Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons

Article22 June 2012free access Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons Peer-Hendrik Kuhn Peer-Hendrik Kuhn DZNE—German Center for Neurodegenerative Diseases, Munich, Germany Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Katarzyna Koroniak Katarzyna Koroniak Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry (IOC), Karlsruhe, Germany Search for more papers by this author Sebastian Hogl Sebastian Hogl Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Alessio Colombo Alessio Colombo DZNE—German Center for Neurodegenerative Diseases, Munich, Germany Search for more papers by this author Ulrike Zeitschel Ulrike Zeitschel Paul Flechsig Institute for Brain Research, University of Leipzig, Leipzig, Germany Search for more papers by this author Michael Willem Michael Willem Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Christiane Volbracht Christiane Volbracht Department of Molecular Neurobiology, H. Lundbeck, Valby, Denmark Search for more papers by this author Ute Schepers Ute Schepers KIT, Institute for Toxicology and Genetics (ITG), Eggenstein-Leopoldshafen, Germany Search for more papers by this author Axel Imhof Axel Imhof Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Albrecht Hoffmeister Albrecht Hoffmeister Division of Gastroenterology and Rheumatology, Department of Medicine and Dermatology, University of Leipzig, Leipzig, Germany Search for more papers by this author Christian Haass Christian Haass DZNE—German Center for Neurodegenerative Diseases, Munich, Germany Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Steffen Roßner Steffen Roßner Paul Flechsig Institute for Brain Research, University of Leipzig, Leipzig, Germany Search for more papers by this author Stefan Bräse Stefan Bräse Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry (IOC), Karlsruhe, Germany Search for more papers by this author Stefan F Lichtenthaler Corresponding Author Stefan F Lichtenthaler DZNE—German Center for Neurodegenerative Diseases, Munich, Germany Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany Technical University of Munich, Munich, Germany Search for more papers by this author Peer-Hendrik Kuhn Peer-Hendrik Kuhn DZNE—German Center for Neurodegenerative Diseases, Munich, Germany Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Katarzyna Koroniak Katarzyna Koroniak Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry (IOC), Karlsruhe, Germany Search for more papers by this author Sebastian Hogl Sebastian Hogl Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Alessio Colombo Alessio Colombo DZNE—German Center for Neurodegenerative Diseases, Munich, Germany Search for more papers by this author Ulrike Zeitschel Ulrike Zeitschel Paul Flechsig Institute for Brain Research, University of Leipzig, Leipzig, Germany Search for more papers by this author Michael Willem Michael Willem Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Christiane Volbracht Christiane Volbracht Department of Molecular Neurobiology, H. Lundbeck, Valby, Denmark Search for more papers by this author Ute Schepers Ute Schepers KIT, Institute for Toxicology and Genetics (ITG), Eggenstein-Leopoldshafen, Germany Search for more papers by this author Axel Imhof Axel Imhof Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Albrecht Hoffmeister Albrecht Hoffmeister Division of Gastroenterology and Rheumatology, Department of Medicine and Dermatology, University of Leipzig, Leipzig, Germany Search for more papers by this author Christian Haass Christian Haass DZNE—German Center for Neurodegenerative Diseases, Munich, Germany Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany Search for more papers by this author Steffen Roßner Steffen Roßner Paul Flechsig Institute for Brain Research, University of Leipzig, Leipzig, Germany Search for more papers by this author Stefan Bräse Stefan Bräse Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry (IOC), Karlsruhe, Germany Search for more papers by this author