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• W2062212050 abstract "Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. The avian sarcoma and leukosis virus envelope glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a cell surface receptor at neutral pH trigger an initial conformational change in the viral glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A avian sarcoma and leukosis viruses envelope glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the disulfide bond more readily than the peptide backbone amide bonds that enabled the identification of disulfide-linked peptides. Seven of nine disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of cysteine residues. The first cysteine of surface and the last cysteine of the transmembrane form a disulfide bond linking the heterodimer. The surface glycoprotein contains a free cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning disulfide bonds in a complex glycoprotein. Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. The avian sarcoma and leukosis virus envelope glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a cell surface receptor at neutral pH trigger an initial conformational change in the viral glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A avian sarcoma and leukosis viruses envelope glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the disulfide bond more readily than the peptide backbone amide bonds that enabled the identification of disulfide-linked peptides. Seven of nine disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of cysteine residues. The first cysteine of surface and the last cysteine of the transmembrane form a disulfide bond linking the heterodimer. The surface glycoprotein contains a free cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning disulfide bonds in a complex glycoprotein. To enter cells and begin replication, enveloped viruses must fuse the membrane coating the viral particle with a cellular membrane to deliver a subviral particle inside the cell (for review, see Refs. 1Harrison S.C. Nat. Struct. Mol. Biol. 2008; 15: 690-698Crossref PubMed Scopus (948) Google Scholar, 2White J.M. Delos S.E. Brecher M. Schornberg K. Crit. Rev. Biochem. Mol. Biol. 2008; 43: 189-219Crossref PubMed Scopus (654) Google Scholar). The fusion of two membranes is thermodynamically favored but comes with a very high kinetic barrier(s). Enveloped viruses have one or more glycoproteins to mediate the fusion process using the energy liberated upon conformational changes of the viral glycoprotein(s) to clear the kinetic barrier(s). Viral entry begins when the viral glycoproteins bind an appropriate cell surface protein initiating one of three possible mechanistic fates. The viral glycoprotein-receptor interaction can serve to enable trafficking of the virion into the endocytic pathway where the low pH environment triggers a conformation change in the viral glycoproteins initiating fusion of the viral and endosome membranes (e.g. influenza virus). With other viruses, for example most retroviruses, the viral glycoprotein-receptor interaction itself triggers a conformational change in the viral glycoproteins at neutral pH at the cell surface sufficient for the ultimate fusion of the viral and cellular membranes. In a third mechanism, the interaction between the viral glycoprotein and receptor at neutral pH at the cell surface triggers an initial conformational change in the viral glycoproteins. The triggered glycoproteins then require exposure to low pH to complete the conformational changes for fusion of the viral and cellular membranes (e.g. avian sarcoma and leukosis viruses (ASLVs)). 3The abbreviations used are: ASLVavian sarcoma and leukosis virusCIDcollision-induced dissociationECDelectron capture dissociationESIelectrospray ionizationFTFourier transformFTICRFourier transform ion cyclotron resonanceMS/MStandem mass spectrometryPNGase FN-glycosidase FSUsurfaceTCEPtris (2-carboxyethyl) phosphineTEVtobacco etch virusTMtransmembrane. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. We are using the homologous group of retroviruses, subgroups A to E ASLV, to study enveloped virus entry because these viruses have evolved from a common ancestor to use different cellular proteins as receptors (3Hunter E. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 71-120Google Scholar, 4Weiss R.A. Levy J.A. The Retroviruses. Plenum Publishing Corp., New York1992: 1-108Google Scholar) and have maintained the fusion process to efficiently enter cells (5Matsuyama S. Delos S.E. White J.M. J. Virol. 2004; 78: 8201-8209Crossref PubMed Scopus (51) Google Scholar, 6Mothes W. Boerger A.L. Narayan S. Cunningham J.M. Young J.A.T. Cell. 2000; 103: 679-689Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 7Smith J.G. Mothes W. Blacklow S.C. Cunningham J.M. J. Virol. 2004; 78: 1403-1410Crossref PubMed Scopus (53) Google Scholar). avian sarcoma and leukosis virus collision-induced dissociation electron capture dissociation electrospray ionization Fourier transform Fourier transform ion cyclotron resonance tandem mass spectrometry N-glycosidase F surface tris (2-carboxyethyl) phosphine tobacco etch virus transmembrane. All retroviral glycoproteins are synthesized as polyprotein precursors consisting of the surface glycoprotein (SU) that contains the domains that bind the cellular receptor and the transmembrane glycoprotein (TM) that anchors the protein to the membrane and contains the domains responsible for the fusion process (for review, see Ref. 8Swanstrom R. Wills J.W. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 263-334Google Scholar). After synthesis, the precursor polyproteins are glycosylated and form trimers through noncovalent interactions between extracellular regions of the TM glycoprotein. The viral glycoprotein precursors form a mature, metastable complex capable of mediating virus entry into the cell only after the SU and TM domains are cleaved by a cellular protease to yield a trimer of SU/TM heterodimers (9Young J.A.T. Knipe D.M. Howley P.M. Fields Virology. Lippincott Williams & Wilkins, Philadelphia2001: 87-103Google Scholar). The interaction of multiple, noncontiguous regions of SU and a specific receptor protein are required to initiate the entry process by triggering a conformational change in the SU glycoproteins, allowing the kinetically trapped TM glycoprotein structure to extend and form a lower energy structure that projects the fusion peptide toward the target membrane. Two domains in TM are critical for the extension and the subsequent refolding of TM: the N-terminal and C-terminal heptad repeats. The lowest energy form of the TM trimer, the six-helix bundle, forms when the C-terminal heptad repeats fold back into grooves created by the N-terminal heptad repeats, bringing the viral and cellular membranes into close proximity. Fusion of the membranes goes through an initial outer lipid leaflet mixing, then hemifusion, initial pore formation, pore widening, and the completion of fusion where the retroviral glycoprotein 6HB may undergo additional structural rearrangements (10Markosyan R.M. Bates P. Cohen F.S. Melikyan G.B. Biophys. J. 2004; 87: 3291-3298Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 11Melikyan G.B. Barnard R.J. Abrahamyan L.G. Mothes W. Young J.A.T. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 8728-8733Crossref PubMed Scopus (82) Google Scholar, 12Tamm L.K. Biochim. Biophys. Acta. 2003; 1614: 14-23Crossref PubMed Scopus (77) Google Scholar). The cooperation of several glycoprotein-receptor interactions is likely required to form a local environment capable of fusion. The subgroup A to E ASLV envelope glycoproteins are highly related: differences in the hypervariable domains of SU (hr1, hr2, and vr3) define receptor usage and suggest that these viruses have evolved from a common ancestor (13Coffin J.M. Curr. Top. Microbiol. Immunol. 1992; 176: 143-164Crossref PubMed Scopus (180) Google Scholar). Tva, the receptor for subgroup A ASLV, is related to the ligand-binding repeat of the low density lipoprotein receptor family (14Bates P. Young J.A. Varmus H.E. Cell. 1993; 74: 1043-1051Abstract Full Text PDF PubMed Scopus (310) Google Scholar, 15Young J.A. Bates P. Varmus H.E. J. Virol. 1993; 67: 1811-1816Crossref PubMed Google Scholar). Tvb, the receptors for the subgroup B, D, and E ASLVs, is a member of the tumor necrosis factor receptor family (16Adkins H.B. Brojatsch J. Naughton J. Rolls M.M. Pesola J.M. Young J.A.T. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 11617-11622Crossref PubMed Scopus (88) Google Scholar, 17Adkins H.B. Brojatsch J. Young J.A.T. J. Virol. 2000; 74: 3572-3578Crossref PubMed Scopus (85) Google Scholar, 18Brojatsch J. Naughton J. Rolls M.M. Zingler K. Young J.A.T. Cell. 1996; 87: 845-855Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Tvc, the receptors for subgroup C ASLVs, is related to yet a third disparate family of proteins, the immunoglobulin Ig proteins (19Elleder D. Stepanets V. Melder D.C. Senigl F. Geryk J. Pajer P. Plachý J. Hejnar J. Svoboda J. Federspiel M.J. J. Virol. 2005; 79: 10408-10419Crossref PubMed Scopus (77) Google Scholar). The normal physiological functions of Tva, Tvb, and Tvc are currently unknown. Engineered secreted forms of these ASLV receptors, the extracellular domain of the receptor alone or fused to a IgG domain, retain biological activity sufficient to bind the viral glycoproteins with high affinity and trigger conformational changes in the viral glycoproteins similar to changes expected to occur during initiation of the infection process (20Damico R. Bates P. J. Virol. 2000; 74: 6469-6475Crossref PubMed Scopus (45) Google Scholar, 21Hernandez L.D. Peters R.J. Delos S.E. Young J.A. Agard D.A. White J.M. J. Cell Biol. 1997; 139: 1455-1464Crossref PubMed Scopus (108) Google Scholar, 22Holmen S.L. Salter D.W. Payne W.S. Dodgson J.B. Hughes S.H. Federspiel M.J. J. Virol. 1999; 73: 10051-10060Crossref PubMed Google Scholar). Engineered secreted forms of the SU glycoproteins either alone or fused to an IgG domain can bind the soluble forms of the receptors in a subgroup-specific manner (23Holmen S.L. Federspiel M.J. Virology. 2000; 273: 364-373Crossref PubMed Scopus (26) Google Scholar). The secreted forms of the ASLV SU or its receptor act as competitive inhibitors, having a significant antiviral effect on the specific ASLV subgroup infection of cells (22Holmen S.L. Salter D.W. Payne W.S. Dodgson J.B. Hughes S.H. Federspiel M.J. J. Virol. 1999; 73: 10051-10060Crossref PubMed Google Scholar, 24Holmen S.L. Melder D.C. Federspiel M.J. J. Virol. 2001; 75: 726-737Crossref PubMed Scopus (36) Google Scholar). We are characterizing the structural and functional organization of the ASLV glycoproteins to understand better how the ASLV fusion proteins mediate the entry process (25Barnard R.J. Young J.A.T. Curr. Top. Microbiol. Immunol. 2003; 281: 107-136PubMed Google Scholar). Because the number and location of the cysteine residues, 14 Cys in SU, 5 in TM, and 13 of 14 possible N-linked glycosylation sites, 12 in SU, 2 in TM, are conserved in the subgroup A to E ASLV glycoproteins, it is likely these glycoproteins share a common structure and glycosylation pattern. Unusual for retroviruses, the ASLV TM glycoprotein has an internal fusion peptide 21 residues downstream of the N terminus with 2 cysteine residues flanking the fusion peptide. The organization of the functional domains of the ASLV TM fusion protein is remarkably similar to the Ebola virus fusion protein, GP2, although the ASLV SU and Ebola virus GP1 glycoproteins are not (26Gallaher W.R. Cell. 1996; 85: 477-478Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 27Weissenhorn W. Calder L.J. Wharton S.A. Skehel J.J. Wiley D.C. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 6032-6036Crossref PubMed Scopus (112) Google Scholar). A disulfide bond reported to be stable throughout the fusion process covalently connects the ASLV SU and TM glycoproteins. If we assume that the disulfide bond links 1 cysteine from SU and 1 cysteine from TM, then there is at least 1 cysteine in SU that is an unbound, free cysteine. Recently, Smith and Cunningham reported that the formation of a reactive cysteine thiolate at residue Cys38 in the subgroup A SU glycoprotein was absolutely required for productive ASLV infection but was not necessary for the glycoprotein binding of the Tva receptor or Tva-triggered conformational changes (28Smith J.G. Cunningham J.M. PLoS Pathog. 2007; 3: e198Crossref PubMed Scopus (25) Google Scholar). Also, there was no evidence that the cysteine thiolate mediated isomerization of the SU-TM disulfide bond. In this study we physically mapped the secondary structure of an engineered secreted form of the subgroup A ASLV envelope glycoprotein (Fig. 1A). Einfeld and Hunter describe a secreted form of the subgroup A envelope glycoprotein truncated at TM residue 129 (SUATM129) (Fig. 1B), deleting the transmembrane and cytoplasmic domains, which was still capable of forming oligomers (likely trimers) detected by sucrose gradient (29Einfeld D.A. Hunter E. J. Virol. 1997; 71: 2383-2389Crossref PubMed Google Scholar). The SUATM129 glycoprotein was expressed in chicken DF-1 cells (30Himly M. Foster D.N. Bottoli I. Iacovoni J.S. Vogt P.K. Virology. 1998; 248: 295-304Crossref PubMed Scopus (343) Google Scholar, 31Schaefer-Klein J. Givol I. Barsov E.V. Whitcomb J.M. VanBrocklin M. Foster D.N. Federspiel M.J. Hughes S.H. Virology. 1998; 248: 305-311Crossref PubMed Scopus (166) Google Scholar), a natural cell substrate for ASLV infection and replication and highly purified from cell culture supernatants for this study. We used this biologically active, complex glycoprotein to develop a simple, automated method for acquiring high resolution mass spectrometry data that enabled the identification of disulfide-linked peptides, the assignment of most of the disulfide bonds, and the identification of the sites of N-linked glycosylation from a single HPLC run. All of the proteases used, trypsin, chymotrypsin, and Asp-N- endoproteinase, were sequencing grade and purchased from Roche Diagnostics. The PNGase F was purchased from New England Biolabs, and the Zwittergent 3-16 from EMD Chemicals (Gibbstown, NJ). The solvents were purchased from Honeywell Burdick and Jackson (Morristown, NJ), and the trifluoroacetic acid (TFA) and formic acid were purchased from Fluka. The tris (2-carboxyethyl) phosphine (TCEP) and N-ethylmorpholine were purchased from Sigma-Aldrich. To aid in the purification of the SUATM129 glycoprotein, a 10-histidine residue tag preceded by the TEV protease cleavage sequence was added in-frame after amino acid 469 of the SR-A enveloped protein (Fig. 1). An adapter plasmid encoding the SUA-rIgG immunoadhesin, PUCCLA112N-SUA-rIgG, was described previously (23Holmen S.L. Federspiel M.J. Virology. 2000; 273: 364-373Crossref PubMed Scopus (26) Google Scholar). The IgG region was replaced with the SalI to MfeI fragment of the SR-A env gene yielding a coding region that begins with a consensus Kozac start site, NcoI site, and encodes the complete leader region and the SR-A envelope to residue 469. Oligonucleotides were then used to add the TEV protease and His10 sequences by cloning as an MfeI to PstI fragment into PUCCLA112-SUATM129. The complete and tagged SUATM129 expression cassette was isolated as a ClaI fragment and cloned into the TFANEO expression plasmid under the transcriptional control of SR-A LTRs (32Federspiel M.J. Crittenden L.B. Hughes S.H. Virology. 1989; 173: 167-177Crossref PubMed Scopus (29) Google Scholar). TFANEO also encodes a neo expression cassette for selection. DF-1 cells (30Himly M. Foster D.N. Bottoli I. Iacovoni J.S. Vogt P.K. Virology. 1998; 248: 295-304Crossref PubMed Scopus (343) Google Scholar, 31Schaefer-Klein J. Givol I. Barsov E.V. Whitcomb J.M. VanBrocklin M. Foster D.N. Federspiel M.J. Hughes S.H. Virology. 1998; 248: 305-311Crossref PubMed Scopus (166) Google Scholar) were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 units of penicillin/ml, and 100 μg of streptomycin/ml (Quality Biological, Gaithersburg, MD) at 39 °C and 5% CO2. DF-1 cells transfected with the TFANEO-SUATM129 plasmid were grown in 500 μg of G418/ml to select for neomycin-resistant cells. Clones were isolated using cloning cylinders (Bellco Glass Inc., Vineland, NJ), expanded, and maintained with standard medium supplemented with 250 μg/ml G418. When the SUATM129-expressing cells were confluent, the medium was changed to a serum-free medium, VP-SFM (Invitrogen) supplemented with 4 mm l-glutamine (Invitrogen), harvested 24 h later, and cleared of debris using a 3 μm capsule filter (12116; PALL). The cleared supernatant was mixed with TALON Metal Affinity Resin (Clontech), prewashed, and equilibrated with wash buffer (50 mm sodium phosphate, pH 7.0, 300 mm NaCl) for 90 min at room temperature to bind the SUATM129 protein His10 tag, and then loaded into a chromatography column by gravity flow. The resin was washed with wash buffer (500 bed volumes) and then washed with 2.5 mm imidazole followed by 10 mm imidazole in wash buffer (100 bed volumes each). The resin was then eluted first with 5 bed volumes of 75 mm imidazole followed by 5 bed volumes of 250 mm imidazole in wash buffer, and the fractions were combined, concentrated and the buffer exchanged with 100 mm sodium phosphate, pH 7.0, using JumboSep concentrators with 10K filters (PALL). To purify the SUATM129 protein further from contaminating proteins, ammonium sulfate was added to the preparation, resulting in a loading buffer of 1 m ammonium sulfate, 100 mm sodium phosphate, pH 7.0, and loaded onto an octyl-Sepharose column. The column was washed with a gradient exchanging the load buffer to 100 mm sodium phosphate, pH 7.0, buffer. The SUATM129 protein was eluted first with water, recovering ∼60–70% of the protein, followed by elution with 20% ethanol that recovered some additional SUATM129 protein. Fractions containing SUATM129 protein were combined and concentrated, and the buffer was exchanged with 10 mm HEPES, pH 7.4, 100 mm NaCl, 5% glycerol for storage. To analyze reduced proteins, the samples were adjusted to 1× Laemmli loading buffer (2% SDS, 10% glycerol, 50 mm Tris-Cl, pH 6.8, 5% β-mercaptoethanol, 0.1% bromphenol blue) and boiled for 5 min. To analyze nonreduced proteins, the samples were adjusted to 1× Laemmli loading buffer without the β-mercaptoethanol (2% SDS, 10% glycerol, 50 mm Tris-Cl, pH 6.8, 0.1% bromphenol blue) and boiled for 5 min. To analyze native proteins, the samples were adjusted to 1× Laemmli loading buffer without the SDS and β-mercaptoethanol (10% glycerol, 50 mm Tris-Cl, pH 6.8, 0.1% bromphenol blue) and loaded directly. Proteins were separated on Criterion 4–15% Tris-HCl gradient polyacrylamide gels (Bio-Rad) and either silver stained directly (33Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3742) Google Scholar), or the proteins were transferred onto nitrocellulose membranes. The Western transfer filters were blocked in phosphate-buffered saline (PBS) with 10% nonfat dry milk for 1 h at 25 °C. The filters were then rinsed briefly in rinse buffer (100 mm NaCl, 10 mm Tris-Cl, pH 8, 1 mm EDTA, 0.1% Tween 20) and incubated with either an unconjugated anti-His antibody (1:3000 dilution) (27471001; GE Healthcare) or anti-ASLV SU(A) monoclonal antibody (34Ochsenbauer-Jambor C. Delos S.E. Accavitti M.A. White J.M. Hunter E. J. Virol. 2002; 76: 7518-7527Crossref PubMed Scopus (14) Google Scholar) (purified from the mc8C5 hybridoma, a kind gift from Christina Ochsenbauer-Jambor and Eric Hunter, University of Alabama at Birmingham, AL) (1:1000 dilution) in rinse buffer containing 1% nonfat dry milk for 1 h at 25 °C. The filters were washed extensively with rinse buffer and then incubated with 50 ng/ml peroxidase-labeled goat anti-mouse IgG (H+L) (Kirkegaard and Perry, Gaithersburg, MD) in rinse buffer with 1% nonfat dry milk for 1 h at 25 °C. After extensive washing with rinse buffer, immunodetection of the protein-antibody-peroxidase complexes was performed with Western blotting Chemiluminescence Reagent (PerkinElmer Life Sciences). The immunoblots were then exposed to Kodak X-Omat film. For alkaline phosphatase assays, DF-1 cell cultures (∼30% confluent) were incubated with 10-fold serial dilutions of the appropriate RCASBP/alkaline phosphatase virus stocks for 36–48 h. The assay for alkaline phosphatase activity was described previously (22Holmen S.L. Salter D.W. Payne W.S. Dodgson J.B. Hughes S.H. Federspiel M.J. J. Virol. 