Title: Glycosylation and Sialylation of Macrophage-derived Human Apolipoprotein E Analyzed by SDS-PAGE and Mass Spectrometry
Abstract: Apolipoprotein E (apoE) is a 34-kDa glycoprotein secreted from various cells including hepatocytes and macrophages and plays an important role in remnant lipoprotein clearance, immune responses, Alzheimer disease, and atherosclerosis. Cellular apoE and plasma apoE exist as multiple glycosylated and sialylated glycoforms with plasma apoE being less glycosylated/sialylated than cell-derived apoE. Some of the glycan structures on plasma apoE are characterized; however, the more complicated structures on plasma and cellular/secreted apoE remain unidentified. We investigated glycosylation and sialylation of cellular and secreted apoE from primary human macrophages by one- and two-dimensional gel electrophoresis and mass spectrometry. Our results identify eight different glycoforms with (HexNAc)2-Hex2-(NeuAc)2 being the most complex glycan detected on Thr194 in both cellular and secreted apoE. Four additional glycans were identified on apoE(283–299), and using β-elimination/alkylation by methylamine in vitro, we identified Ser290 as a novel site of glycan attachment. Comparison of plasma and cellular/secreted apoE from the same donor confirmed that cell-derived apoE is more extensively sialylated than plasma apoE. Given the importance of the C terminus of apoE in regulating apoE solubility, stability, and lipid binding, these results may have important implications for our understanding of apoE biochemistry. Apolipoprotein E (apoE) is a 34-kDa glycoprotein secreted from various cells including hepatocytes and macrophages and plays an important role in remnant lipoprotein clearance, immune responses, Alzheimer disease, and atherosclerosis. Cellular apoE and plasma apoE exist as multiple glycosylated and sialylated glycoforms with plasma apoE being less glycosylated/sialylated than cell-derived apoE. Some of the glycan structures on plasma apoE are characterized; however, the more complicated structures on plasma and cellular/secreted apoE remain unidentified. We investigated glycosylation and sialylation of cellular and secreted apoE from primary human macrophages by one- and two-dimensional gel electrophoresis and mass spectrometry. Our results identify eight different glycoforms with (HexNAc)2-Hex2-(NeuAc)2 being the most complex glycan detected on Thr194 in both cellular and secreted apoE. Four additional glycans were identified on apoE(283–299), and using β-elimination/alkylation by methylamine in vitro, we identified Ser290 as a novel site of glycan attachment. Comparison of plasma and cellular/secreted apoE from the same donor confirmed that cell-derived apoE is more extensively sialylated than plasma apoE. Given the importance of the C terminus of apoE in regulating apoE solubility, stability, and lipid binding, these results may have important implications for our understanding of apoE biochemistry. Apolipoprotein E (apoE) 1The abbreviations used are:apoEapolipoprotein ELTQlinear ion trap quadrupole1-DEone-dimensional gel electrophoresis2-DEtwo-dimensional gel electrophoresisIPimmunoprecipitationMAAM. amurensis lectin IISNAS. nigra bark lectinHMDMhuman monocyte-derived macrophageEasialylated apoEEssialylated apoEHexhexoseHexNAcN-acetylhexosamineDDAdata-dependent acquisitionXICextracted ion chromatogramTICtotal ion chromatogramBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. is a 34-kDa glycosylated apolipoprotein of 299 amino acids. ApoE is synthesized and secreted by most cells including hepatocytes, smooth muscle cells, neuronal cells, and macrophages (1.Mahley R.W. Rall Jr., S.C. Apolipoprotein E: far more than a lipid transport protein.Annu. Rev. Genomics Hum. Genet. 2000; 1: 507-537Crossref PubMed Scopus (1311) Google Scholar, 2.Greenow K. Pearce N.J. Ramji D.P. The key role of apolipoprotein E in atherosclerosis.J. Mol. Med. 2005; 83: 329-342Crossref PubMed Scopus (184) Google Scholar, 3.Kockx M. Jessup W. Kritharides L. Regulation of endogenous apolipoprotein E secretion by macrophages.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1060-1067Crossref PubMed Scopus (63) Google Scholar) and demonstrates extraordinary functional diversity. It has important roles in remnant lipoprotein clearance, the immune response, Alzheimer disease, cell proliferation, and lymphocyte activation (4.Kothapalli D. Fuki I. Ali K. Stewart S.A. Zhao L. Yahil R. Kwiatkowski D. Hawthorne E.A. FitzGerald G.A. Phillips M.C. Lund-Katz S. Puré E. Rader D.J. Assoian R.K. Antimitogenic effects of HDL and APOE mediated by Cox-2-dependent IP activation.J. Clin. Investig. 2004; 113: 609-618Crossref PubMed Scopus (42) Google Scholar, 5.van den Elzen P. Garg S. León L. Brigl M. Leadbetter E.A. Gumperz J.E. Dascher C.C. Cheng T.Y. Sacks F.M. Illarionov P.A. Besra G.S. Kent S.C. Moody D.B. Brenner M.B. Apolipoprotein-mediated pathways of lipid antigen presentation.Nature. 2005; 437: 906-910Crossref PubMed Scopus (301) Google Scholar). More recent studies suggest that elevated plasma apoE precedes elevation of C-reactive protein and confers increased risk of cardiovascular death in the elderly (6.Mooijaart S.P. Berbée J.F. van Heemst D. Havekes L.M. de Craen A.J. Slagboom P.E. Rensen P.C. Westendorp R.G. ApoE plasma levels and risk of cardiovascular mortality in old age.PLoS Med. 2006; 3: e176Crossref PubMed Scopus (99) Google Scholar). Proteomics-based approaches have identified elevated high density lipoprotein (HDL)-apoE as being associated with coronary disease (7.Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. Chea H. Knopp R.H. Brunzell J. Geary R. Chait A. Zhao X.Q. Elkon K. 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The mechanisms by which macrophage apoE is antiatherogenic may include stimulating the removal of excess cholesterol from macrophage foam cells as well as anti-inflammatory, antiproliferative, and immunomodulatory properties (4.Kothapalli D. Fuki I. Ali K. Stewart S.A. Zhao L. Yahil R. Kwiatkowski D. Hawthorne E.A. FitzGerald G.A. Phillips M.C. Lund-Katz S. Puré E. Rader D.J. Assoian R.K. Antimitogenic effects of HDL and APOE mediated by Cox-2-dependent IP activation.J. Clin. Investig. 2004; 113: 609-618Crossref PubMed Scopus (42) Google Scholar, 5.van den Elzen P. Garg S. León L. Brigl M. Leadbetter E.A. Gumperz J.E. Dascher C.C. Cheng T.Y. Sacks F.M. Illarionov P.A. Besra G.S. Kent S.C. Moody D.B. Brenner M.B. Apolipoprotein-mediated pathways of lipid antigen presentation.Nature. 2005; 437: 906-910Crossref PubMed Scopus (301) Google Scholar, 10.Mazzone T. Apolipoprotein E secretion by macrophages: its potential physiological functions.Curr. Opin. 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An accurate understanding of the structure of apoE secreted from macrophages is important for our understanding of its properties and its role in the atherosclerotic process. apolipoprotein E linear ion trap quadrupole one-dimensional gel electrophoresis two-dimensional gel electrophoresis immunoprecipitation M. amurensis lectin II S. nigra bark lectin human monocyte-derived macrophage asialylated apoE sialylated apoE hexose N-acetylhexosamine data-dependent acquisition extracted ion chromatogram total ion chromatogram 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Structural studies on apoE have provided important insights into its biological properties (13.Hatters D.M. Peters-Libeu C.A. Weisgraber K.H. Apolipoprotein E structure: insights into function.Trends Biochem. Sci. 2006; 31: 445-454Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). Crystallography has demonstrated that the N-terminal domain is structured in a globular four-helix bundle with the helices orientated in an antiparallel alignment (14.Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. The amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function.J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar). The structure of the C terminus has not been resolved by crystallography, but circular dichroism spectroscopy indicates it to be highly α-helical (14.Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. The amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function.J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar). Recently, NMR studies of monomeric, full-length human apoE indicated that the C-terminal domain in the intact protein adopts a more defined structure than it does as an isolated fragment (15.Zhang Y. Vasudevan S. Sojitrawala R. Zhao W. Cui C. Xu C. Fan D. Newhouse Y. Balestra R. Jerome W.G. Weisgraber K. Li Q. Wang J. A monomeric, biologically active, full-length human apolipoprotein E.Biochemistry. 2007; 46: 10722-10732Crossref PubMed Scopus (59) Google Scholar). Lipid binding occurs at the C terminus (residues 244–272), resulting in unfolding of the molecule into a helical hairpin with the binding region for the low density lipoprotein (LDL) receptor contained within the N terminus at its apex (16.Peters-Libeu C.A. Newhouse Y. Hatters D.M. Weisgraber K.H. Model of biologically active apolipoprotein E bound to dipalmitoylphosphatidylcholine.J. Biol. Chem. 2006; 281: 1073-1079Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Mucin-type O-glycosylation is a particularly common, complex, and important post-translational modification of secreted and cell surface glycoproteins (17.Van den Steen P. Rudd P.M. Dwek R.A. Opdenakker G. Concepts and principles of O-linked glycosylation.Crit. Rev. Biochem. Mol. Biol. 1998; 33: 151-208Crossref PubMed Scopus (619) Google Scholar, 18.Hanisch F.G. O-Glycosylation of the mucin type.Biol. Chem. 2001; 382: 143-149Crossref PubMed Scopus (270) Google Scholar) that is difficult to accurately characterize; however, several recent reports have facilitated analysis (19.Wells L. Vosseller K. Cole R.N. Cronshaw J.M. Matunis M.J. Hart G.W. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications.Mol. Cell. Proteomics. 2002; 1: 791-804Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 20.Mirgorodskaya E. Hassan H. Clausen H. Roepstorff P. Mass spectrometric determination of O-glycosylation sites using beta-elimination and partial acid hydrolysis.Anal. Chem. 2001; 73: 1263-1269Crossref PubMed Scopus (51) Google Scholar). Cellular apoE and plasma apoE exist as multiple glycoforms, which vary in charge because of variable sialylation. The initial analysis of the carbohydrate content of plasma very low density lipoprotein (VLDL)-apoE by colorimetric methods and gas chromatography demonstrated that the major unmodified hexose in apoE was galactose and that N-acetylglucosamine, N-acetylgalactosamine, and sialic acid were present (21.Jain R.S. Quarfordt S.H. The carbohydrate content of apolipoprotein E from human very low density lipoproteins.Life Sci. 1979; 25: 1315-1323Crossref PubMed Scopus (28) Google Scholar, 22.Zannis V.I. vanderSpek J. Silverman D. Intracellular modifications of human apolipoprotein E.J. Biol. Chem. 1986; 261: 13415-13421Abstract Full Text PDF PubMed Google Scholar). Two-dimensional gel electrophoresis (2-DE) identified up to six sialylated apoE (Es) glycoforms in cells for any given genotype and fewer sialylated glycoforms in plasma (22.Zannis V.I. vanderSpek J. Silverman D. Intracellular modifications of human apolipoprotein E.J. Biol. Chem. 1986; 261: 13415-13421Abstract Full Text PDF PubMed Google Scholar). ApoE does not contain the consensus sequence (NX(T/S/C)) required for N-linked glycans, and carbohydrate residues are attached to apoE via an O-linkage to residue Thr194 (23.Kritharides L. Jessup W. Mander E.L. Dean R.T. Apolipoprotein A-I-mediated efflux of sterols from oxidized LDL-loaded macrophages.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 276-289Crossref PubMed Scopus (89) Google Scholar, 24.Kontula K. Aalto-Setälä K. Kuusi T. Hämäläinen L. Syvänen A.C. Apolipoprotein E polymorphism determined by restriction enzyme analysis of DNA amplified by polymerase chain reaction: convenient alternative to phenotyping by isoelectric focusing.Clin. Chem. 1990; 36: 2087-2092Crossref PubMed Scopus (79) Google Scholar, 25.Mancone C. Amicone L. Fimia G.M. Bravo E. Piacentini M. Tripodi M. Alonzi T. Proteomic analysis of human very low-density lipoprotein by two-dimensional gel electrophoresis and MALDI-TOF/TOF.Proteomics. 2007; 7: 143-154Crossref PubMed Scopus (44) Google Scholar). More recent studies using 2-DE and MALDI-TOF/TOF (23.Kritharides L. Jessup W. Mander E.L. Dean R.T. Apolipoprotein A-I-mediated efflux of sterols from oxidized LDL-loaded macrophages.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 276-289Crossref PubMed Scopus (89) Google Scholar) confirmed previous results and identified five glycosylated glycoforms of apoE in plasma VLDL with the most complex sugar structures containing two sialic acid residues (HexNAc-Hex-NeuAc-NeuAc). There were more negatively charged glycoforms present on 2-DE than were distinguished by MALDI-TOF/TOF, raising the possibility that complex structures containing more than two sialic acid residues may be inherently unstable during MS analysis. Importantly, this recent study did not analyze apoE glycoforms in, or secreted from, cells. The purpose of this study was to undertake the first detailed characterization of the glycan structures of apoE from primary human macrophages by 1-DE, 2-DE, and mass spectrometry. We found that cellular and secreted apoE in human macrophages has at least eight different glycoforms with (HexNAc)2-Hex2-(NeuAc)2 being the most complex glycan identified. We extend previous studies by the identification of a novel site of glycan attachment on Ser290 near the functionally important apoE C terminus in addition to glycosylation of Thr194 and show that a major glycoform is present in each of the spots separated by 2-DE. Protein A-Sepharose™ LC-4B was purchased from GE Healthcare. Goat anti-apolipoprotein E polyclonal antibodies to human apoE were obtained from Chemicon International Inc. ZOOM strips (5 × 70 mm, pH 4–7), carrier ampholyte pH 4–7, thiourea, urea, CHAPS, ultrapure dithiothreitol (DTT), ultrapure agarose, DNase I, SilverQuest™ silver staining kit, and SimplyBlue™ SafeStain were purchased from Invitrogen. Biotinylated Maackia amurensis lectin II (MAA), biotinylated Sambucus nigra bark lectin (SNA), and horseradish peroxidase (HRP)-avidin D were purchased from Vector Laboratories. α-(2→3,6,8,9)-Neuraminidase, α-(2→3)-neuraminidase, BSA, and RNase A were supplied by Sigma. LDL, acetylated LDL (AcLDL), and lipoprotein-deficient serum were prepared as described (23.Kritharides L. Jessup W. Mander E.L. Dean R.T. Apolipoprotein A-I-mediated efflux of sterols from oxidized LDL-loaded macrophages.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 276-289Crossref PubMed Scopus (89) Google Scholar). Human monocytes were isolated from white cell buffy coat concentrates from healthy donors using density gradient centrifugation after layering on Ficoll-Paque Plus (GE Healthcare). Purified monocytes were differentiated in 6-well Primaria plates (BD Biosciences) by culturing in RPMI 1640 medium containing 50 units/ml penicillin G, 50 μg/ml streptomycin, 2 mm l-glutamine, 10% heat-inactivated human serum, and 25 ng/ml macrophage colony-stimulating factor (PreproTech) for 3 days followed by culturing in the same medium without macrophage colony-stimulating factor for up to 7 days. After differentiation, the cells were washed and enriched with cholesterol by incubation in RPMI 1640 medium including 10% lipoprotein-deficient serum and 50 μg/ml acetylated LDL for 2 days. After enrichment, the cultures were washed twice with prewarmed RPMI 1640 medium and incubated in RPMI 1640 medium for between 1 and 24 h. At the indicated time points, the cells and medium samples were harvested. Cells were lysed using radioimmune precipitation assay buffer (50 mm Tris-Cl, pH 7.5, 150 mm NaCl, 0.1% SDS, 1% Triton X-100, 0.5% deoxycholate, and protease inhibitors). White cell buffy coat concentrates and human serum were supplied by the New