To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation - an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Müller cell-specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Müller cell genes by quantitative real time PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Müller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Müller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Müller glia specific proteins, which were validated as markers and for their functional impact in glial physiology. This provides the basis to allow the discovery of novel glial specializations and will enable us to elucidate the role of Müller cells in retinal pathologies — a topic still controversially discussed. To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation - an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Müller cell-specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Müller cell genes by quantitative real time PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Müller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Müller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Müller glia specific proteins, which were validated as markers and for their functional impact in glial physiology. This provides the basis to allow the discovery of novel glial specializations and will enable us to elucidate the role of Müller cells in retinal pathologies — a topic still controversially discussed. For many years, research on retinal diseases mainly concentrated on investigations of functional deficits of retinal neurons. Müller cells, the dominant macroglia cells of the retina, were considered passive bystanders. However, owing to their distinct morphology spanning the whole thickness of the retina and being in contact with virtually all retinal cell types enables them to fulfil a plethora of functions which are absolutely essential for neuronal well-being. Experimental deletion of Müller cells results in disorganization of retinal layers, photoreceptor degeneration, and malformation of the retinal vasculature (1.Shen W. Fruttiger M. Zhu L. Chung S.H. Barnett N.L. Kirk J.K. Lee S. Coorey N.J. Killingsworth M. Sherman L.S. Gillies M.C. Conditional Mullercell ablation causes independent neuronal and vascular pathologies in a novel transgenic model.J. Neurosci. 2012; 32: 15715-15727Crossref PubMed Scopus (171) Google Scholar). Moreover, recent studies on Müller cells in the pathologically altered retina clearly indicate that gene expression changes and functiol constraints in Müller cells, because of their response to tissue damage, are very likely to affect neuronal survival in the diseased retina (2.Pannicke T. Frommherz I. Biedermann B. Wagner L. Sauer K. Ulbricht E. Hartig W. Krugel U. Ueberham U. Arendt T. Illes P. Bringmann A. Reichenbach A. Grosche A. Differential effects of P2Y1 deletion on glial activation and survival of photoreceptors and amacrine cells in the ischemic mouse retina.Cell Death Disease. 2014; 5: e1353Crossref PubMed Scopus (19) Google Scholar, 3.Pannicke T. Iandiev I. Uckermann O. Biedermann B. Kutzera F. Wiedemann P. Wolburg H. Reichenbach A. Bringmann A. A potassium channel-linked mechanism of glial cell swelling in the postischemic retina.Mol. Cell. Neurosci. 2004; 26: 493-502Crossref PubMed Scopus (196) Google Scholar, 4.Wurm A. Pannicke T. Iandiev I. Wiedemann P. Reichenbach A. Bringmann A. The developmental expression of K+ channels in retinal glial cells is associated with a decrease of osmotic cell swelling.Glia. 2006; 54: 411-423Crossref PubMed Scopus (43) Google Scholar). However, strikingly little is known about the mechanisms and modulatory factors of this Müller cell reaction termed Müller cell gliosis. Additionally, there is an ongoing discussion whether Müller cell gliosis has primarily detrimental or also beneficial effects on retinal neurons (5.Bringmann A. Iandiev I. Pannicke T. Wurm A. Hollborn M. Wiedemann P. Osborne N.N. Reichenbach A. Cellular signaling and factors involved in Muller cell gliosis: neuroprotective and detrimental effects.Prog. Retin. Eye Re.s. 2009; 28: 423-451Crossref PubMed Scopus (487) Google