title: Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo.
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.
authors.affiliation: From the ‡German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; §Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany;
authors.forename: Bastian
authors.initials: B
authors.lastname: Dislich
authors.affiliation: From the ‡German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; §Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany;
authors.forename: Felix
authors.initials: F
authors.lastname: Wohlrab
authors.affiliation: ¶Adolf Butenandt Institute, Ludwig-Maximilians University, Munich Biochemistry, Munich Germany;
authors.forename: Teresa
authors.initials: T
authors.lastname: Bachhuber
authors.affiliation: From the ‡German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; §Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany;
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Müller
authors.affiliation: §Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; **Institute for Advanced Study, Technische Universität München, Garching, Germany;
authors.forename: Peer-Hendrik
authors.initials: PH
authors.lastname: Kuhn
authors.affiliation: From the ‡German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; §Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany;
authors.forename: Sebastian
authors.initials: S
authors.lastname: Hogl
authors.affiliation: ‖Neurocenter, Department of Neurology, University of Freiburg, Freiburg, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Meyer-Luehmann
authors.affiliation: From the ‡German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; §Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; **Institute for Advanced Study, Technische Universität München, Garching, Germany; ‡‡Munich Center for Systems Neurology (SyNergy), Munich, Germany stefan.lichtenthaler@dzne.de.
authors.forename: Stefan F
authors.initials: SF
authors.lastname: Lichtenthaler
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000134:cerebrospinal fluid* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000134:cerebrospinal fluid* / Q000235:genetics / Q000378:metabolism*; D015415:Biomarkers / Q000134:cerebrospinal fluid / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism
chemical_list: D015415:Biomarkers; C558538:PLXDC2 protein, human; D011956:Receptors, Cell Surface; C102889:PTPRN2 protein, human; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D010727:Phosphoric Diester Hydrolases; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse