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. 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. Cerebrospinal fluid (CSF)1 consists of interstitial fluid that is in continuous exchange with the central nervous system and the peripheral blood system. It represents the only body fluid in humans that is in direct contact with brain tissue and accessible in a routine clinical setting. Thus, the easy accessibility from the periphery renders CSF perfectly suited to study pathologic neurological processes (1.Romeo M.J. Espina V. Lowenthal M. Espina B.H. Petricoin 3rd, E.F. Liotta L.A. CSF proteome: a protein repository for potential biomarker identification.Expert Rev. Proteomics. 2005; 2: 57-70Crossref PubMed Scopus (109) Google Scholar). Human CSF has a relatively low protein content (∼ 0.4 mg/ml), but features a highly diverse proteome. It is thus increasingly studied by modern mass spectrometry based proteomics (2.Craft G.E. Chen A. Nairn A.C. Recent advances in quantitative neuroproteomics.Methods. 2013; 61: 186-218Crossref PubMed Scopus (106) Google Scholar). The proteomic analysis of human CSF typically involves various protein concentration and fractionation steps as well as the depletion of highly abundant proteins, such as serum albumin. This allows the identification of several hundred up to 2600 proteins from several milliliters of human CSF (3.Schutzer S.E. Liu T. Natelson B.H. Angel T.E. Schepmoes A.A. Purvine S.O. Hixson K.K. Lipton M.S. Camp D.G. Coyle P.K. Smith R.D. Bergquist J. Establishing the proteome of normal human cerebrospinal fluid.PLoS ONE. 2010; 5: e10980Crossref PubMed Scopus (160) Google Scholar). Mice are the most popular animal model in preclinical research, because of their similarity to humans in genetics and physiology, their unlimited supply and their ease of genetic engineering. The study of their CSF can provide valuable insights into disease mechanisms and biomarker discovery and may allow the rapid translation of preclinical findings into human patients. However, the proteomic study of murine CSF has been limited because of several shortcomings. The low total CSF volume of ∼30 μl and an average yield of only ∼10 μl 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.DeMattos R.B. Bales K.R. Parsadanian M. O'Dell M.A. Foss E.M. Paul S.M. Holtzman D.M. Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimer's disease.J. Neurochem. 2002; 81: 229-236Crossref PubMed Scopus (224) Google Scholar, 5.Rudick R.A. Zirretta D.K. Herndon R.M. Clearance of albumin from mouse subarachnoid space: a measure of CSF bulk flow.J. Neurosci. Meth. 1982; 6: 253-259Crossref PubMed Scopus (49) Google Scholar). 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.Cunningham R. Jany P. Messing A. Li L. Protein changes in immunodepleted cerebrospinal fluid from a transgenic mouse model of Alexander disease detected using mass spectrometry.J. Proteome Res. 2013; 12: 719-728Crossref PubMed Scopus (17) Google Scholar). 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.Smith J.S. Angel T.E. Chavkin C. Orton D.J. Moore R.J. Smith R.D. Characterization of individual mouse cerebrospinal fluid proteomes.Proteomics. 2014; 14: 1102-1106Crossref PubMed Scopus (21) Google Scholar). Here we show that label-free quantitative proteomics in murine CSF can be achieved in unprecedented depth in individual animals using single ultra HPLC runs on the benchtop Q Exactive mass spectrometer. We demonstrate the feasibility of our approach by comparing the CSF of BACE1 (β-site amyloid precursor protein (APP) cleaving enzyme 1) −/− mice with their wild-type littermates. BACE1 is a membrane bound aspartyl protease that is essential in the pathogenesis of Alzheimer's disease. It is the rate-limiting enzyme in a proteolytic cascade leading to the liberation of the neurotoxic Aβ peptide from the much larger amyloid precursor protein (APP) into the extracellular space (8.Dislich B. Lichtenthaler S.F. The Membrane-Bound Aspartyl Protease BACE1: Molecular and Functional Properties in Alzheimer's Disease and Beyond.Front. Physiol. 2012; 3: 8Crossref PubMed Scopus (59) Google Scholar, 9.Haass C. Selkoe D.J. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.Nat. Rev. Mol. Cell Biol. 2007; 8: 101-112Crossref PubMed Scopus (3876) Google Scholar). Inhibition of BACE1 abolishes Aβ generation, rendering BACE1 a prime drug target for the therapy of Alzheimer's disease (10.Ghosh A.K. Osswald H.L. BACE1 (beta-secretase) inhibitors for the treatment of Alzheimer's disease.Chem. Soc. Rev. 2014; 43: 6765-6813Crossref PubMed Google Scholar). Besides APP, BACE1 processes numerous other substrates in vivo and in vitro, which raises concerns about mechanism based side effects on the therapeutic inhibition of this protease (11.Vassar R. Kuhn P.H. Haass C. Kennedy M.E. Rajendran L. Wong P.C. Lichtenthaler S.F. Function, therapeutic potential and cell biology of BACE proteases: current status and future prospects.J. Neurochem. 2014; 130: 4-28Crossref PubMed Scopus (243) Google Scholar). Although BACE1 expression levels are the highest in the brain, it is currently unknown whether BACE1 substrate levels besides APP can be monitored in the CSF as a read-out of BACE1 activity. This would be desirable, as it would allow the longitudinal monitoring of BACE1 substrate levels on therapeutic inhibition of BACE1 in humans and thus an effective screening for possible adverse effects. Our approach allows the accurate identification and quantification of several hundred proteins in as little as 2 μl of murine CSF in ∼4.5 h per sample, at a much greater speed and proteomic depth than in previous studies, despite using lower sample amounts (6.Cunningham R. Jany P. Messing A. Li L. Protein changes in immunodepleted cerebrospinal fluid from a transgenic mouse model of Alexander disease detected using mass spectrometry.J. Proteome Res. 2013; 12: 719-728Crossref PubMed Scopus (17) Google Scholar, 7.Smith J.S. Angel T.E. Chavkin C. Orton D.J. Moore R.J. Smith R.D. Characterization of individual mouse cerebrospinal fluid proteomes.Proteomics. 2014; 14: 1102-1106Crossref PubMed Scopus (21) Google Scholar). Overall, 715 proteins were identified with at least two unique peptides and 522 proteins were quantified in at least three biological replicates of both BACE1−/− and wild-type mice. We provide evidence that BACE1 activity is reflected in the composition of the CSF, as the secreted ectodomains of well-known BACE1 substrates were reduced in BACE1−/− animals. In addition, we identified and validated a previously unknown BACE1 substrate candidate and confirmed two recently described novel BACE1 substrates. The three proteins may represent novel prognostic or diagnostic biomarkers and may aid in the development of APP-specific BACE1 inhibitors. The following antibodies were used: pAb APLP1 antibody (Proteintech, Chicago, IL; 12305–2-AP), pAb APLP2 antibody (Calbiochem; 171617), mouse mAb HA.11 (Covance, Emeryville, CA), rat mAb HA 3F10 (Roche, Rotkreutz, Switzerland), mouse mAb FLAG M2 (Sigma, St. Louis, MO), mouse mAb 3D5 (specific for BACE1, kind gift of R. Vassar)(12.Zhao J. Fu Y. Yasvoina M. Shao P. Hitt B. O'Connor T. Logan S. Maus E. Citron M. Berry R. Binder L. Vassar R. Beta-site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques: implications for Alzheimer's disease pathogenesis.J. Neurosci. 2007; 27: 3639-3649Crossref PubMed Scopus (304) Google Scholar), rabbit pAb calnexin (Stressgen, Enzo Life Sciences, Lörrach, Germany), HRP- coupled anti-rabbit and anti-mouse antibody (DAKO, Glostrup, Denmark), HRP coupled anti- rat antibody (Santa Cruz, Dallas, TX). The following reagents were used: Acetonitrile, water, formic acid (all LC-MS/MS grade), dithiothreitol and iodoacetamide were purchased from Sigma-Aldrich. BACE Inhibitor C3 (β-Secretase Inhibitor IV, Calbiochem)(13.Stachel S.J. Coburn C.A. Steele T.G. Jones K.G. Loutzenhiser E.F. Gregro A.R. Rajapakse H.A. Lai M.T. Crouthamel M.C. Xu M. Tugusheva K. Lineberger J.E. Pietrak B.L. Espeseth A.S. Shi X.P. Chen-Dodson E. Holloway M.K. Munshi S. Simon A.J. Kuo L. Vacca J.P. Structure-based design of potent and selective cell-permeable inhibitors of human beta-secretase (BACE-1).J. Med. Chem. 2004; 47: 