Scholar, 6.Bringmann A. Pannicke T. Grosche J. Francke M. Wiedemann P. Skatchkov S.N. Osborne N.N. Reichenbach A. Muller cells in the healthy and diseased retina.Prog. Retin. Eye Res. 2006; 25: 397-424Crossref PubMed Scopus (1261) Google Scholar, 7.Hauck S.M. von Toerne C. Ueffing M. The neuroprotective potential of retinal Muller glial cells.Adv. Exp. Med. Biol. 2014; 801: 381-387Crossref PubMed Scopus (12) Google Scholar). To answer these questions, there is an urgent need of in-depth, comprehensive characterization of Müller cell protein expression to better understand how they intimately interact with retinal neurons, microglia, and retinal vasculature. Modern techniques for determining expression profiles from biological samples have evolved into powerful, highly sensitive, quantitative tools that are extensively applied to generate huge sets of data. These techniques include proteomic methods such as mass spectrometry with ever-increasing sensitivity to analyze protein expression, translating gene expression into the effector level. Combined with a cell fractionation sample preparation approach, information about subcellular localization of proteins can be gained, enabling a better understanding of the underlying mechanisms. Comprehensive proteomic data have been previously collected from whole retinal tissue samples (8.Ethen C.M. Reilly C. Feng X. Olsen T.W. Ferrington D.A. The proteome of central and peripheral retina with progression of age-related macular degeneration.Invest. Ophthalmol. Vis. Sci. 2006; 47: 2280-2290Crossref PubMed Scopus (93) Google Scholar, 9.Kim S.J. Jin J. Kim Y.J. Kim Y. Yu H.G. Retinal proteome analysis in a mouse model of oxygen-induced retinopathy.J. Proteome Res. 2012; 11: 5186-5203Crossref PubMed Scopus (22) Google Scholar, 10.Ly A. Scheerer M.F. Zukunft S. Muschet C. Merl J. Adamski J. de Angelis M.H. Neschen S. Hauck S.M. Ueffing M. Retinal proteome alterations in a mouse model of type 2 diabetes.Diabetologia. 2014; 57: 192-203Crossref PubMed Scopus (25) Google Scholar, 11.Zhang P. Dufresne C. Turner R. Ferri S. Venkatraman V. Karani R. Lutty G.A. Van Eyk J.E. Semba R.D. The proteome of human retina.Proteomics. 2015; 15: 836-840Crossref PubMed Scopus (27) Google Scholar), however, major limitations with respect to assigning altered protein expression levels to functional changes at cellular resolution remain. The retina comprises multiple highly specialized cell types, with neurons largely outnumbering Müller cells which make up only 1.5% of the cell population of the murine retina (12.Jeon C.J. Strettoi E. Masland R.H. The major cell populations of the mouse retina.J. Neurosci. 1998; 18: 8936-8946Crossref PubMed Google Scholar). To identify expression of Müller cell proteins, it is therefore inevitable and logical to reconsider current approaches and to switch from whole tissue expression analysis to (Müller) cell type-specific data generation. To date, only very few studies have performed cell type-specific mRNA expression analysis of Müller cells. Enrichment of Müller cells from the adult retinal tissue is highly challenging because of their intricate and fragile morphology and huge cell size. Picking single Müller cells from dissociated murine retinal tissue under the microscope, Roesch et al. (13.Roesch K. Jadhav A.P. Trimarchi J.M. Stadler M.B. Roska B. Sun B.B. Cepko C.L. The transcriptome of retinal Muller glial cells.J. Comp. Neurol. 2008; 509: 225-238Crossref PubMed Scopus (292) Google Scholar) performed single-cell microarrays analyses using very limited numbers of cells (2–5 cells per cell type). Another study reported microarray data from murine Müller glia that were enriched by fluorescence-activated cell sorting (FACS) 1The abbreviations used are:FACSfluorescence-activated cell sorting2Dtwo-dimensionalABCammoniumbicarbonateACNacetonitrileANOVAanalysis of varianceAnxaAnnexinCM-H2DCFDA5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester, general oxidative stress indicatorCRALBPglutamine synthetase and cellular retinaldehyde binding proteinDMSOdimethyl sulfoxideDAPI4′,6-diamidino-2-phenylindoleECSextracellular solutionGABAglutamate and γ-amino butyric acidGdmClguanidinium chlorideGePSGenomatix Pathway SytemGLASTglutamate/aspartate transporterGlulglutamine synthetase (mRNA)GSglutamine synthetase (protein)GSTM5glutathione S-transferase mu 5GSTT1glutathione S-transferase T1FASPfilter aided sample preparationFDRfalse discovery rateFOLH1folate hydrolase 1Iba1ionized calcium binding adapter molecule 1iSTin-StageTip protein sample preparationItgb1integrin beta-1LC-MSMShigh resolution mass spectrometry coupled to liquid chromatographyMACSmagnetic-activated cell sortingmgfMascot generic filesOPLouter plexiform layerPBSphosphate-buffered salinePKCαprotein kinase C alphaPon2paraoxonase 2PrdxperoxiredoxinPVDFpolyvinylidenfluorideqRT-PCRquantitative real time polymerase chain reactionROSreactive oxygen speciesRPEretinal pigment epitheliumSEMstandard error of the meanTBStris-buffered salineTCEPtris(2-carboxyethyl)phosphineTFAtrifluoroacetic acid. (14.Xue W. Cojocaru R.I. Dudley V.J. Brooks M. Swaroop A. Sarthy V.P. Ciliary neurotrophic factor induces genes associated with inflammation and gliosis in the retina: a gene profiling study of flow-sorted, Muller cells.PLoS ONE. 2011; 6: e20326Crossref PubMed Scopus (44) Google Scholar). However, because FACS sorting severely distorts cell morphology by tearing apart cell processes leaving rounded Müller cells, this enrichment method is not well suited for proteomic studies because it likely alters detectable levels of proteins, especially proteins that allocate specifically to fragile cellular processes. fluorescence-activated cell sorting two-dimensional ammoniumbicarbonate acetonitrile analysis of variance Annexin 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester, general oxidative stress indicator glutamine synthetase and cellular retinaldehyde binding protein dimethyl sulfoxide 4′,6-diamidino-2-phenylindole extracellular solution glutamate and γ-amino butyric acid guanidinium chloride Genomatix Pathway Sytem glutamate/aspartate transporter glutamine synthetase (mRNA) glutamine synthetase (protein) glutathione S-transferase mu 5 glutathione S-transferase T1 filter aided sample preparation false discovery rate folate hydrolase 1 ionized calcium binding adapter molecule 1 in-StageTip protein sample preparation integrin beta-1 high resolution mass spectrometry coupled to liquid chromatography magnetic-activated cell sorting Mascot generic files outer plexiform layer phosphate-buffered saline protein kinase C alpha paraoxonase 2 peroxiredoxin polyvinylidenfluoride quantitative real time polymerase chain reaction reactive oxygen species retinal pigment epithelium standard error of the mean tris-buffered saline tris(2-carboxyethyl)phosphine trifluoroacetic acid. Protein expression data from Müller cells are so far restricted to studies of cultured cells (15.Hauck S.M. Suppmann S. Ueffing M. Proteomic profiling of primary retinal Muller glia cells reveals a shift in expression patterns upon adaptation to in vitro conditions.Glia. 2003; 44: 251-263Crossref PubMed Scopus (92) Google Scholar, 16.Merl J. Ueffing M. Hauck S.M. von Toerne C. Direct comparison of MS-based label-free and SILAC quantitative proteome profiling strategies in primary retinal Muller cells.Proteomics. 2012; 12: 1902-1911Crossref PubMed Scopus (100) Google Scholar). Unfortunately, taking Müller glia into cell selective culture leads to their rapid dedifferentiation. A price for obtaining relatively pure Müller cells at high numbers, the cells gain stem cell-like characteristics after a few days in culture (15.Hauck S.M. Suppmann S. Ueffing M. Proteomic profiling of primary retinal Muller glia cells reveals a shift in expression patterns upon adaptation to in vitro conditions.Glia. 2003; 44: 251-263Crossref PubMed Scopus (92) Google Scholar). Thus, expression profiles from cultured Müller glia do not adequately mirror those of Müller cells in native tissue. Accordingly, comprehensive data on protein expression from differentiated native Müller glia are missing completely to date. We set out to first establish and validate a novel rapid method of isolating and enriching adult Müller cells from intact tissue, which yields sufficient cell numbers for proteomic profiling while keeping cells viable and fully intact morphologically as well as physiologically. From these preparations we collected a comprehensive data set of the proteome of native adult murine Müller cells. Because many Müller cell functions depend on cell-cell interactions occurring at the plasma membrane level, we additionally included a subcellular fractionation approach that enabled discrimination of proteins primarily residing in the cytosol or the nucleus from those allocated in the plasma membrane and thus generate for the first time a Müller cell surfaceome study. We consider these data a first important step to better understanding the complex crosstalk between various retinal cell types and Müller cells as key players in health and ultimately also under disease conditions. All experiments were done in accordance with the European Communities Council Directive 86/609/EEC, and were approved by the local authorities. Animals were maintained with free access to water and food in an air-conditioned room on a 12-hour light-dark cycle. Adult (2–4 months old) C57Bl6/J mice were used for isolation of native Müller cells. Isolated retinae were incubated with papain (0.2 mg/ml, Roche, Mannheim, Germany) in Ca2+-/Mg2+-free phosphate-buffered saline containing 11 mm glucose, pH 7.4, for 30 min at 37 °C, followed by several washing steps with saline. After short incubation in saline supplemented with DNase I (200 U/ml), the tissue was triturated in extracellular solution (ECS, that contained (mm) 135 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 1 Na2HPO4, 10 HEPES, and 11 glucose, adjusted to pH 7.4 with Tris) to obtain isolated retinal cells. After centrifugation, the supernatant was removed and the cells were resuspended and incubated in ECS containing biotinylated hamster anti-CD29 (clone Ha2/5, BD Biosciences, Heidelberg, Germany) for 15 min at 4 °C. After washes in ECS and centrifugation, cells were taken up in ECS containing anti-biotin MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated for 10 min at 4 °C. After an additional washing step in ECS, cell populations were separated using MACS® cell separation large cell columns (Miltenyi Biotec) according to the manufacturer's recommendation. If microglia cells were isolated in addition to Müller cells, the retinal suspension was incubated with CD11b-microbeads (Miltenyi Biotec) for 15 min at 4 °C and positively selected using MACS® cell separation large cell columns (Miltenyi Biotec) before Müller cells were surface-labeled for MACS sorting. 10 μl from both samples (positive and negative fraction) were transferred to a counting chamber (Labor Optik, Friedrichsdorf, Germany) to assess information about obtained cell numbers using a standard protocol. Another 50 μl of each sample were fixed in 4% paraformaldehyde, washed in buffered saline, and then cell nuclei and Müller cells (using labeling against glutamine synthetase and CRALBP as marker protein) were fluorescently labeled on slide. We assessed the ratio of identified Müller cells (based on glutamine synthetase labeling and the intricate morphology of the cells) and total counts of cell nuclei to determine the purity of the respective cell subpopulations. Remaining sample volumes were centrifuged and the cell pellet was prepared either for proteomic analysis or RNA extraction. The Müller cell enriched-samples contained 500,000 and 800,000 cells (representing two biological replicates) obtained by pooling isolated cell subpopulations from four retinae derived from two animals per replicate and <1 500,000 cells in the neuronal fraction, respectively. Cells were separated into cell surface fraction, nuclear fraction and a crude "cytosolic" fraction as follows. To achieve a specific biotinylation of sialylated surface proteins, a periodate oxidation and aniline-catalyzed oxime ligation-based cell surface labeling procedure (17.Zeng Y. Ramya T.N. Dirksen A. Dawson P.E. Paulson J.C. High-efficiency labeling of sialylated glycoproteins on living cells.Nat. Methods. 