterine cancer and lung cancer. Taxol induces cytotoxicity by promoting stabilization of tubulin polymers, resulting in microtubule dysfunction. Nerve cells with their long axonal processes are particularly vulnerable to the disruption of cytoskeletal function by taxol. The clinical consequence is that for many patients, taxol treatment is often associated with neuronal degeneration and painful peripheral neuropathy. This peripheral neuropathy can be recapitulated in rats that receive taxol. Therefore, our goal is to define the pain mechanism induced by taxol treatment and to identify the molecules that play crucial roles in pain pathways.
Two types of samples will be collected from untreated and taxol-treated rats. These samples include the cerebrospinal fluid and spinal cord tissues micro-dissected from dorsal horn region of the cervical and lumbosacral somatomes which has been shown to be pivotal for pain processing. We wish to employ the accurate mass and time tag (AMT) proteomics approach developed at PNNL to identify key proteins that are altered in our neuropathic pain model.
Initially, we will trypsin digest our proteins (500ug per sample) and then use strong cation exchange to create a combined total of 60 fractions from each sample set (2 sample sets = 120 total fractions). We would like to have these fractions analyzed using cLC-ESI tandem MS to create a potential mass and time tag (PMT) database. We will then use 16O/18O water in a trypsin exchange reaction to isotopically label the samples (three from each study group) for duplicate analyses by FTICR MS (24 runs total). The MS data would then be analyzed to identify which proteins are differentially regulated in rats with neuropathic pain.
Project Details
Project type
Exploratory Research
Start Date
2005-08-01
End Date
2007-03-20
Status
Closed
Released Data Link
Team
Principal Investigator