6447-6450Crossref PubMed Scopus (277) Google Scholar), Lipofectamine 2000 (Invitrogen). BACE1−/− mice that are commercially available from the Jackson Laboratory (www.jax.org, strain B6.129-Bace1tm1Pcw/J) were maintained according to the European community council directive (86/609/ECC). Wild-type and BACE1−/− mice were both on a C57BL/6 background and obtained from BACE1 ± x BACE1 ± matings. Animals were kept under a 12/12 h light-dark cycle with food and water ad libitum. All animal procedures were carried out in accordance with the European Communities Council Directive (86/609/EEC) and with an animal protocol approved by the Ludwigs-Maximilians-University Munich and the government of Upper Bavaria. For the analysis of murine CSF, 7 μl of CSF plus 2.3 μl of 4x Laemmli buffer per sample were incubated at 95 °C for 5 min and loaded on 8% acrylamide gels for immunoblot analysis and detection of APLP1 and APLP2. PVDF membranes (Millipore) were incubated overnight at 4 °C. Blots were developed using horseradish peroxidase-conjugated secondary antibodies and the ECL chemiluminescence system (Millipore). Immunoblots were quantified using the LAS-4000 Fujifilm chemiluminescence camera and software (Fuji Film, Inc.) and quantification was based on three independent replicates. For analysis of HEK293T cell lysates and conditioned media, samples were boiled in reducing Laemmli buffer for 5 min at 95 °C and subsequently applied to 8% SDS-polyacrylamide-gel electrophoresis. Nitrocellulose membranes pore size 0.45 μm were used for transfer, emulsified nonfat dry milk for blocking. Primary antibodies were incubated overnight at 4 °C. The full-length proteins were detected with HA.11 antibody and FLAG M2 antibody. The soluble proteins were detected with HA 3F10 antibody. Blots were developed and quantified as described above for the CSF samples. Full-length and soluble protein levels were normalized to the protein levels of calnexin, which was used as a loading control. Six independent experiments were included for the statistical analysis, applying a one-way ANOVA test. The CSF was isolated from the cisterna magna of 4 month old animals according to the protocol from DeMattos et al. (4.DeMattos R.B. Bales K.R. Parsadanian M. O'Dell M.A. Foss E.M. Paul S.M. Holtzman D.M. Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimer's disease.J. Neurochem. 2002; 81: 229-236Crossref PubMed Scopus (224) Google Scholar). Mice were anesthetized via intraperitoneal injection of a mixture containing ketamine (Bayer, 100 mg/kg body weight) and Rompun (Ratiopharm, 10 mg/kg body weight). A dorsal excision along the base of the skull to the dorsal thorax up to Th1 was made. The musculature was displaced and the meninges on top of the cisterna magna were exposed. The area was cleaned using cotton swabs. The cisterna magna was punctuated and the CSF collected using glass micropipettes (Stoelting, Wood Dale, IL, #50614). CSF samples were subjected to centrifugation on a benchtop centrifuge and visually inspected for the presence of blood in the form of pelleted residual erythrocytes. A total of 5–20 μl of blood-free CSF was collected from each animal and stored at −80 °C. The animals were sacrificed afterward. Samples affected by visible blood contamination were excluded from the analysis. Overall, five BACE1−/− and eight wild-type mice (males and females) were used for the analysis. Human CSF samples were provided by the Karolinska University Hospital & Institute, Dept. of Clinical Neuroscience, Neuroimmunology Unit. The ethical review board of the Karolinska Institute approved the study (Diary Number: 2009/2107–31-2) and written informed consent was obtained from all patients. Six human CSF samples of patients with minor symptoms were chosen for the analysis. The in-solution digestion was performed according to Olsen et al. (14.Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2810) Google Scholar). All steps were performed at room temperature. Briefly, 5 μl of CSF were solubilized in denaturation buffer (6 m urea (Sigma, U5128) in 10 mm HEPES pH 8.0, sample to buffer ratio 1:10) in low protein binding tubes (Eppendorf). 