1999; 73: 10051-10060Crossref PubMed Google Scholar). Trypsin digestions were done as follows. 100 μg of SUATM129 was initially digested with 2.5 μg of trypsin in 100 mm Tris, pH 8.4, 0.004% Zwittergent 3-16 buffer at 55 °C for 2 h. An additional 2.5 μg of trypsin in 100 mm Tris, pH 8.4, 0.004% Zwittergent 3-16 buffer was then added to the reaction and incubated at 37 °C overnight. Chymotrypsin digestions were done as follows. 100 μg of purified SUATM was digested with 5 μg of chymotrypsin in 100 mm Tris, pH 8.5, 0.004% Zwittergent 3-16 buffer at room temperature overnight. To remove all N-linked glycosylation, PNGase F (500 units/μl) (3000 units) was added to the postdigest reactions and incubated at 37 °C for 3 h. Each digest mixture was split into two aliquots, with the disulfide bonds reduced in one aliquot by adding TCEP (25 mm final) and incubating at 55 °C for 2 h. The nonreduced or reduced peptide mixtures were each separated with a Zorbax 300 SB-C18, 5-μm, 150 × 0.5-mm reverse phase column using an Agilent 1100 Series Capillary HPLC system at a flow rate of 15 μl/min and collecting 1-min fractions using gradients described in Table 1. Detection was by UV absorbance at 214 nm. All fractions were then dried down using a SpeedVac spinning concentrator and stored at −80 °C. Fractions were reconstituted with 40% acetonitrile, 5% isopropyl alcohol, and 0.2% formic acid prior to analysis by direct chip-based infusion Fourier Transform ion cyclotron resonance mass spectrometry using an LTQ-FTICR-MS with electron capture dissociation (ECD) and an Advion Nanomate 100.TABLE 1HPLC running conditions for peptide separationProteaseGradientTime% BminSUATM/trypsinPhase A: 5% acetonitrile/0.1% TFAPhase B: 80% acetonitrile/0.1% TFA0515511545117801208012251355SUATM/chymotripsinPhase A: 5% acetonitrile/0.1% TFAPhase B: 80% acetonitrile/0.1% TFA0252108593069807280732782 Open table in a new tab Endoproteinase Asp-N was utilized for the fractions found to contain adjacent cysteines. The digests were performed on dried HPLC fractions that were resolubilized in 100 mm Tris, pH 8.4, with 4 ng of Asp-N and digested at 37 °C overnight prior to analysis by nanoLC-ESI-MS/MS. The fractions that were analyzed by chip-based direct infusion FTICR-ECD-MS were redried and solubilized in 0.2% N-ethylmorpholine buffer plus 1 ng of Asp-N and digested at 37 °C for 1.5 h. The HPLC fractions were resolubilized in 30 μl of 40% acetonitrile, 0.2% formic acid, 5% isopropyl alcohol. All disulfide-linked peptides were identified by screening the fractions with chip-based ESI infusion using an Advion Nanomate 100 coupled to a ThermoFinnigan LTQ-FT Hybrid Ion Cyclotron Resonance Mass Spectrometer upgraded with the ECD capability. The Nanomate was set to pick up 6 μl of sample and spray at 1.5 kV with N2 back-pressure of 0.3 pound/square inch. Each fraction was screened for 3 min, performing data-dependent acquisitions consisting of a FT full scan from 300 to 2000 m/z with the resolving power set at 100,000 @ 400 m/z followed by an ECD scan from 100–2000 m/z (3 microscans, 100,000 R, energy = 4.0, delay = 10 ms, duration = 100 ms) on ions with charge states of [M+2H]2+ or higher, plus a linear ion trap collision-induced dissociation (CID) scan on ions with charge states of [M+2H]2+ and [M+3H]3+. Each ion was analyzed twice then placed on an exclusion list for 60 s. All ECD spectra were analyzed manually by looking for the presence of theoretical trypsin-cleaved peptide ions. Non-cysteine-containing peptide ions were identified from analysis of the CID-MS/MS spectra. ThermoFinnigan Xcalibur QualBrowser software was used to determine the theoretical isotopic distributions