South Wales Red Cross blood transfusion service, Sydney, Australia. Donors were genotyped for apoE by the laboratory of Prof. D. Sullivan, Royal Prince Alfred Hospital, Sydney, Australia, by restriction enzyme analysis (24.Kontula K. Aalto-Setälä K. Kuusi T. Hämäläinen L. Syvänen A.C. Apolipoprotein E polymorphism determined by restriction enzyme analysis of DNA amplified by polymerase chain reaction: convenient alternative to phenotyping by isoelectric focusing.Clin. Chem. 1990; 36: 2087-2092Crossref PubMed Scopus (79) Google Scholar). Blood samples in EDTA-containing tubes were obtained from a healthy volunteer with an apoE3/3 genotype. Monocytes were isolated as described above. After density gradient centrifugation, plasma supernatant was collected. Total plasma proteins were prepared as described (25.Mancone C. Amicone L. Fimia G.M. Bravo E. Piacentini M. Tripodi M. Alonzi T. Proteomic analysis of human very low-density lipoprotein by two-dimensional gel electrophoresis and MALDI-TOF/TOF.Proteomics. 2007; 7: 143-154Crossref PubMed Scopus (44) Google Scholar). Briefly, 12 μl of plasma was mixed with 20 μl of a 10% SDS and 2.3% DTT solution boiled at 95 °C for 5 min. The sample was diluted to 500 μl with rehydration buffer (9 m urea, 2 m thiourea, 4% CHAPS, and trace bromophenol blue). 30 μl of the sample was separated by 2-DE, and apoE was detected by Western blot. To isolate apoE from cholesterol-enriched HMDMs, cell lysates and medium were immunoprecipitated using a goat antibody to human apoE and protein A-Sepharose. 1.2 mg of cell lysates and medium samples was precleared for 30 min by the addition of 50 μl of protein A-Sepharose, then mixed with 5 μl of goat anti-apoE antibody, and incubated for 1 h with rotation. After 1 h, 50 μl of protein A-Sepharose was added, and the samples were incubated for another 1 h with rotation. Beads were spun down and washed five times with radioimmune precipitation assay buffer. ApoE was eluted using rehydration buffer. To detect apoE protein bands in HMDMs, 9 mg of cell lysates and corresponding medium samples were immunoprecipitated, eluted in 150 μl of sample buffer (50 mm Tris-HCl, pH 6.8, 100 mm DTT, 2% SDS, 0.1% bromphenol blue, and 10% glycerol), and separated by Tris-glycine SDS-PAGE using 10% polyacrylamide gels. ApoE was detected by Coomassie staining. To detect individual apoE glycoforms, 40 μl of immunoprecipitated apoE was subjected to 2-DE. For the first dimension, isoelectric focusing was performed with a ZOOM IPGRunner system (Invitrogen) using 7-cm, pH 4–7 strips at 2000 V-h at room temperature. Samples were then reduced in 1× NuPAGE sample reducing agent for 15 min and alkylated with 125 mm iodoacetamide for 15 min after which second-dimension SDS-PAGE was performed using NuPAGE Novex 4–12% Bis-Tris ZOOM gels. After electrophoresis, the gels were fixed, and protein spots were visualized using a SilverQuest silver staining kit (Invitrogen) and SimplyBlue SafeStain (Invitrogen) for mass spectrometry analysis. Preliminary experiments confirmed that this separation clearly distinguished apoE 2, 3, and 4 glycoforms as described (26.Zannis V.I. Breslow J.L. Utermann G. Mahley R.W. Weisgraber K.H. Havel R.J. Goldstein J.L. Brown M.S. Schonfeld G. Hazzard W.R. Blum C. Proposed nomenclature of apoE isoproteins, apoE genotypes, and phenotypes.J. Lipid Res. 1982; 23: 911-914Abstract Full Text PDF PubMed Google Scholar), consistent with calculated pI values of 5.65 (apoE3), 5.81 (apoE4), and 5.52 (apoE2) (ExPASy Compute pI/Mw tool). For all experiments described herein, apoE3/3 donor macrophages were used exclusively. Protein spots were excised from one- or two-dimensional gels and destained to remove Coomassie stain by incubation with 100 mm NH4HCO3 in CH3CN for 1 h. Reduction and alkylation were then performed to maximize digestion efficiency. For reduction, gel spots were incubated with 10 mm DTT in 20 mm NH4HCO3 at 37 °C for 1 h, and this was followed by incubation with 25 mm iodoacetamide in 20 mm NH4HCO3 