1 μl of reduction buffer (10 mm dithiothreitol in 0.05 m ammonium bicarbonate) was added for 10 μl of digestion buffer, followed by incubation for 30 min. Then, 1 μl of iodoacetamide solution for 10 μl of digestion buffer was added, followed by incubation for 20 min. 0.5 μg Lys-C solution (Promega, Life Technologies, Darmstadt, Germany) was added and incubated for 4 h. The sample was diluted 4-fold with 50 mm ammonium bicarbonate, 0.4 μg trypsin (Promega, Life Technologies, Darmstadt, Germany) was added and incubated for 16 h. The digestion reaction was stopped using 1 μl of 100% trifluoroacetic acid. Homemade C18 STAGE Tips were used for purification of the in-solution digested samples. Cleanup was done according to Rappsilber et al. (15.Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2570) Google Scholar). The eluted peptides from one digestion reaction (5 μl of CSF) were split into two aliquots and processed on individual STAGE Tips, each representing one technical replicate. Peptides were eluted in 60% acetonitrile, dried by speedvac, resuspended in 10 μl 0.1% trifluoroacetic acid in water and loaded onto the autosampler. 8 μl of the peptide solution was injected per run. Thus, each technical replicate contained the peptides of 2 μl of digested CSF. Pooled human CSF from six individuals was used to test the efficiency and reproducibility of in-solution digestion and FASP. Three replicates, 5 μl human CSF each, was digested with the in-solution digestion protocol described above and with FASP according to Wisniewski et al. (16.Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5043) Google Scholar). A double digestion with 0.1 μg LysC and 0.1 μg trypsin was performed. Samples were analyzed by LC-MS/MS, coupling an Easy nLC1000 nanoflow HPLC system to the Q Exactive benchtop mass spectrometer (both Thermo Fisher Scientific). A two-column setup was used. The pre-column (Acclaim Pep Map 100, 75 μm × 2 cm, nano Viper C18, 3 μm, Thermo Fisher Scientific), waste line and the analytical column (Pep Map RSLC, C18, 2 μm, 75 μm × 50 cm, Thermo Fisher Scientific) were interconnected using a three way tee connector. Peptides were eluted in a trilinear gradient at 50 °C at a maximum pressure of 800 bar. The aqueous solvent (solvent A) consisted of HPLC grade 0.1% formic acid in water, whereas the organic solvent was pure HPLC grade acetonitrile (solvent B) (both Sigma). The column was equilibrated with at least 10 column volumes of solvent A, followed by loading of the sample at maximum pressure of 800 bar (in solvent A). The trilinear gradient consisted of the following linear increases in solvent B: 5–25% 175 min, 25–35% 45 min, 35–60% 20 min. The layout of the gradient was provided by Nagarjuna Nagaraj (Max Planck Institute of Biochemistry, Martinsried, Germany). At the end of the gradient, the column was washed with at least 10 column volumes of 95% solvent B in order to avoid sample carryover. The analysis of BACE1−/− and wild-type samples was alternated. Online electrospray of the eluting peptides into the Q Exactive mass spectrometer was achieved with the Easy Spray ion source (Thermo Fisher Scientific). 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 × 106 and a maximum injection time of 50 ms was allowed. The 10 most intense peptide ions were chosen for fragmentation. 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. A mass window of 2.0 m/z was applied to precursor selection. Normalized collisional energy for the higher-energy collision-induced dissociation fragmentation was set to 25%. A dynamic exclusion with a time window of 40 s was applied. Singly charged molecules were not selected for fragmentation and the monoisotopic precursor selection was enabled. The underfill ratio (minimum percentage of the estimated target value at maximum fill time) was set to 0.1%. Overall, 26 LC-MS/MS files (five BACE1−/− 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). The implemented Andromeda search engine was used for matching the peak lists against a concatenated forward and reverse database including the complete UniProt-SwissProt