A new sample preparation workflow for proteome profiling of very limited-volume murine cerebrospinal fluid

Cerebrospinal fluid (CSF) is a clear, colourless body fluid present in the brain and spinal cord. CSF performs vital functions since it protects the brain from injury and helps in maintaining a stable chemical environment for the central nervous system (CNS), acting as waste removal.

Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides insight into the status of the CNS.

The analysis of changes in CSF proteome of murine models could help researchers in understanding the pathophysiology of several neurological diseases, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis. However, the small volume of CSF in mice has limited individual mouse proteome characterization.

Moreover, the wide dynamic range of protein concentrations in CSF requires specific sample preparation for label-free untargted proteomics; the removal of the most abundant proteins, such as albumin and immunoglobulins, from CSF samples significantly enhances the ability to detect and quantify low-abundance proteins, providing a deeper and more accurate proteomic profile. This process is known as depletion, and several immunodepletion kits are available on the market, specifically designed for the depletion of high-abundance proteins in murine CSF. To overcome issues related to the low protein concentration in murine CSF (typically ranges from 0.3 to 0.5 mg/mL), and to the limited amount of CSF which can be collected from each mouse, pooling CSF from multiple mice is a common practice in proteomics to increase the protein content available for the analysis.

Recently, PreOmics, a company specialized in sample preparation solutions for proteomics research, developed the ENRICH-iST kit, that enables the identification and the quantification of low-abundant proteins by using only 20 µL of murine CSF. In this way, we were able to perform label-free quantitative proteomic analysis on CSF collected from single biological replicate thus preserving the inter-individual variability of the experiment.

We identified and quantified a total of 502 proteins by comparing 20 µL of CSF collected from 8 colitis-like mice to the same volume collected from control mice. Of these proteins, corresponding to 465 genes, we found 431 genes that are already known to be present in human CSF. Moreover, we found some proteins (about 1/5 of the total quantified proteins) that are significantly dysregulated in the condition of interest in comparison to the control.

With these results, we demonstrated that the Enrich-iST is perfectly suited for the analysis of 20 µL murine CSF, without the need of pooling samples.