Stefan F Lichtenthaler Corresponding Author Stefan F Lichtenthaler DZNE—German Center for Neurodegenerative Diseases, Munich, Germany Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany Technical University of Munich, Munich, Germany Search for more papers by this author Author Information Peer-Hendrik Kuhn1,2, Katarzyna Koroniak3, Sebastian Hogl2, Alessio Colombo1, Ulrike Zeitschel4, Michael Willem2, Christiane Volbracht5, Ute Schepers6, Axel Imhof2, Albrecht Hoffmeister7, Christian Haass1,2, Steffen Roßner4, Stefan Bräse3 and Stefan F Lichtenthaler 1,2,8 1DZNE—German Center for Neurodegenerative Diseases, Munich, Germany 2Biochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University, Munich, Germany 3Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry (IOC), Karlsruhe, Germany 4Paul Flechsig Institute for Brain Research, University of Leipzig, Leipzig, Germany 5Department of Molecular Neurobiology, H. Lundbeck, Valby, Denmark 6KIT, Institute for Toxicology and Genetics (ITG), Eggenstein-Leopoldshafen, Germany 7Division of Gastroenterology and Rheumatology, Department of Medicine and Dermatology, University of Leipzig, Leipzig, Germany 8Technical University of Munich, Munich, Germany *Corresponding author. DZNE and Technical University Munich, Munich 80336, Germany. Tel.:+49 89 218075453; Fax:+49 89 218075415; E-mail: [email protected] The EMBO Journal (2012)31:3157-3168https://doi.org/10.1038/emboj.2012.173 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cell surface proteolysis is essential for communication between cells and results in the shedding of membrane-protein ectodomains. However, physiological substrates of the contributing proteases are largely unknown. We developed the secretome protein enrichment with click sugars (SPECS) method, which allows proteome-wide identification of shedding substrates and secreted proteins from primary cells, even in the presence of serum proteins. SPECS combines metabolic glycan labelling and click chemistry-mediated biotinylation and distinguishes between cellular and serum proteins. SPECS identified 34, mostly novel substrates of the Alzheimer protease BACE1 in primary neurons, making BACE1 a major sheddase in the nervous system. Selected BACE1 substrates—seizure-protein 6, L1, CHL1 and contactin-2—were validated in brains of BACE1 inhibitor-treated and BACE1 knock-out mice. For some substrates, BACE1 was the major sheddase, whereas for other substrates additional proteases contributed to total substrate shedding. The new substrates point to a central function of BACE1 in neurite outgrowth and synapse formation. SPECS is also suitable for quantitative secretome analyses of primary cells and may be used for the discovery of biomarkers secreted from tumour or stem cells. Introduction Proteolysis is an irreversible post-translational modification mediated by over 500 different proteases in man. Proteases control the function or mediate the degradation of virtually all proteins in the cell, but the biological functions of many proteases are unknown, because no or only few physiological substrates have been identified. This is particularly true for a large class of membrane-bound proteases, referred to as sheddases, which mostly cleave single-span membrane proteins at the extracellular surface of cellular membranes. This process is termed as ectodomain shedding and is essential for the communication between cells (Reiss and Saftig, 2009; Bai and Pfaff, 2011; Lichtenthaler et al, 2011). Shedding is involved in various physiological and pathophysiological conditions, including Alzheimer's disease. One of the sheddases is the aspartyl protease BACE1 (β-secretase), which is a key drug target for Alzheimer's disease, as it mediates the shedding of amyloid precursor protein (APP) and catalyses the first step in the generation of the pathogenic Aβ peptide (Vassar et al, 2009). Possible side effects of BACE1 inhibition in patients may result from a reduced cleavage of additional, largely unknown physiological BACE1 substrates. Besides APP, BACE1 also cleaves neuregulin-1 and contributes to myelination in the peripheral nervous system (Hu et al, 2006; Willem et al, 2006). Additionally, several new phenotypes of BACE1-deficient mice were reported recently, such as schizophrenic symptoms, increased mortality, epileptic seizures, hyperactivity, anxiety, impaired axon guidance and protection against diet-induced obesity (Harrison et al, 2003; Dominguez et al, 2005; Laird et al, 2005; Savonenko et al, 2008; Wang et al, 2008; Hu et al, 2010; Farah et al, 2011; Meakin et al, 2011; Rajapaksha et al, 2011). These phenotypes mostly affect brain and pancreas, where BACE1 expression is highest (Vassar et al, 1999), but it remains unclear which substrates are affected in these tissues. The secretome of a cell comprises soluble, secreted proteins and the membrane protein ectodomains proteolytically released by sheddases (sheddome). Proteomic identification of secretome proteins from the conditioned medium of cells is in principle possible by mass spectrometry, but has been difficult due to three fundamental limitations (Makridakis and Vlahou, 2010). First, secretome proteins have low concentrations in conditioned media. Second, the use of media supplements such as fetal calf serum or the neuronal supplement B27 introduces albumin and other serum proteins at concentrations (up to 5 g/l) much higher than the secretome proteins (Price and Brewer, 2001). Third, secretome proteins can be masked by highly abundant cytosolic proteins released from broken or apoptotic cells. Thus, the mass spectrometer used for protein identification predominantly identifies albumin, other serum proteins and cytosolic proteins, but not the cell-derived secretome proteins. To circumvent these limitations, previous studies used serum- or protein-free cell culture conditions. However, cellular stress and incompatibility with the culture of many cell types are major drawbacks of this approach, making identification and quantification of secreted proteins in primary cells, such as neurons, impossible. Additionally, many sheddases are less active in the absence of serum. As a consequence, the desired protease is frequently overexpressed or added exogenously in vitro, as carried out for example for BACE1, meprin β and MT1-MMP (Tam et al, 2004; Hemming et al, 2009; Jefferson et al, 2011). While this type of approach can demonstrate which substrates may in principle be cleaved by a protease, false positive substrate identification is a major risk of protease overexpression, for example because of mislocalization of the protease (Huse et al, 2002). Here, we developed a novel technique for quantitative proteomics of cell culture supernatants containing serum or albumin, called secretome protein enrichment with click sugars (SPECS), which solves the challenges mentioned above. SPECS distinguishes between secretome proteins and exogenous serum proteins within the conditioned medium. We used SPECS to determine the secretome of human embryonic kidney 293 (HEK293T) cells and of primary, murine neurons in the presence of serum proteins. Additionally, SPECS was used to identify novel, physiological BACE1 substrates in primary neurons. Selected BACE1 substrates—seizure-protein 6, L1, CHL1 and contactin-2—were validated in brains of BACE1 inhibitor-treated and BACE1 knock-out mice. Results Development of the SPECS method SPECS exploits the fact that the majority of secreted proteins (66%) and potential shedding substrates (87% of type I and type II transmembrane proteins) is glycosylated as annotated in Uniprot. SPECS consists of metabolic labelling of cellular glycoproteins with azido sugars followed by copper-free click chemistry-mediated biotinylation of cellular, but not of serum glycoproteins (Figure 1A). The click-chemistry reaction consists of the bioorthogonal, chemical [3+2] cycloaddition of an azide moiety with a strained cycloalkyne (Jewett and Bertozzi, 2010). We used the biotinylated, strained cycloalkyne dibenzylcyclooctyne (DBCO-PEG12-biotin) (Figure 1B) and tetraacetyl-N-azidoacetyl-mannosamine (ManNAz), which is metabolically converted to N-azidoacetyl-sialic acid and incorporated into terminal positions in N- and O-linked glycans (Sletten and Bertozzi, 2011). Due to the lack of an active transport of N-acetylamino sugars across the plasma membrane of mammalian cells, the hydroxy-groups of the sugar are peracetylated to permit passive diffusion of the sugars into the cytosol, where the acetyl groups are cleaved off by cellular esterases. Figure 1.Overview and validation of SPECS method. (A) Detailed description of the work flow of the SPECS method including a timeline. (B) Schematic representation of the click reaction between the azide group (blue) of 1,3,4,6-acetyl-N-acetyl-azido-mannosamine (ManNAz) and the strained alkyne (red) of dibenzylcycloocytne-PEG12-Biotin. (C) APP and APLP2 shedding were analysed in the presence of the BACE1 inhibitor C3 and ManNAz or DMSO as a control. Subsequently, click-chemistry reaction was performed in the conditioned medium. Full-length APP (APPfl) and APLP2 (APLP2fl) in the lysates as well as secreted APP and APLP2 (sAPPtotal, sAPPβ, sAPLP2) were detected by immunoblot. The analysis shows no significant changes of the shedding of APP and APLP2 upon addition of ManNAz. Biotinylated proteins (specific bands and broad smear) were detected with Streptavidin-HRP only when ManNAz was present. Acetylated (Ac) tubulin serves as a loading control. (D) Purification of glycoproteins by streptavidin pull down after click-chemistry reaction. Left: aliquot of conditioned medium is directly loaded (input). Right: upon streptavidin pull down, sAPPtotal is enriched by about two-fold, whereas serum albumin in the coomassie gel is reduced over 50-fold, leading to a specific enrichment of the glycosylated sAPPtotal by over 100-fold. (E) Distribution of glycoprotein types among all glycoproteins identified in the HEK293T (293T) secretome and the neuronal secretome. Only such proteins were included, which were detected by at least two peptides. Detailed listing of identified proteins and their topology is in Supplementary Tables 1, 2, 5 and 6. Download figure Download PowerPoint After ManNAz labelling the conditioned medium contained the labelled ectodomains released by shedding together with cell-derived secreted proteins (Figure 1A). Free ManNAz was removed by ultrafiltration followed by in-vitro click reaction, resulting in biotinylation of secretome proteins (Figure 1A). Subsequent ultrafiltration removed excessive biotinylated cyclooctyne. After streptavidin pull down, the protein samples were separated by SDS–PAGE, followed by in-gel trypsinization. The resulting peptides were analysed by nanoLC coupled to high-resolution mass spectrometry. Peptide identification was done by Andromeda. The SPECS method can be carried out with little hands-on time within 6 days, which includes 2 days of metabolic sugar labelling and 2 days of mass spectrometric analysis. To exclude any interference of metabolic glycan labelling with cellular physiology, we investigated in primary neurons the known shedding of the APP and its homologue, the amyloid precursor-like protein 2 (APLP2), by the protease BACE1, which is a main drug target in Alzheimer's disease (Vassar et al, 2009) and is also involved in myelination (Hu et al, 2006; Willem et al, 2006). The presence of ManNAz neither altered shedding of APP and APLP2 or its response to the specific BACE1 inhibitor C3 (Stachel et al, 2004; Figure 1C) nor did it affect neuronal viability, in agreement with a previous study (Almaraz et al, 2012). Secreted APP (sAPPtotal) was enriched by over 100-fold relative to albumin (Figure 1D). These results demonstrate that SPECS works successfully and is compatible with primary cells, such as neurons. Determination of cellular secretomes and sheddomes Next, we used SPECS to determine the secretome of neurons and of HEK293T cells, an immortalized cell line. HEK293T cells were grown in the presence of serum with