2009; 6: 207-209Crossref PubMed Scopus (307) Google Scholar) was performed on vital cells with cell membranes still being intact. The experiment was performed as described (18.Uhl P.B. Szober C.M. Amann B. Alge-Priglinger C. Ueffing M. Hauck S.M. Deeg C.A. In situ cell surface proteomics reveals differentially expressed membrane proteins in retinal pigment epithelial cells during autoimmune uveitis.J. Proteomics. 2014; 109: 50-62Crossref PubMed Scopus (15) Google Scholar, 19.Graessel A. Hauck S.M. von Toerne C. Kloppmann E. Goldberg T. Koppensteiner H. Schindler M. Knapp B. Krause L. Dietz K. Schmidt-Weber C.B. Suttner K. A combined omics approach to generate the surface atlas of human naive CD4+ T cells during early TCR activation.Mol. Cell. Proteomics. 2015; 14: 2085-2102Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Briefly, after MACS sorting, cells were pelleted by centrifugation and resuspended in phosphate-buffered saline (PBS), pH 6.7, containing 11 mm glucose before the cells were transferred to the labeling solution (11 mm glucose, 500 μm aminooxy-biotin, 1 mm NaIO4, 0.94 mg/ml aniline, in PBS, pH 6.7) and incubated under constant rotation for 30 min at 4 °C. The reaction was stopped by adding glycerol to a final concentration of 1 mm and after additional washes in TBS (30 mm Tris, 150 mm NaCl) the cells were pelleted by centrifugation and resuspended in low-salt lysis buffer (10 mm Tris/HCl pH 7.6, 10 mm NaCl, 1% Nonidet P-40, 1× protease inhibitors) in order to keep the nuclei intact. After careful sonication, the nuclei were separated by centrifugation at 6000 × g for 10 min at 4 °C, briefly washed with 10 mm Tris/HCl pH 7.6/10 mm NaCl and stepwise resuspended in high-salt buffer (10 mm Tris/HCl pH 7.6, 500 mm NaCl) without or with 1% Triton X-100 and total protein content measured by a Bradford assay (Bio-Rad, Munich, Germany) following the manufacturer's instructions. Each 10 μg of extracted nuclear proteins were subjected to tryptic digest applying a modified FASP procedure (20.Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5042) Google Scholar) as follows. For protein reduction, 1 μl of 1 m DTT was added to the samples and incubated for 30 min at 60 °C. After cooling the samples to room temperature, the samples were diluted with UA buffer (8 m urea in 0.1 m Tris/HCl pH 8.5) and 10 μl of freshly prepared 300 mm iodacetamide solution were added for 30 min at room temperature in the dark. Samples were centrifuged through a 30 kDa cut-off filter device (PALL) and washed thrice with UA buffer and twice with 50 mm ammoniumbicarbonate (ABC). Proteins were digested in 40 μl of 50 mm ABC for 2 h at room temperature using 1 μg Lys-C (Wako Chemicals, Neuss, Germany) and for 16h at 37 °C using 2 μg trypsin (Sigma Aldrich, Taufkirchen, Germany). Peptides were collected by centrifugation and filters were washed with 20 μl 50 mm ABC/2% ACN. Samples were acidified with 0.5% trifluoroacetic acid (TFA) prior to mass spectrometric analysis. The supernatant of the centrifuged cell lysate containing cytoplasmic and surface proteins was diluted fivefold with TBS and biotinylated proteins were extracted by binding to 80 μl (50% bead slurry) equilibrated strep-tactin superflow beads (suspension beads, IBA, Göttingen, Germany) at 4 °C for 2h. The flow-through containing the crude cytosolic fraction was measured for total protein content using a Bradford assay, and 10 μg were proteolysed applying a modified FASP procedure similar to the nuclear fraction (see above). The beads with bound surface proteins were washed with TBS/0.2% Nonidet P-40 pH 7.4 and 0.5%SDS/TBS pH 7.4. Beads were incubated with 0.5%SDS/100 mm DTT/TBS pH 7.4 for 30 min at room temperature and washed with UC buffer (6 m urea/100 mm Tris-HCl, pH 8.5). Beads were incubated with UC buffer containing 50 mm iodoacetamide for 30 min at room temperature and then washed sequentially with UC buffer, 5 m NaCl, 100 mm Na2CO3 (pH 11.5), 50 mm Tris-HCl pH 8.5. Bound proteins were subjected to tryptic digest directly on the affinity matrix in 50 mm Tris-HCl pH 8.5 with 1 μg trypsin for 16h at 37 °C. Tryptic peptides