Determination of the Disulfide Bond Arrangement of Dengue Virus NS1 Protein
2004; Elsevier BV; Volume: 279; Issue: 20 Linguagem: Inglês
10.1074/jbc.m312907200
ISSN1083-351X
AutoresTristan P. Wallis, Chang‐Yi Huang, Subodh Nimkar, Paul R. Young, Jeffrey J. Gorman,
Tópico(s)Viral Infections and Vectors
ResumoThe 12 half-cystines of NS1 proteins are absolutely conserved among flaviviruses, suggesting their importance to the structure and function of these proteins. In the present study, peptides from recombinant Dengue-2 virus NS1 were produced by tryptic digestion in 100% H216O, peptic digestion in 50% H218O, thermolytic digestion in 50% H218O, or combinations of these digestion conditions. Peptides were separated by size exclusion and/or reverse phase high performance liquid chromatography and examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry, matrix-assisted laser desorption ionization post-source decay, and matrix-assisted laser desorption ionization tandem mass spectrometry. Where digests were performed in 50% H218O, isotope profiles of peptide ions aided in the identification and characterization of disulfide-linked peptides. It was possible to produce two-chain peptides containing C1/C2, C3/C4, C5/C6, and C7/C12 linkages as revealed by comparison of the peptide masses before and after reduction and by post-source decay analysis. However, the remaining four half-cystines (C8, C9, C10, and C11) were located in a three-chain peptide of which one chain contained adjacent half-cystines (C9 and C10). The linkages of C8/C10 and C9/C11 were determined by tandem mass spectrometry of an in-source decay fragment ion containing C9, C10, and C11. This disulfide bond arrangement provides the basis for further refinement of flavivirus NS1 protein structural models. The 12 half-cystines of NS1 proteins are absolutely conserved among flaviviruses, suggesting their importance to the structure and function of these proteins. In the present study, peptides from recombinant Dengue-2 virus NS1 were produced by tryptic digestion in 100% H216O, peptic digestion in 50% H218O, thermolytic digestion in 50% H218O, or combinations of these digestion conditions. Peptides were separated by size exclusion and/or reverse phase high performance liquid chromatography and examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry, matrix-assisted laser desorption ionization post-source decay, and matrix-assisted laser desorption ionization tandem mass spectrometry. Where digests were performed in 50% H218O, isotope profiles of peptide ions aided in the identification and characterization of disulfide-linked peptides. It was possible to produce two-chain peptides containing C1/C2, C3/C4, C5/C6, and C7/C12 linkages as revealed by comparison of the peptide masses before and after reduction and by post-source decay analysis. However, the remaining four half-cystines (C8, C9, C10, and C11) were located in a three-chain peptide of which one chain contained adjacent half-cystines (C9 and C10). The linkages of C8/C10 and C9/C11 were determined by tandem mass spectrometry of an in-source decay fragment ion containing C9, C10, and C11. This disulfide bond arrangement provides the basis for further refinement of flavivirus NS1 protein structural models. Globally, Dengue virus is an important human pathogen, with perhaps half of the world's population geographically at risk of infection, and with an estimated 50–100 million new infections, 500,000 hospitalizations, and 25,000 deaths annually (1Halstead S.B. Science. 1988; 239: 476-481Crossref PubMed Scopus (1310) Google Scholar, 2Jacobs M.G. Young P.R. Curr. Opin. Infect. Dis. 1998; 11: 319-324Crossref PubMed Scopus (19) Google Scholar, 3Gubler D.J. Granoff A. Webster R.G. Encyclopedia of Virology. 