or without ManNAz. In all, 254 proteins were specifically identified in the presence but not in the absence of ManNAz and constitute the secretome of HEK293T cells. The secretome comprised 142 membrane protein ectodomains, which constitute the sheddome, and 112 secreted proteins (Figure 1E; Supplementary Tables 1, 2, 3 and 4). To determine the neuronal secretome, primary E15/E16 wild-type neurons were cultured in the presence of ManNAz. Additionally, the neurons were incubated with or without the BACE1 inhibitor C3 to determine which of the secretome proteins are BACE1 substrates (next paragraph). Relative label-free protein quantification of identified unique protein groups was performed using the MaxQuant software suite. In all, 427 glycoproteins were identified in total, with 283 of them being identified in at least four out of five experiments without inhibitor. These 283 proteins comprise 97 secreted proteins and 186 shed membrane proteins (sheddome) and constitute the secretome of primary neurons (Figure 1E; Supplementary Tables 5 and 6, raw data and peptides are given in Supplementary Tables 7 and 8). While a previous study identified 34 proteins as the neuronal secretome using serum-free conditions (Thouvenot et al, 2008), SPECS identified 283 proteins with high sequence coverage, demonstrating the advantage of the SPECS method. It is possible that neurons and HEK293T cells secrete additional proteins, in particular low-abundant proteins below the detection limit of the SPECS method or proteins with a molecular mass <30 kDa, which would be lost during the filtration steps of the SPECS method. However, smaller molecular mass cut-off columns may also be used, which would allow detection of small-sized glycosylated secretome proteins. In all, 112 proteins of the neuronal secretome (47%) were shared with the HEK293T cell secretome, whereas the remaining proteins include many proteins with neuronal functions, such as neuroligins and LINGO-1 (Supplementary Table 5). Identification of neuronal BACE1 sheddome While the majority of the neuronal secretome was not affected by C3 treatment, 40 proteins showed a reduced shedding or secretion (Table I, top part). Proteins were included into this hit list, if their levels were quantifiable in at least four out of the five biological replicates and had a variance score ≤0.35 in the C3-treated samples. The 40 proteins include 34 membrane proteins (Table I), which we refer to as the neuronal BACE1 substrates (BACE1 sheddome), even though it remains possible that the shedding of some substrates may have been reduced indirectly. In all, 8 of the 34 proteins had previously been identified as candidate substrates in BACE1 overexpressing cells (Hemming et al, 2009). The 23 remaining proteins besides APP and its homologues are novel BACE1 substrates. Table 1. Changes in protein secretion and shedding upon BACE1 inhibitor C3 treatment Protein name IPIa Meanb s.e.m.c VSd Pepte Protein typef Membrane proteins showing reduced shedding upon C3 treatment Seizure protein 6-like 1 IPI00674241 0.04 0.01 1.28E−02 13 Type I Seizure protein 6 IPI00380749 0.08 0.03 3.08E−02 19 Type I Amyloid precursor-like protein 1 IPI00129249 0.11 0.02 2.32E−02 27 Type I VWFA and cache domain-containing protein 1 IPI00350425 0.12 0.12 1.31E−01 6 Type I Golgi apparatus protein 1 IPI00122399 0.21 0.05 6.69E−02 11 Type I L1 IPI00785371 0.21 0.03 3.79E−02 24 Type I Leucine-rich repeat neuronal protein 1 IPI00126070 0.25 0.06 7.69E−02 7 Type I Plexin domain-containing protein 2 IPI00471179 0.25 0.10 1.38E−01 3 Type I Neurotrimin IPI00417005 0.33 0.14 2.08E−01 6 GPI Cell adhesion molecule with homology to L1CAM IPI00831546 0.35 0.05 7.25E−02 49 Type I Peptidyl-glycine α-amidating monooxygenase IPI00323974 0.36 0.12 1.83E−01 23 Type I Alpha-1,4-N-acetylhexosaminyltransferase EXTL2 IPI00112900 0.36 0.16 2.47E−01 5 Type II Protocadherin γ A11 IPI00129686 0.42 0.15 2.56E−01 7 Type I Amyloid precursor-like Protein 2 IPI00121338 0.43 0.09 1.57E−01 20 Type I ST3GAL-I sialyltransferase