at 37 °C for 1 h for alkylation. Gel spots were dehydrated with 100 μl of 100% CH3CN for 10 min. Sequencing grade trypsin (Promega) was added to the dehydrated gel spots at 10 ng/μl in 15 mm NH4HCO3, and samples were incubated overnight at 37 °C. Digested peptides were extracted from gel spots with 1% formic acid for 10 min followed by incubation with 100% CH3CN for another 10 min. Peptide mixtures were evaporated in a SpeedVac for 1 h and finally dissolved in 0.05% heptafluorobutyric acid and 1% formic acid. Digest peptides were separated by nano-LC using a CapLC and autosampler system (Waters). Samples (5 μl) were concentrated and desalted onto a micro-C18 precolumn (500 μm × 2 mm; Michrom Bioresources, Auburn, CA) with H2O:CH3CN (98:2, 0.05% heptafluorobutyric acid) at 15 μl/min. After a 4-min wash, the precolumn was switched (Valco 10-port valve, Dionex) in line with a fritless nanocolumn (75 μm × ∼10 cm) containing C18 medium (5 μm, 200 Å; Magic Bioresources, Michrom) manufactured according to Gatlin et al. (27.Gatlin C.L. Kleemann G.R. Hays L.G. Link A.J. Yates 3rd, J.R. Protein identification at the low femtomole level from silver-stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry.Anal. Biochem. 1998; 263: 93-101Crossref PubMed Scopus (351) Google Scholar). Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (65:35, 0.1% formic acid) at ∼300 nl/min over 30 or 60 min using a CapLC system (Waters). The precolumn was connected via a fused silica capillary (25 μm × 10 cm) to a low volume tee (Upchurch Scientific) where high voltage (2400 V) was applied, and the column tip was positioned ∼1 cm from the Z-spray inlet of a Q-Tof Ultima API hybrid tandem mass spectrometer (Micromass, Manchester, UK). Positive ions were generated by electrospray, and the Q-Tof Ultima was operated in data-dependent acquisition (DDA) mode. A TOF MS survey scan was acquired (m/z 350–1700; 1 s), and the two largest multiply charged ions (counts >20) were sequentially selected by Q1 for MS/MS analysis. Argon was used as the collision gas, and an optimum collision energy was chosen (based on charge state and mass). Tandem mass spectra were accumulated for up to 2 s (m/z 50–2000). Peak lists were generated by MassLynx (version 4.0 SP4, Micromass) using the Mass Measure program and submitted to the database search program Mascot (version 2.2, Matrix Science, London, UK). Search parameters were as follows: precursor and product ion tolerances were ±0.25 and 0.2 Da, respectively; Met(O) and carboxyamidomethyl-Cys were specified as variable modifications; enzyme specificity was trypsin; one missed cleavage was possible; and the non-redundant protein database from NCBI (March 2008) was searched. Digest peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex, Amsterdam, Netherlands) as described above. Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 350 nl/min over 30 or 60 min. High voltage (1800 V) was applied to a low volume tee (Upchurch Scientific), and the column tip was positioned ∼0.5 cm from the heated capillary (T = 200 °C) of an LTQ-FT Ultra (Thermo Electron, Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray, and the LTQ-FT Ultra was operated in DDA mode. A survey scan (m/z 350–1750) was acquired in the FT ICR cell (resolution = 100,000 at m/z 400 with an accumulation target of 1,000,000 ions). Up to six of the most abundant ions (>3000 counts) with charge states >2+ were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with a normalized collision energy of 25 V, activation q of 0.25, and activation time of 30 ms at a target value of 30,000 ions. m/z values selected for MS/MS were dynamically excluded for 30 s. Peak lists were generated using Mascot Daemon/extract_msn (Matrix Science, Thermo) using the default parameters and submitted to the database search program Mascot (version 2.2, Matrix Science). General search parameters were as follows: precursor tolerance was 4 ppm, product ion tolerance was ±0.4 Da, Met(O) and Cys carboxyamidomethylation were