2nd Ed. Academic Press, San Diego, CA1999: 375-384Crossref Google Scholar, 4Gubler D.J. Trends Microbiol. 2002; 10: 100-103Abstract Full Text Full Text PDF PubMed Scopus (1169) Google Scholar, 5Clarke T. Nature. 2002; 416: 672-674Crossref PubMed Scopus (44) Google Scholar). Dengue virus belongs to the flavivirus genus, one of three genera within the Flaviviridae family of viruses, which include pestiviruses and hepaciviruses (6Ruggli N. Rice C.M. Adv. Virus Res. 1999; 53: 183-207Crossref PubMed Google Scholar). Flavivirus virions consist of a single positive stranded RNA genome of ∼11 kb (7Rice C.M. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. 3rd Ed. Lippencott-Raven, Philadelphia, PA1996: 931-959Google Scholar). Flavivirus RNA encodes a single large polypeptide NH2-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS5-COOH (7Rice C.M. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. 3rd Ed. Lippencott-Raven, Philadelphia, PA1996: 931-959Google Scholar), which is cotranslationally and post-translationally processed into mature viral proteins (7Rice C.M. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. 3rd Ed. Lippencott-Raven, Philadelphia, PA1996: 931-959Google Scholar). The RNA is encapsidated within an icosahedral core of capsid (C) 1The abbreviations used are: C, capsid protein; NS, non-structural; capHPLC, capillary high performance liquid chromatography; CHCA, α-cyano-4-hydroxycinnamic acid; DHAP, 2,6-dihydroxyacetophenone; FPLC, fast protein liquid chromatography; Fuc, fucose; ISD, in-source decay; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MVE, Murray Valley encephalitis; PBS, phosphate-buffered saline; PSD, post-source decay; q, collision quadrupole; Q, quadrupole; rpHPLC, reverse phase high performance liquid chromatography; secHPLC, size exclusion high performance liquid chromatography; TCEP, tris(2-carboxyethyl)phosphine. proteins (8Schlesinger S. Schlesinger M.J. Fields B.N. Knipe D.M. Replication of Togaviridae and Flaviviridae. 1. Lippincott-Raven, New York1990: 697-711Google Scholar, 9Kuhn R.J. Zhang W. Rossmann M.G. Pletnev S.V. Corver J. Lenches E. Jones C.T. Mukhopadhyay S. Chipman P.R. Strauss E.G. Baker T.S. Strauss J.H. Cell. 2002; 108: 717-725Abstract Full Text Full Text PDF PubMed Scopus (1212) Google Scholar), and further enveloped in a host cell-derived lipid bilayer studded with membrane and envelope glycoproteins (10Russell P.K. Brandt W.E. Dalrymple J.M. Schlesinger R.W. The Togaviruses: Biology, Structure and Replication. Academic Press, New York1980: 503-529Crossref Google Scholar). The remaining proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are non-structural and are involved in various aspects of viral replication (7Rice C.M. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. 3rd Ed. Lippencott-Raven, Philadelphia, PA1996: 931-959Google Scholar). Flavivirus NS1 proteins exhibit a high degree of sequence homology (11Mackow E. Makino Y. Zhao B. Zhang Y.-M. Markoff I. Buckler-White A. Guiler M. Chanock R. Lai C.-J. Virology. 1987; 159: 217-228Crossref PubMed Scopus (110) Google Scholar), and contain no amino- or carboxyl-terminal membrane anchoring sequences (12Speight G. Coia A. Parker M.D. Westaway E.G. J. Gen. Virol. 1988; 69: 23-24Crossref PubMed Scopus (83) Google Scholar, 13Wright P.J. Cauchi M.R. Ng M.L. Virology. 1989; 171: 61-67Crossref PubMed Scopus (33) Google Scholar). However, the precise roles of NS1 in the flavivirus life cycle remain unclear. Immature NS1 exists as a hydrophilic monomer in the endoplasmic reticulum lumen, and is rapidly processed into a stable hydrophobic non-covalent homodimer, with the subunits interacting via their carboxyl termini (14Winkler G. Randolph V.B. Cleaves G.R. Ryan T.E. Stollar V. Virology. 