IPI00108849 0.44 0.17 2.98E−01 3 Type II Latrophilin-1 IPI00918724 0.45 0.10 1.79E−01 19 Polytopic Neuroligin 4 IPI00858277 0.45 0.08 1.38E−01 22 Type I Semaphorin-6D IPI00396759 0.47 0.09 1.70E−01 3 Type I Lysosomal membrane glycoprotein 1 IPI00469218 0.48 0.16 3.14E−01 2 Type I Neurexin I-α IPI00468539 0.51 0.04 7.94E−02 30 Type I Protocadherin-20 IPI00222278 0.52 0.16 3.22E−01 10 Type I Latrophilin-3 IPI00411157 0.53 0.10 2.09E−01 7 Polytopic Latrophilin-2 IPI00876558 0.56 0.10 2.17E−01 10 Polytopic Sodium/potassium-dependent ATPase subunit β-1 IPI00121550 0.57 0.14 3.33E−01 3 Type II Delta and Notch-like epidermal growth factor-related receptor IPI00170342 0.57 0.09 2.19E−01 3 Type I Interferon α/β receptor 2 IPI00395209 0.58 0.12 2.84E−01 4 Type I Neuroligin-2 IPI00468605 0.58 0.11 2.55E−01 16 Type I Seizure 6-like protein 2 IPI00128454 0.60 0.14 3.41E−01 13 Type I Leucine-rich repeat and fibronectin type-III domain-containing protein 2 IPI00330152 0.62 0.13 3.37E−01 4 Type I CX3C membrane-anchored chemokine IPI00127811 0.64 0.11 2.97E−01 4 Type I Contactin-2 IPI00119970 0.64 0.06 1.81E−01 39 GPI Amyloid precursor protein IPI00114389 0.67 0.08 2.44E−01 30 Type I Neuroligin-1 IPI00309113 0.73 0.08 2.86E−01 13 Type I Transmembrane protein 132A IPI00464151 0.82 0.06 3.45E−01 38 Type I Soluble proteins reduced upon C3 treatment Activin β-B chain IPI00355134 0.14 0.08 8.87E−02 7 Secreted Adamts3 IPI00672899 0.30 0.18 2.53E−01 7 Secreted Insulin-like growth factor-binding protein 2 IPI00313327 0.41 0.12 2.10E−01 2 Secreted Extracellular matrix protein 1 IPI00889948 0.49 0.13 2.63E−01 5 Secreted Neuronal olfactomedin-related ER localized protein IPI00136712 0.50 0.12 2.28E−01 10 Secreted Reelin IPI00121421 0.85 0.04 2.87E−01 21 Secreted Selection of proteins unaltered upon C3 treatment Hepatocyte growth factor receptor IPI00130420 0.83 0.13 7.60E−01 8 Type I Neogenin IPI00129159 0.90 0.09 8.56E−01 33 Type I Protocadherin-γ C3 IPI00129613 0.81 0.13 6.86E−01 12 Type I Receptor-type tyrosine-protein phosphatase sigma IPI00230067 0.90 0.19 1.90E+00 20 Type I Leucine-rich repeat-containing protein 4B IPI00381059 0.91 0.18 2.13E+00 20 Type I Netrin receptor DCC IPI00137347 1.00 0.18 1.38E+02 31 Type I Prostaglandin F2 receptor-negative regulator IPI00515319 0.85 0.15 1.04E+00 29 Type I Neural cell adhesion molecule 1 IPI00122971 0.99 0.16 1.12E+01 28 Type I a IPI accession number of the protein. b Mean ratio between BACE1 inhibitor treatment (C3) and control (DMSO) conditions of the summed unique peptides intensities identified for a unique protein group for five biological replicates (C3/DMSO) shows remaining ectodomain levels upon BACE1 inhibition. SPECS values for remaining shedding of APP (0.67=67%), APLP1 (0.11=11%) and APLP2 (0.42=42%) correspond well to the literature. In neurons, APLP1 is mainly cleaved by BACE1 (Sala Frigerio et al, 2010), whereas APLP2 shedding is mediated to about 60% by BACE1 (Hogl et al, 2011). In contrast, total APP shedding was only mildly inhibited with C3, because it is known that inhibition of BACE1 cleavage of APP is accompanied by an increase in the ADAM10-mediated cleavage, resulting in only a moderate inhibition of total APP shedding upon BACE1 inhibition (Vassar et al, 1999; May et al, 2011). c Standard error of the mean for five biological replicates. d Variance score was calculated for all proteins. Proteins with a variance score of ≤0.35 were considered as proteins with a consistent change under BACE1 inhibition. e Number of identified peptides of the protein group. f Protein type: Secreted: Secreted, soluble protein, Type I: type I membrane protein, Type II: type II membrane protein, Polytopic: membrane protein with multiple transmembrane domains, GPI: GPI-anchored membrane protein. The protein list of BACE1 substrates includes APP and its homologues APLP1 and APLP2 (Table I), which are all three known BACE1 substrates and validate the SPECS analysis. Importantly, the extent to which the shedding of APP and its homologues was