specified as variable modifications, enzyme specificity was trypsin, one missed cleavage was possible, and the non-redundant protein database from NCBI or Swiss-Prot (March 2008) was searched. Additional searches with variable modifications N-acetylation, Cys-sulfenic acid, Cys-sulfonic acid, deamidation, and phosphorylation were performed. All peptides assigned by Mascot had Mowse scores >20, and spectra of glycopeptides were interpreted and validated manually. Extracted ion chromatograms (XICs) were derived from either MS or MS/MS spectra from the calculated monoisotopic mass of each ion (±m/z 0.1) using the QualBrowser in XCalibur (version 2.07), and abundances were calculated from the area of each extracted ion. Peptide digests were treated with NH2CH3 vapor as described (20.Mirgorodskaya E. Hassan H. Clausen H. Roepstorff P. Mass spectrometric determination of O-glycosylation sites using beta-elimination and partial acid hydrolysis.Anal. Chem. 2001; 73: 1263-1269Crossref PubMed Scopus (51) Google Scholar). Briefly, a portion of the digests (∼15%) was dried (SpeedVac), tubes were placed in scintillation vials together with a microcentrifuge tube containing NH2CH3 (50 μl), and the vial was flushed with N2. After capping, the vials were left at 70 °C for 60 min. Peptides were solubilized and analyzed by nano-LC MS/MS using the Q-Tof Ultima as described above. Lectin blot analysis was performed as described (28.Glass 2nd, W.F. Briggs R.C. Hnilica L.S. Use of lectins for detection of electrophoretically separated glycoproteins transferred onto nitrocellulose sheets.Anal. Biochem. 1981; 115: 219-224Crossref PubMed Scopus (191) Google Scholar). This is based on the differing relative affinities for α-(2→6) and α-(2→3) linkages of SNA and MAA, respectively (29.Shibuya N. Goldstein I.J. Broekaert W.F. Nsimba-Lubaki M. Peeters B. Peumans W.J. The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac(alpha 2–6)Gal/GalNAc sequence.J. Biol. Chem. 1987; 262: 1596-1601Abstract Full Text PDF PubMed Google Scholar, 30.Wang W.C. Cummings R.D. The immobilized leukoagglutinin from the seeds of Maackia amurensis binds with high affinity to complex-type Asn-linked oligosaccharides containing terminal sialic acid-linked alpha-2,3 to penultimate galactose residues.J. Biol. Chem. 1988; 263: 4576-4585Abstract Full Text PDF PubMed Google Scholar). In short, after 2-DE and protein transfer, nitrocellulose membranes were incubated with 2% periodate-oxidized BSA buffer containing 0.1% Tween 20 in PBS (BSA-Tween buffer) at 40 °C for 1 h. Membranes were subsequently incubated for 1 h with biotinylated lectins in BSA-Tween buffer containing 0.5 μg/ml SNA or 1 μg/ml MAA. After five 5-min washes in PBS containing 0.5% Tween 20, the membranes were incubated with 1 μg/ml HRP-avidin D for 1 h, washed five times with PBS containing 0.5% Tween 20, and revealed by ECL. For neuraminidase cleavage of sialic acid residues, HMDM cell lysates and medium samples were treated with α-(2→3,6,8,9)-neuraminidase or α-(2→3)-neuraminidase in 50 mm sodium phosphate, pH 5.0 at 37 °C for 3 h. A high resolution EPR structure of lipid-bound apoE was recently published, and the Protein Data Bank file was a kind gift from Prof K. Weisgraber (31.Hatters D.M. Voss J.C. Budamagunta M.S. Newhouse Y.N. Weisgraber K.H. Insight on the molecular envelope of lipid-bound apolipoprotein E from electron paramagnetic resonance spectroscopy.J. Mol. Biol. 2009; 386: 261-271Crossref PubMed Scopus (16) Google Scholar). Analysis of the glycosylation site on Ser290 was performed using Discovery Studio 2.0 (Accelrys Software Inc.) on this structure. To analyze the sugar structures of apoE glycoforms, cellular and secreted apoE was immunoprecipitated and separated by 1-DE (Fig. 1, inset). After Coomassie staining, 34-kDa apoE bands were excised, destained, and treated with trypsin, and peptides were analyzed by nano-LC data-dependent tandem mass spectrometry. Total ion chromatograms (TICs) and XICs derived from all MS/MS spectra u