mouse database (release 2012–08, with a total of 59345 database entries) and the standard MaxQuant contaminant database. The following settings were chosen for the MaxQuant software environment: Oxidation of methionines and N-terminal acetylation were set as variable modifications. Mass deviation was set to 20 ppm for the first and 6 ppm for the main search. The maximum number of peptide modifications was set to 5, the maximum number of missed cleavages was set to 2. Peptide and site false discovery rate (FDR) were set to 0.01. The search for co-fragmented peptides in the MS/MS spectra was enabled (“second peptides” option). Quantification was achieved using the LFQ (Label-Free Quantification) and iBAQ (intensity Based Absolute Quantification) algorithms (17.Luber C.A. Cox J. Lauterbach H. Fancke B. Selbach M. Tschopp J. Akira S. Wiegand M. Hochrein H. O'Keeffe M. Mann M. Quantitative proteomics reveals subset-specific viral recognition in dendritic cells.Immunity. 2010; 32: 279-289Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 18.Schwanhäusser B. Busse D. Li N. Dittmar G. Schuchhardt J. Wolf J. Chen W. Selbach M. Global quantification of mammalian gene expression control.Nature. 2011; 473: 337-342Crossref PubMed Scopus (4059) Google Scholar). Razor and unique peptides were used for LFQ quantification. 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. Human CSF samples were searched with the same settings against a human database including all known isoforms from UniProt (reference database, release 2014–04-14). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD001514 (19.Vizcaino J.A. Deutsch E.W. Wang R. Csordas A. Reisinger F. Rios D. Dianes J.A. Sun Z. Farrah T. Bandeira N. Binz P.A. Xenarios I. Eisenacher M. Mayer G. Gatto L. Campos A. Chalkley R.J. Kraus H.J. Albar J.P. Martinez-Bartolomé S. Apweiler R. Omenn G.S. Martens L. Jones A.R. Hermjakob H. ProteomeXchange provides globally coordinated proteomics data submission and dissemination.Nat. Biotechnol. 2014; 32: 223-226Crossref PubMed Scopus (2071) Google Scholar). Annotated MS/MS spectra are available at the UCSF MS-Viewer (20.Baker P.R. Chalkley R.J. MS-Viewer: A Web-based Spectral Viewer for Proteomics Results.Mol. Cell. Proteomics. 2014; 13: 1392-1396Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) (http://prospector2.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer) under following search key: “sxmp8fxdhm.” Statistical evaluation of the data was performed with the freely available Perseus statistics software (version 1.2.0.17) and Microsoft Excel. Common contaminants and reverse decoy matches were removed from the protein identification list. At least 2 unique peptides per protein were required for a protein identification. Only proteins that were identified and quantifiable in at least one technical of at least three biological replicates in each group were used for relative quantification. The arithmetic mean was used to obtain the average LFQ intensity within each biological group. For statistical evaluation a two-sided t test was used. The p value was corrected using false discovery rate (FDR) based multiple hypothesis testing. Both t test and FDR based multiple hypothesis testing were carried out with the default settings of the Perseus statistics software. Plasmids pcDNA3.1-CD5-SLIC-FLAG was generated by exchanging RHA-MBP (EcoRV/Not1) of pcDNA3.1-CD5-SLIC-MBP for the PCR product RHA-FLAG amplified with CMV-F and RHA FLAG Not1 rev RHA FLAG Not1 C-terminal from pcDNA3.1-CD5-SLIC-MBP. Subsequently, pcDNA3.1-CD5-HA-SLIC-FLAG was generated by exchanging CD5-LHA in between (HindIII/EcoRV) of pcDNA3.1-CD5-SLIC-FLAG for 5′UTR-CD5-HA-SLIC generated with CMV-F and CD5-HA-LHA rev from pcDNA3.1-HA-IL1R2-FLAG template (21.Kuhn P.H. Marjaux E. Imhof A. De Strooper B. Haass C. Lichtenthaler S.F. Regulated intramembrane proteolysis of the interleukin-1 receptor II by alpha-, beta-, and gamma-secretase.J. Biol. Chem. 2007; 282: 11982-11995Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). pcDNA3.1-CD5-HA-SLIC-FLAG-ENPP5, pcDNA3.1-CD5-HA-SLIC-FLAG-PTPRN2 and pcDNA3.1-HA-CD5-SLIC-FLAG-PLXDC2 