Protein Changes in Immunodepleted Cerebrospinal Fluid from a Transgenic Mouse Model of Alexander Disease Detected Using Mass Spectrometry

Abstract

Cerebrospinal fluid (CSF) is a low protein content biological fluid with dynamic range spanning at least nine orders of magnitude in protein content and is in direct contact with the brain. A modified IgY-14 immunodepletion treatment was performed to enhance analysis of the low volumes of CSF that are obtainable from mice. As a model system in which to test this approach, we utilized transgenic mice that over-express the intermediate filament glial fibrillary acidic protein (GFAP). These mice are models for Alexander disease (AxD), a severe leukodystrophy in humans. From the CSF of control and transgenic mice we report the identification of 289 proteins, with relative quantification of 103 proteins. Biological and technical triplicates were performed to address animal variability as well as reproducibility in mass spectrometric analysis. Relative quantitation was performed using distributive normalized spectral abundance factor (dNSAF) spectral counting analysis. A panel of biomarker proteins with significant changes in the CSF of GFAP transgenic mice has been identified with validation from ELISA and microarray data, demonstrating the utility of our methodology and providing interesting targets for future investigations on the molecular and pathological aspects of AxD.

Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo

“…Moreover, our workflow increases proteomic depth, as the number of protein identifications were two-to threefold higher compared with previous studies (6,7). This is a clear step forward toward the goal of determining the whole mouse CSF proteome.…”

“…Full MS spectra were recorded at a resolution of 70,000 over a mass range between m/z 400 -1800. The automatic gain control target was set to 3 ϫ 10 6 and a maximum injection time of 50 ms was allowed. The 10 most intense peptide ions were chosen for fragmentation.…”

“…Our approach allows the accurate identification and quantification of several hundred proteins in as little as 2 l of murine CSF in ϳ4.5 h per sample, at a much greater speed and proteomic depth than in previous studies, despite using lower sample amounts (6,7). Overall, 715 proteins were identified with at least two unique peptides and 522 proteins were quantified in at least three biological replicates of both BACE1Ϫ/Ϫ and wild-type mice.…”

“…The low total CSF volume of ϳ30 l and an average yield of only ϳ10 l blood-free CSF pose a challenge for various protein concentration and depletion steps that are routinely applied to human CSF, where the sample volume is up to 1,000-fold more (4,5). One study reported the identification of 289 proteins and the quantification of 103 proteins using pooled immunodepleted CSF from 10 -12 mice per sample (6). A second study reported the identification of 566 proteins in murine CSF of individual mice, relying on time consuming fractionation by two dimensional liquid chromatography tandem MS (2D-LC-MS/MS) (7).…”

Glial fibrillary acidic protein is a body fluid biomarker for glial pathology in human disease

“…Mass-spectrometry has been used to detect small amounts of GFAP and better characterise the post-translational modifications of GFAP (Herskowitz et al, 2010;Cunningham et al, 2013;Hanrieder et al, 2009;Ishigami et al, 2005;Joachim et al, 2007;Nomura et al, 2009;Ekegren et al, 2006). An important finding from these studies was that compared to other proteins GFAP is an extremely stable and robust candidate biomarker for massspectrometry (Ekegren et al, 2006).…”