were collected by centrifugation. Residual peptides were eluted with an additional elution in 50 mm Tris-HCl pH 8.5 and pooled with the first eluate. Beads were washed with 50 mm sodium phosphate pH 7.5 and glycopeptides were released using 500 Units PNGaseF (New England Biolabs) in 50 mm sodium phosphate pH 7.5 for 6 h at 37 °C. Glycopeptides were collected by centrifugation and residual peptides were eluted with 50 mm sodium phosphate pH 7.5. All eluates were pooled, acidified with TFA and analyzed on the OrbitrapXL. There was an input of ∼200,000 cells from the Müller cell-enriched and 500,000 cells from the Müller cell-depleted fraction obtained by pooling cell subpopulations from two retinae derived from one animal per replicate; three biological replicates were analyzed. Cells were processed for LC-MS/MS with the in-StageTip (iST) method as described (21.Kulak N.A. Pichler G. Paron I. Nagaraj N. Mann M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.Nat. Methods. 2014; 11: 319-324Crossref PubMed Scopus (990) Google Scholar) with slight adaptations. Briefly, cells were lysed in lysis buffer (6 m GdmCl, 10 mm TCEP, 40 mm chloroacetamide, 100 mm Tris pH 8.5), boiled for 8 min at 95 °C and subsequently sonicated for 5 × 1 min using a waterbath sonicator (Elma Transsonic T310-H). Cell lysates were diluted 1:10 with dilution buffer (10% (v/v) ACN, 25 mm Tris pH 8.5) containing 1 μg Trypsin and Lys-C each and digested overnight at 37 °C. After digestion peptides were acidified to an end-concentration of 1% TFA. Prior to peptide loading to the StageTip, activation of material (14-gauge plug SDB-RPS, EmporeTM, 3M Bioanalytical Technologies, Neuss, Germany) was performed by successively applying 50 μl acetone, isopropanol, methanol and 0.2% TFA (v/v) followed by centrifugation in between (1 min, 1000 × g). Acidified peptides were transferred to the StageTip and loaded onto activated SDB-RPS material by centrifugation for 1–2 min for up to 1500 × g. The loading procedure was repeated three times. Then the loaded StageTip was washed three times using 100 μl 0.2% TFA (v/v). Peptide elution was performed in four fractions by successive application of 60 μl elution buffers 1–4 (supplemental Table S7) and centrifugation for 1–2 min for up to 1500 × g. Eluates were collected and evaporated using a SpeedVac centrifuge. Peptides were resuspended in 50 μl loading buffer (2% ACN, 0.5% TFA), briefly sonicated and used for LC-MS/MS analysis. LC-MS/MS analysis was performed as described previously (22.Merl J. Deeg C.A. Swadzba M.E. Ueffing M. Hauck S.M. Identification of autoantigens in body fluids by combining pull-downs and organic precipitations of intact immune complexes with quantitative label-free mass spectrometry.J. Proteome Res. 2013; 12: 5656-5665Crossref PubMed Scopus (14) Google Scholar, 23.Hauck S.M. Dietter J. Kramer R.L. Hofmaier F. Zipplies J.K. Amann B. Feuchtinger A. Deeg C.A. Ueffing M. Deciphering membrane-associated molecular processes in target tissue of autoimmune uveitis by label-free quantitative mass spectrometry.Mol. Cell. Proteomics. 2010; 9: 2292-2305Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) on an LTQ OrbitrapXL (Thermo Fisher Scientific Inc., Waltham, MA). Approximately 0.5 μg per sample were automatically loaded to the HPLC system. A nano trap column was used (300 μm inner diameter × 5 mm, packed with Acclaim PepMap100 C18. 5 μm, 100 Å; LC Packings, Sunnyvale, CA) before separation by reversed phase chromatography (PepMap, 25 cm, 75 μm ID, 2 μm/100 Å pore size, LC Packings) operated on a RSLC (Ultimate 3000, Dionex, Sunnyvale, CA). Peptides were eluted with the following gradient of increasing ACN concentrations in 0.1% formic acid over 170 min: 135 min of 6% to 31% followed by 10 min from 31% to 72% ACN. Between each gradient the ACN in 0.1% FA concentration was set back to starting conditions for 20 min. From the high resolution MS prescan, the 10 most abundant peptide ions were selected for fragmentation in the linear ion trap if they were at least doubly charged and if they exceeded an intensity of at least 200 counts, with a dynamic