Abstract
Analysis of murine cerebrospinal fluid (CSF) by quantitative mass spectrometry is challenging because of low CSF volume, low total protein concentration, and the presence of highly abundant proteins such as albumin. We demonstrate that the CSF proteome of individual mice can be analyzed in a quantitative manner to a depth of several hundred proteins in a robust and simple workflow consisting of single ultra HPLC runs on a benchtop mass spectrometer. The workflow is validated by a comparative analysis of BACE1-/- and wild-type mice using label-free quantification. The protease BACE1 cleaves the amyloid precursor protein (APP) as well as several other substrates and is a major drug target in Alzheimer's disease. We identified a total of 715 proteins with at least 2 unique peptides and quantified 522 of those proteins in CSF from BACE1-/- and wild-type mice. Several proteins, including the known BACE1 substrates APP, APLP1, CHL1 and contactin-2 showed lower abundance in the CSF of BACE1-/- mice, demonstrating that BACE1 substrate identification is possible from CSF. Additionally, ectonucleotide pyrophosphatase 5 was identified as a novel BACE1 substrate and validated in cells using immunoblots and by an in vitro BACE1 protease assay. Likewise, receptor-type tyrosine-protein phosphatase N2 and plexin domain-containing 2 were confirmed as BACE1 substrates by in vitro assays. Taken together, our study shows the deepest characterization of the mouse CSF proteome to date and the first quantitative analysis of the CSF proteome of individual mice. The BACE1 substrates identified in CSF may serve as biomarkers to monitor BACE1 activity in Alzheimer patients treated with BACE inhibitors.
Highlights
From the ‡German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; §Neuroproteomics, Klinikum rechts der Isar, Technische Universitat Munchen, Munich, Germany; ¶Adolf Butenandt Institute, Ludwig-Maximilians University, Munich Biochemistry, Munich Germany; ʈNeurocenter, Department of Neurology, University of Freiburg, Freiburg, Germany, **Institute for Advanced Study, Technische Universitat Munchen, Garching, Germany; ‡‡Munich Center for Systems Neurology (SyNergy), Munich, Germany
For the quantitative proteomic analysis of murine Cerebrospinal fluid (CSF) from adult mice, we first evaluated the performance of in-solution digestion versus Filter Aided Sample Preparation (FASP) (16, 23)
Five l each of a pooled human CSF sample were used in three replicates for in-solution digestion and FASP
Summary
Materials—The following antibodies were used: pAb APLP1 antibody (Proteintech, Chicago, IL; 12305–2-AP), pAb APLP2 antibody (Calbiochem; 171617), mouse mAb HA. (Covance, Emeryville, CA), rat mAb HA 3F10 (Roche, Rotkreutz, Switzerland), mouse mAb FLAG M2 The eluted peptides from one digestion reaction (5 l of CSF) were split into two aliquots and processed on individual STAGE Tips, each representing one technical replicate. Each technical replicate contained the peptides of 2 l of digested CSF. The MS/MS spectra were recorded at a resolution of 17,500 with the automatic gain control target set to 100,000 and a maximum injection time of 50 ms. Data Analysis—Overall, 26 LC-MS/MS files (five BACE1Ϫ/Ϫ and eight wild-type CSF samples with two technical replicates each) were subjected to data analysis using the MaxQuant software environment (version 1.3.0.5). Reproducibility of retention times was checked manually According to this inspection the match between runs option was enabled, allowing a time window of 2 min to search for already identified peptides in all obtained chromatograms. The samples were boiled in reducing Laemmli buffer and applied to Western blot analysis as described above
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