1988; 162: 187-196Crossref PubMed Scopus (177) Google Scholar, 15Parrish C.R. Woo W.S. Wright P.J. Arch. Virol. 1991; 117: 279-286Crossref PubMed Scopus (14) Google Scholar). NS1 exists predominantly in the dimeric form, which is associated with intracellular and cell surface membranes (16Winkler G. Maxwell S.E. Ruemmler C. Stoller V. Virology. 1989; 171: 302-305Crossref PubMed Scopus (186) Google Scholar). A component of this cell surface association has been shown to be covalently linked via a glycosylphosphatidylinositol anchor (17Jacobs M.G. Robinson P.J. Bletchly C. Mackenzie J.M. Young P.R. FASEB J. 2000; 14: 1603-1610Crossref PubMed Google Scholar). NS1 is also secreted from infected cells as a soluble, detergent-labile hexamer (18Flamand M. Megret F. Mathieu M. Lepault J. Rey F.A. Deubel V. J. Virol. 1999; 73: 6104-6110Crossref PubMed Google Scholar, 19Crooks A.J. Lee J.M. Easterbrook L.M. Tinofeev A.V. Stephenson J.R. J. Gen. Virol. 1994; 75: 3453-3460Crossref PubMed Scopus (72) Google Scholar). Maturation of NS1 involves N-linked glycosylation with Dengue virus NS1 from all four serotypes containing two conserved N-linked glycosylation sites. When expressed in mammalian cells the cell-associated form has both of these sites occupied by high mannose moieties, whereas the secreted form comprises complex glycans on one site (implying post-translational glycan processing in the Golgi) with the other remaining in a high mannose form. Other flavivirus NS1 proteins may contain an additional occupied site (14Winkler G. Randolph V.B. Cleaves G.R. Ryan T.E. Stollar V. Virology. 1988; 162: 187-196Crossref PubMed Scopus (177) Google Scholar, 16Winkler G. Maxwell S.E. Ruemmler C. Stoller V. Virology. 1989; 171: 302-305Crossref PubMed Scopus (186) Google Scholar). Glycosylation of NS1 contributes to dimer stability and resultant membrane association because of increased hydrophobicity of the dimer (14Winkler G. Randolph V.B. Cleaves G.R. Ryan T.E. Stollar V. Virology. 1988; 162: 187-196Crossref PubMed Scopus (177) Google Scholar, 20Pryor M.J. Wright P.J. J. Gen. Virol. 1994; 75: 1183-1187Crossref PubMed Scopus (75) Google Scholar). Mutation of glycosylation sites has been shown to reduce viral RNA production and attenuate neurovirulence, suggesting a role for glycosylated NS1 dimers in viral RNA replication (20Pryor M.J. Wright P.J. J. Gen. Virol. 1994; 75: 1183-1187Crossref PubMed Scopus (75) Google Scholar, 21Pryor M.J. Wright P.J. Virology. 1993; 194: 769-780Crossref PubMed Scopus (74) Google Scholar, 22Pryor M.J. Gualano R.C. Lin B. Davidson A. Wright P.J. J. Gen. Virol. 1998; 79: 2631-2639Crossref PubMed Scopus (54) Google Scholar, 23Muylaert I.A. Chambers T.J. Galler R. Rice C.M. Virology. 1996; 222: 159-168Crossref PubMed Scopus (137) Google Scholar, 24Pletnev A.G. Bray M. Lai C.J. J. Virol. 1993; 67: 4956-4963Crossref PubMed Google Scholar, 25Mackenzie J. Jones M. Young P.R. J. Virol. Methods. 1996; 220: 232-240Google Scholar). NS1 dimers have been shown to interact with a number of other non-structural viral proteins and, via this association, with the viral RNA, and may be involved in assembly of the viral replicase complex and its localization to cytoplasmic membranes (23Muylaert I.A. Chambers T.J. Galler R. Rice C.M. Virology. 1996; 222: 159-168Crossref PubMed Scopus (137) Google Scholar, 26Muylaert I.R. Galler R. Rice C.M. J. Virol. 1997; 71: 291-298Crossref PubMed Google Scholar, 27Lindenbach B.D. Rice C.M. J. Virol. 1997; 71: 9608-9617Crossref PubMed Google Scholar, 28Lindenbach B.D. Rice C.M. J. Virol. 