Proteomic Characterization of cerebrospinal fluid (CSF) by high resolution LC-MS/MS
EMSL Project ID
11093
Abstract
This NCRR collaborative project will focus on the proteomic characterization of human cerebrospinal fluid (CSF). CSF is routinely sampled in clinical medicine to help determine and monitor the presence of illness in the brain or spinal cord. Clinicians usually measure the total protein concentration for this purpose since very little is known about the protein composition, or proteome, of CSF in normal or disease states. Much of our current knowledge is based upon 2D gel analysis techniques which are severely limited in both sensitivity and throughput. The overall goals of the work proposed in this study are twofold: 1. determine, as possible, the content and distribution of proteins in "normal" human CSF for the creation of a mass and time tag database for future quantitative LC-FTICR studies; and 2. Evaluate how the content and abundance of proteins in CSF are altered in specific diseases of the brain and spinal cord. The project will proceed in three phases: 1. In phase 1 CSF will be obtained from 1-2 patients at the MGH that are selected to reflect normal human CSF as much as is possible. The IRB permission has been obtained for "discarded" CSF, (CSF that is obtained in the routine care of patients for analysis, but then is slated to be discarded); therefore, CSF will not be specifically obtained in this initial study for the research and there will not be a need for informed consent. Samples will be sent to PNNL for proteomic analysis by high resolution liquid chromatography (LC) coupled by electrospray ionization to a ThermoElectron Finnigan 2-dimensional linear ion trap MS/MS instrument (MS/MS). The purpose of the analysis of these first 1-2 samples will be to assess that the fluid is suitable for LC-MS/MS analysis, to troubleshoot the process, and to obtain preliminary data as an indicator of the feasibility of the project. After this initial analysis, all of the investigators will discuss the results in person or via conference call with the goal of modifying and improving the process and to decide on how to further proceed. At this point it may be appropriate to consult a statistical analysis expert to determine how best to analyze the data.
2. Depending upon the initial LC-MS/MS results, an additional number of different patient samples will be obtained in phase 2 from "normal" patient populations at the MGH to complete the first phase of the study. With IRB approvals in place from both participating institutions, MGH and PNNL, the samples will be shipped to the EMSL facility to further extend the mass and time tag database for normal CSF. We anticipate that this might include 10-15 samples, although the appropriate sample number will need to be determined from both the initial results and with statistical help. The goal of phase 2 will be to attempt to define in a limited way the proteome in normal human CSF by LC-MS/MS. After this analysis, all of the investigators will discuss the results in person or via conference call with the goal of modifying and improving the process to decide how to further proceed with phase 3 of the project. After collecting proteomic data from several samples in phase 2, we anticipate seeking funding support for an expanded CSF project from the National Institutes of Health.
3. Depending upon the results obtained in phase 2, the phase 3 goal will be to select a well defined disease of the brain or spinal cord for quantitative comparative analysis using high resolution LC coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICR). At this point careful discussions about the advantages and disadvantages of selecting the first disease type to be characterized will ensue, but we presently anticipate that this will be an infectious disease. If so, we would attempt to detect the microbial proteins from the pathogen involved by LC-MS/MS in addition to characterizing changes in the human host proteins in CSF in terms of both protein identifications and protein quantitation using LC-FTICR. An additional number of CSF samples will be obtained from this population of diseased patients and will be analyzed using this same approach. We anticipate that this might include 10-15 samples, although the appropriate sample number will again need to be determined from both the initial results and with statistical help.
Project Details
Project type
Exploratory Research
Start Date
2004-09-02
End Date
2007-06-28
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members