exclusion of 60 s. During fragment analysis, a high-resolution (60,000 full width at half-maximum) MS spectrum was acquired with a mass range from 300 to 1500 Da. The acquired spectra of the different samples were loaded and analyzed using Progenesis QI software for proteomics (Version 2.0, Nonlinear Dynamics, Waters, Newcastle upon Tyne, U.K.) for label-free quantification as previously described (23.Hauck S.M. Dietter J. Kramer R.L. Hofmaier F. Zipplies J.K. Amann B. Feuchtinger A. Deeg C.A. Ueffing M. Deciphering membrane-associated molecular processes in target tissue of autoimmune uveitis by label-free quantitative mass spectrometry.Mol. Cell. Proteomics. 2010; 9: 2292-2305Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The profile data of the MS scans were transformed into peak lists with respective m/z values, intensities, abundances, and m/z width. MS/MS spectra were transformed similarly and then stored in peak lists comprising m/z and abundance. Using one sample as reference, the retention times of the other samples were aligned by automatic alignment to a maximal overlay of the 2D features. Features with one or more than seven charges were masked at this point and excluded from further analyses. After alignment and feature exclusion, samples were allocated to their respective experimental groups (Müller cell-enriched versus -depleted fractions), and raw abundances of all features were normalized. Normalization corrects for factors resulting from experimental variation and was automatically calculated over all features in all samples to correct for technical variation. All MS/MS spectra were exported from the Progenesis QI software as Mascot generic files (mgf) and used for peptide identification with Mascot (version 2.5) using the Ensembl Mouse protein database (mus musculus; release 75, containing 51772 sequences). Search parameters used were 10 ppm peptide mass tolerance, 0.6 Da f
The delivery of HIV-1 envelope (Env) trimer-based immunogens on the surface of nanoparticles holds promise to promote immunogenicity with the aim of inducing a potent, durable and broad neutralizing antibody (bnAb) response. Towards that goal, we examined the covalent conjugation of Env to 100 nm and 200 nm silica nanoparticles (SiNPs) to optimize conjugation density and attachment stability. Env was redesigned to enable site-specific cysteine-mediated covalent conjugation while maintaining its structural integrity and antigenicity. Env was anchored to different sized SiNPs with a calculated spacing of 15 nm between adjacent trimers. Both particle sizes exhibited high in vitro stability over a seven-day period. After attachment, 100 nm particles showed better colloidal stability compared to 200 nm particles. Importantly, the antigenic profile of Env was not impaired by surface attachment, indicating that the quaternary structure was maintained. In vitro Env uptake by dendritic cells was significantly enhanced when Env was delivered on the surface of nanoparticles compared to soluble Env. Furthermore, multivalent Env displayed efficiently activated B cells even at Env concentrations in the low nanomolar range. In mice, antibody responses to nanoparticle-coupled Env were stronger compared to the free protein and had equivalent effects at lower doses and without adjuvant.
Stabilization of the HIV-1 Envelope glycoprotein trimer (Env) in its native pre-fusion closed conformation is regarded as one of several requirements for the induction of neutralizing antibody (nAb) responses, which, in turn, will most likely be a prerequisite for the development of an efficacious preventive vaccine. Here, we systematically analyzed how the stepwise stabilization of a clade C consensus (ConC) Env immunogen impacts biochemical and biophysical protein traits such as antigenicity, thermal stability, structural integrity, and particle size distribution. The increasing degree of conformational rigidification positively correlates with favorable protein characteristics, leading to optimized homogeneity of the protein preparations, increased thermal stability, and an overall favorable binding profile of structure-dependent broadly neutralizing antibodies (bnAbs) and non-neutralizing antibodies (non-nAbs). We confirmed that increasing the structural integrity and stability of the Env trimers positively correlates with the quality of induced antibody responses by the immunogens. These and other data contribute to the selection of ConCv5 KIKO as novel Env immunogens for use within the European Union’s H2020 Research Consortium EHVA (European HIV Alliance) for further preclinical analysis and phase 1 clinical development.