1999; 73: 4611-4621Crossref PubMed Google Scholar). NS1 also induces a protective host immune response, however, anti-NS1 antibodies do not function in a neutralizing capacity as NS1 is not present in flavivirus virions. It is likely that antibodies targeted to cell surface-associated NS1 potentiate complement mediated lysis of infected cells (29Schlesinger J.J. Brandriss M.W. Putnak J.R. Walsh E.E. J. Gen. Virol. 1990; 71: 593-599Crossref PubMed Scopus (82) Google Scholar). The importance of NS1 to the flavivirus life cycle and immune response makes it an attractive target for development of immune-based and structure/function-based antiviral therapeutics. Mature Dengue virus NS1 contains 352 amino acid residues in a base polypeptide of ∼40 kDa, with glycosylation increasing the apparent mass of the protein on SDS-PAGE (30Deubel V. Kinney R.M. Trent D.W. Virology. 1988; 165: 234-244Crossref PubMed Scopus (129) Google Scholar). Dengue virus NS1 contains 12 cysteine residues that are absolutely conserved among all flavivirus NS1 proteins (7Rice C.M. Fields B.N. Knipe D.M. Howley P.M. Fields Virology. 3rd Ed. Lippencott-Raven, Philadelphia, PA1996: 931-959Google Scholar, 31Gibson C.A. Schlesinger J.J. Barrett A.D.T. Vaccine. 1988; 6: 7-9Crossref PubMed Scopus (28) Google Scholar, 32Wang P. Geng L. Qin E. Tu M. Zhao W. J. Biochem. Mol. Biol. 2001; 17: 148-154Google Scholar), indicating their importance to the structure and function of the protein. The ability to form intramolecular disulfide linkages, particularly in the carboxyl terminus of the protein, appears to be crucial for NS1 dimer formation and subsequent trafficking within and secretion from the cell (21Pryor M.J. Wright P.J. Virology. 1993; 194: 769-780Crossref PubMed Scopus (74) Google Scholar). Disulfide-bonded NS1 monomers form non-covalent dimers that are stable to reduction but can be dissociated by heating, indicating that disulfide bonds are required to stabilize the structure of NS1 monomers for them to initially associate as dimers, but are not required for subsequent dimer stability (21Pryor M.J. Wright P.J. Virology. 1993; 194: 769-780Crossref PubMed Scopus (74) Google Scholar). In the absence of crystallographic data, the disulfide bond configuration and glycosylation pattern may provide valuable information on protein folding and hence facilitate refinement of molecular models (33Wedemeyer W.J. Welker E. Narayan M. Scheraga H.A. Biochemistry. 2000; 39: 4207-4216Crossref PubMed Scopus (530) Google Scholar, 34Pitt J.J. Da Silva E. Gorman J.J. J. Biol. Chem. 2000; 275: 6469-6478Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The disulfide linkage arrangement of the NS1 protein of the related Murray Valley encephalitis (MVE) flavivirus has been partially described (35Blitvich B.J. Scanlon D. Shiell B.J. Mackenzie J.S. Pham K. Hall R.A. J. Gen. Virol. 2001; 82: 2251-2256Crossref PubMed Scopus (24) Google Scholar) by a combination of tryptic cleavage, reverse phase high performance liquid chromatography (rpHPLC) to isolate tryptic disulfide-linked peptides, and peptide analysis by Edman protein sequencing and/or electrospray ionization mass spectrometry (ESI-MS). Using this technique, the disulfide linkage pattern of the 6 half-cystines from the amino terminus of MVE NS1 was determined to be C1/C2, C3/C4, and C5/C6. The disulfide linkage pattern of the 6 halfcystines from the carboxyl terminus was not determined, because of the lack of amenable tryptic cleavage sites in this region of the protein, and a consequent inability to obtain disulfide-linked tryptic peptides. Similar approaches were used in this study to confirm that the 3 amino-terminal disulfide linkages of Dengue virus NS1 were the same as for MVE NS1. In addition, pepsin and thermolysin were used to overcome the resistance of