Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid
December 4th, 2017
A method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF) is presented. By employing molecular weight cut-off filtration, chromatographic pre-fractionation, mass spectrometric analysis and a subsequent combination of peptide identification strategies, it was possible to expand the known CSF peptidome nearly ten-fold compared to previous studies.
The goal of this protocol is to prepare and analyze human cerebrospinal fluid samples for LC-MS analysis of endogenous peptides.A peptide pre-fractionation step prior to LC-MS increases the number of identified peptides and enables detection of relatively low abundance CSF peptides.Endogenous peptides in CSF are calcium molecules that have not been extensively explored and may potentially include important new biomarkers of neurodegenerative disorders.This method enables extensive characterization of the CSF peptidome by mass spectrometry.The main advantages of this method are that it enables injection of low abundance CSF peptides and is compatible with multiplex isobaric labeling techniques, making it applicable for clinical studies.So far we have mainly applied this method to the analysis of cerebrospinal fluid.But we have preliminary data indicating that with minor modifications, it can also be used for analysis of plasma and tissue samples.Begin by thawing 1.5 milliliter aliquots of CSF at room temperature on roller mixer.Once thawed, transfer the contents to 10 milliliter polypropylene tubes.And add 80 microliters of one molar TAB as a buffering agent.Then add 0.65 milliliters of a eight molar guanidinium hydrochloride to dissolve protein aggregates and vortex gently at room temperature for 10 minutes.Next, at 60 microliters of 200 millimolar aqueous TCEP, and incubate at 55
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Overview
This study presents a method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF). By utilizing molecular weight cut-off filtration, chromatographic pre-fractionation, and mass spectrometric analysis, the research significantly expands the known CSF peptidome, enhancing the identification of low abundance peptides that could serve as biomarkers for neurodegenerative disorders.
Key Study Components
Area of Science
- Neuroscience
- Mass Spectrometry
- Biomarker Discovery
Background
- Endogenous peptides in CSF are potentially significant in identifying neurodegenerative disorders.
- Low abundance CSF peptides have not been extensively studied.
- Existing methods have provided limited data on the CSF peptidome.
Purpose of Study
- To enhance the identification and characterization of endogenous peptides in human CSF.
- To provide a more comprehensive view of the CSF peptidome.
- To explore the potential for new biomarkers in neurodegenerative conditions.
Methods Used
- Mass spectrometry with pre-fractionation of CSF samples.
- Involves thawing and processing 1.5 milliliter aliquots of CSF with specific reagents.
- Compatible with multiplex isobaric labeling techniques.
- Can be adapted for plasma and tissue sample analysis with minor modifications.
Main Results
- The method allows for the detection of a significantly larger number of peptides compared to previous studies.
- Key findings indicate the presence of previously unexplored low abundance peptides.
- Provides insights that may lead to the discovery of new biomarkers.
Conclusions
- This study demonstrates an efficient approach to characterizing the CSF peptidome.
- The method's versatility opens avenues for clinical studies on neurodegenerative disorders.
- Potential implications for understanding neuronal mechanisms and identifying disease markers.
Frequently Asked Questions
Chapters in this video
0:05
Title
1:12
Pre-treatment of Bulk Volume CSF without Quantification
2:24
Pre-treatment of Small Volume CSF Samples with Isobaric Labeling
4:00
MWCO Filtration, Desalting, and Sample Clean-up by Solid Phase Extraction
6:00
Offline High-pH Reverse Phase HPLC Sample Fractionation
7:09
LC-MS Analysis
7:47
Results: Peptide Identification After LC-MS Analysis
9:13
Conclusion