were generated by cloning PCR products of murine ENPP5, PTPRN2, and PLXDC2 lacking the respective signal peptide and containing the left homology arm (LHA) and the right homology arm (RHA) into EcoRV linearized pcDNA3.1-CD5-HA-SLIC-FLAG vector via Gibson assembly. The resulting plasmids are coding for respectively PLXDC2, PTPRN2 and ENPP5 with an HA epitope tag (YPYDVPDYA) at the N terminus and a FLAG (DYKDDDDK) epitope tag at the C terminus. The generation of peak12-BACE1 and peak12-control vectors has been described (22.Lichtenthaler S.F. Dominguez D.I. Westmeyer G.G. Reiss K. Haass C. Saftig P. De Strooper B. Seed B. The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1.J. Biol. Chem. 2003; 278: 48713-48719Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Human embryonic kidney 293T cells (HEK293T) were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco, Life Technologies, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS/Gibco) and 1% Penicillin/Streptomycin (P/S). Transfections of HEK293T cells were done with Lipofectamine 2000. Selection was achieved with Zeocin (Invitrogen) at a concentration of 200 μg/ml. 2 days after transient BACE1 transfection the medium was replaced with either BACE1 inhibitor C3 (2 μm) containing selection medium or DMSO containing selection medium as a control. After 48 h of treatment, conditioned media and cell lysates (150 mm NaCl, 50 mm Tris pH 7.5, 1% Triton X-100, protease inhibitor mixture from Roche) were collected. Protein measurement assay was performed and volumes for immunoprecipitation and immunoblotting were calculated accordingly. To enrich the secreted ectodomains from the supernatants immunoprecipitation using HA.7 agarose (Sigma) was performed overnight at 4 °C. Recombinant proteins HA-PLXDC2-FLAG, HA-PTPRN2-FLAG, and HA-ENPP5-FLAG were purified from cell lysates of stably expressing HEK293T cells using anti-HA-agarose (Sigma). Proteins were eluted by adding 0.1 μg/μl HPLC purified HA peptide (Sigma). The eluted proteins were incubated with recombinant mouse BACE1 (R&D Systems, Minneapolis, MN) in 50 mm Na+ acetate buffer pH 4.4 from 4 to 16 h as described (22.Lichtenthaler S.F. Dominguez D.I. Westmeyer G.G. Reiss K. Haass C. Saftig P. De Strooper B. Seed B. The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1.J. Biol. Chem. 2003; 278: 48713-48719Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The samples were boiled in reducing Laemmli buffer and applied to Western blot analysis as described above. For the quantitative proteomic analysis of murine CSF from adult mice, we first evaluated the performance of in-solution digestion versus FASP (16.Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5043) Google Scholar, 23.Wiśniewski J.R. Ostasiewicz P. Mann M. High recovery FASP applied to the proteomic analysis of microdissected formalin fixed paraffin embedded cancer tissues retrieves known colon cancer markers.J. Proteome Res. 2011; 10: 3040-3049Crossref PubMed Scopus (225) Google Scholar). Given that the amount of blood-free CSF ranged between 10–15 μl per mouse, we used human CSF for the initial comparison of in-solution digestion and FASP. Human CSF has a similar protein concentration as murine CSF, but is available in milliliter quantities. Five μl each of a pooled human CSF sample were used in three replicates for in-solution digestion and FASP. Results were analyzed independently with MaxQuant. In-solution digestion gave slightly better results than FASP regarding the number of identified and quantified proteins (Fig. 1A). On average, 297 and 273 proteins were identified by at least two unique peptides with in-solution digestion and FASP, respectively. The number of quantified proteins in three out of three replicates was 270 for in-solution digestion and 255 for FASP. The Venn diagram (Fig. 1B) shows that FASP is rather complementary to in-solution digestion as the overlap of quantified proteins was only 61%. We also tested the reproducibility of protein quantification using the LFQ intensity values. Both methods performed equally well with an average correlation coeffi