Abstract Understanding the balance between epitope shielding and accessibility on HIV-1 envelope (Env) trimers is essential to guide immunogen selection for broadly neutralizing antibody (bnAb) based vaccines. To investigate the antigenic space of Env immunogens, we created a strategy based on synthetic, high diversity, Designed Ankyrin Repeat Protein (DARPin) libraries. We show that DARPin Antigenicity Analysis (DANA), a purely in vitro screening tool, has the capability to extrapolate relevant information of antigenic properties of Env immunogens. DANA screens of stabilized, soluble Env trimers revealed that stronger trimer stabilization led to the selection of highly mutated DARPins with length variations and framework mutations mirroring observations made for bnAbs. By mimicking heterotypic prime-boost immunization regimens, DANA may be used to select immunogen combinations that favor the selection of trimer-reactive binders. This positions DANA as a versatile strategy for distilling fundamental antigenic features of immunogens, complementary to preclinical immunogenicity testing.
Introduction The generation of an HIV-1 vaccine able to induce long-lasting protective immunity remains a main challenge. Here, we aimed to modify next-generation soluble, prefusion-stabilized, close-to-native, glycan-engineered clade C gp140 envelope (Env) trimers (sC23v4 KIKO and ConCv5 KIKO) for optimal display on the cell surface following homologous or heterologous vector delivery. Methods A combination of the following modifications scored best regarding the preservation of closed, native-like Env trimer conformation and antigenicity when using a panel of selected broadly neutralizing (bnAb) and non-neutralizing (nnAb) monoclonal antibodies for flow cytometry: i) replacing the natural cleavage site with a native flexible linker and introducing a single amino acid substitution to prevent CD4 binding (*), ii) fusing a heterologous VSV-G-derived transmembrane moiety to the gp140 C-terminus, and iii) deleting six residues proximal to the membrane. Results When delivering membrane-tethered sC23v4 KIKO* and ConCv5 KIKO* via DNA, VSV-GP, and NYVAC vectors, the two native-like Env trimers provide differential antigenicity profiles. Whereas such patterns were largely consistent among the different vectors for either Env trimer, the membrane-tethered ConCv5 KIKO* trimer adopted a more closed and native-like structure than sC23v4 KIKO*. In immunized mice, VSV-GP and NYVAC vectors expressing the membrane-tethered ConCv5 KIKO* administered in prime/boost combination were the most effective regimens for the priming of Env-specific CD4 T cells among all tested combinations. The subsequent booster administration of trimeric ConCv5 KIKO* Env protein preserved the T cell activation levels between groups. The evaluation of the HIV-1-specific humoral responses induced in the different immunization groups after protein boosts showed that the various prime/boost protocols elicited broad and potent antibody responses, preferentially of a Th1-associated IgG2a subclass, and that the obtained antibody levels remained high at the memory phase. Discussion In summary, we provide a feasible strategy to display multiple copies of native-like Env trimers on the cell surface, which translates into efficient priming of sustained CD4 + T cell responses after vector delivery as well as broad, potent, and sustained antibody responses following booster immunizations with the homologous, prefusion-stabilized, close-to-native ConCv5 KIKO* gp140 Env trimer.
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