for the isotope clusters. Fractions containing peptides with adjacent cysteines were analyzed by nanoLC-ESI-MS/MS using a ThermoFinnigan LTQ Orbitrap Hybrid Mass Spectrometer (Thermo Fisher Scientific) coupled to a nanoLC-two-dimensional HPLC system (Eksigent, Dublin, CA). The chymotrypsin fraction containing the C1 and C17C18C19 peptides was treated with Asp-N protease, after which an aliquot was diluted with 0.15% formic acid and 0.05% TFA and loaded onto a 250-nl OPTI-PAK trap (Optimize Technologies, Oregon City, OR) custom packed with Michrom Magic C8 solid phase (Michrom Bioresources, Auburn, CA). Chromatography was performed using 0.2% formic acid in both the A solvent (98% water and 2% acetonitrile) and B solvent (80% acetonitrile, 10% isopropyl alcohol, and 10% water), and a 10% B to 50% B gradient over 45 min at 325 nl/min through a hand-packed PicoFrit (New Objective, Woburn, MA) nanobore (Michrom Magic C18 3-μm 75 μm × 200 mm) column. The LTQ Orbitrap mass spectrometer experiment consisted of a FT full scan from 300 to 1200 m/z with resolution set at 60,000 (at 400 m/z), followed by Orbitrap CID MS/MS scans on the top three ions at 30,000 resolution. Dynamic exclusion was set to 2, and selected ions were placed on an exclusion list for 30 s. The lock-mass option was enabled for the FT full scans using the ambient air polydimethylcyclosiloxane ion of m/z = 445.120024 or a common phthalate ion m/z = 391.284286 for real-time internal calibration (35Olsen J.V. de Godoy L.M. Li G. Macek B. Mortensen P. Pesch R. Makarov A. Lange O. Horning S. Mann M. Mol. Cell Proteomics. 2005; 4: 2010-2021Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar). The MS/MS spectra ions were assigned manually. Relative abundance of the disulfide-linked peptides was determined for some of the HPLC fractions using Edman degradation N-terminal chemical sequencing on a Procise cLC Protein Sequencer (Applied Biosystems, Foster City, CA). The amino acid assignments were made manually, and the relative amounts of each peptide were determined from the amino acid yields of the first few sequencing cycles. The SUATM129 glycoprotein was produced in chicken cells, normal host cells for ASLV replication, to ensure appropriate envelope glycoprotein folding, glycosylation, and transport. DF-1 cell lines were established that stably produced SUATM129. SUATM129 was expressed as a secreted protein with a histidine tag to allow a relatively simple purification procedure of biologically active protein from cell culture supernatants in good yield (see “Experimental Procedures”). Biological activity was demonstrated by the ability of the SUATM129 protein to bind a soluble form of the ASLV subgroup A Tva receptor as shown by immunoprecipitation (data not shown) and the ability of SUATM129 to inhibit cell surface expressed Tva and specifically block ASLV(A) infection of normally susceptible cells by greater than 10–50-fold compared with another ASLV subgroup (data not shown). The calculated molecular mass of SUATM129 is 53,496 Da; the SUA subunit (37,265 Da) connected to the TM129 subunit (16,249 Da) by a disulfide bond. Previously published work on the secreted SUA-rIgG immunoadhesin, the complete SUA subunit fused to a rabbit IgG domain, identified that 10 of 11 potential N-linked glycosylation sites in SUA were glycosylated with ∼23 kDa of carbohydrate. If the SUATM129 glycoprotein contains similar levels of glycosylation, the SUATM129 glycosylated molecular mass would be a" @default.
• W2062212050 created "2016-06-24" @default.
• W2062212050 creator A5004040305 @default.
• W2062212050 creator A5021191650 @default.
• W2062212050 creator A5057920575 @default.
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• W2062212050 date "2011-05-01" @default.
• W2062212050 modified "2023-10-01" @default.
• W2062212050 title "Simple, Automated, High Resolution Mass Spectrometry Method to Determine the Disulfide Bond and Glycosylation Patterns of a Complex Protein" @default.
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