reduced (Table I) corresponds well to previous results obtained by quantitative immunoblots in neurons or brain (Vassar et al, 1999; Sala Frigerio et al, 2010; Hogl et al, 2011; May et al, 2011). For example, total APP shedding was only mildly inhibited with C3, because inhibition of BACE1 cleavage of APP has been shown to be accompanied by an increase in the ADAM10-mediated cleavage, resulting in only a moderate inhibition of total APP shedding upon BACE1 inhibition (Vassar et al, 1999; May et al, 2011). These results demonstrate that SPECS is well suited for the quantitative analysis of protein shedding. For other proteins on the BACE1 sheddome list shedding was reduced by about 20% (transmembrane protein 132A) to over 95% (seizure 6-like protein) (Table I, top part), suggesting that for some substrates (transmembrane protein 132A) BACE1 only contributes to a small extent to total shedding, whereas other substrates are nearly exclusively cleaved by BACE1 in neurons. Peptides identified from individual membrane proteins were exclusively derived from their ectodomains (Supplementary Figure 1A, shown in yellow), indicating that they derive from true ectodomain shedding and not from full-length proteins released by broken cells. Additionally, for three BACE1 substrates (APP, APLP1, seizure 6-like protein 1 (SEZ6L1)) semi-tryptic peptides were identified (Supplementary Figure 1A, shown in green), which were reduced after BACE1 inhibition (Supplementary Figure 1B), suggesting that they derive from direct BACE1 cleavage. Indeed, the peptide from APP corresponds exactly to the known BACE1 cleavage site (Supplementary Figure 1B) and demonstrates that SPECS allows cleavage site determination. Six of the forty proteins were soluble proteins and showed an inhibition of secretion by about 20–90% (Table I, middle part), including the TGFβ superfamily member activin β and insulin-like growth factor-binding protein 2. Because these are known soluble proteins, their reduced secretion is likely to be a secondary consequence of BACE1 inhibition. However, they may be useful as diagnostic markers to monitor the efficacy of BACE1 inhibitors in Alzheimer's patients. Validation of BACE1 substrates in primary neurons To further validate the SPECS data, the shedding inhibition by C3 was analysed in the absence of ManNAz and quantified by immunoblot for four novel BACE1 substrates besides APP, APLP1 and APLP2. For most of the novel substrates, no antibodies are available which allow detection of the endogenous, shed ectodomain by immunoblot. We chose four proteins, where suitable antibodies were available and which showed mild, moderate and strong shedding inhibition by C3 in the SPECS measurement (Table I, top part). The quantitative comparison of SPECS and immunoblots yielded the following shedding inhibition upon C3 treatment: 12.3/7.8% (SPECS/immunoblot) for seizure protein 6 (SEZ6) (Figure 2A and quantification in Figure 2B); 23/21% for cell adhesion protein L1; 49/35% for close homologue of L1 (CHL1); 56/64% for contactin-2, a GPI-anchored protein. The values obtained by immunoblots were similar to the SPECS measurements and demonstrate the quantitative accuracy of SPECS. The reduced shedding was accompanied by increased levels of the full-length proteins in the cell lysate (Figure 2A and quantification in Figure 2B), showing that C3 reduced substrate shedding and not simply substrate expression. Interestingly, a strong reduction in shedding was not always accompanied by a strong increase in full-length protein levels in the lysate, suggesting that additional cellular mechanisms besides shedding control the full-length protein levels. Figure 2.Validation of BACE1 substrates in primary neurons. (A) Primary neurons were treated with DMSO or with the BACE1 inhibitor C3 overnight. Sez6, CHL1, L1 and contactin-2 showed reduced shedding into the cell supernatant (Sup) and accumulation of their membrane-tethered precursors in the lysate (Lys) after treatment with C3. The specific BACE1 cleavage produc