the carboxylterminal half of NS1 to tryptic digestion. Recombinant Dengue-2 virus NS1 was digested with these enzymes in buffers containing 50% H 182O to identify disulfide-linked peptide ions during mass spectrometry (MS), by virtue of their characteristic isotope profiles (36Wallis T.P. Pitt J.J. Gorman J.J. Protein Sci. 2001; 10: 2251-2271Crossref PubMed Scopus (42) Google Scholar, 37Gorman J.J. Wallis T.P. Pitt J.J. Mass Spectrom. Rev. 2002; 21: 183-216Crossref PubMed Scopus (224) Google Scholar). Disulfide-linked peptides were isolated by size exclusion HPLC (secHPLC), further purified where necessary by capillary HPLC (capHPLC), and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), matrix-assisted laser desorption ionization post-source decay (MALDI-PSD), and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS). Together these data confirmed the previously determined disulfide linkages of the NS1 amino terminus and allowed determination of the remaining carboxyl-terminal disulfide linkages. These techniques enabled the definition of the complete disulfide linkage arrangement of Dengue virus NS1 and will contribute to the refinement of molecular models of this important protein. All reagents used in this study were of analytical or HPLC grade. Generation of Baculovirus Shuttle Vector Encoding NS1—C6/36 insect cells were infected with Dengue-2 virus strain PR159 at a multiplicity of infection of 1.0 and incubated at 32 °C for 3 days. Infected cell RNA was then extracted with RNazol B (Tel-Test Inc.) according to the manufacturer's instructions. cDNA was generated from this infected cell RNA extract using the NS1RBglII reverse primer 5′-ATTAGATCTCAGGCTGTGACCAAGGAGTTGAC-3′ (restriction sites are underlined), which was then used as template in a PCR employing this primer and NS1FBglII, 5′-ATTAGATCTCGGATAGTGGTTGCGTTGTGAGC-3′. The resulting PCR product comprised the full-length NS1 sequence flanked by BglII sites for cloning into the baculovirus shuttle vector pVTBac.His (generated and kindly provided by Dr. A. Khromykh). Cloning into the BglII site of this vector results in the expression by the final recombinant baculovirus of a fusion protein comprising a melittin signal sequence followed immediately by a 6-histidine tag and then the cloned insert. Such fusion proteins are targeted to the endoplasmic reticulum of infected insect cells for secretion as NH2-terminal His-tagged species. The PCR product was digested with BglII and ligated into BglII cut pVTBac.His. Selected recombinants were analyzed by automated sequence analysis to confirm correct orientation and sequence. Generation of Recombinant Baculovirus, and Expression and Purification of NS1—The pVTBac.His.NS1 shuttle vector was co-transfected with linearized (digested with Bsu36I) BacPak6 DNA (Clontech) into Spodoptera frugiperda (Sf9) cells according to the manufacturer's instructions. Recombinant baculoviruses liberated into the culture media were isolated by plaque analysis and blue-white selection using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). Several plaque picks were amplified in culture and expression of NS1 assessed by immunoblot analysis using NS1-specific monoclonal antibodies with the final selected recombinant designated AcHis.D2.NS1. For high level recombinant protein expression, multiple 800-ml suspension cultures of HIGH FIVE (Invitrogen, Australia) cells in 2-liter flasks and at an initial concentration of 2 × 106 cells/ml were infected with AcHis.D2.NS1 virus at a multiplicity of infection of 1.0. Cells were grown in Hyclone HyQ SFX (Hyclone) serum-free insect cell media at 28 °C and shaken at 120 rpm on an orbital shaker. The cells were harvested and collected by centrifugation at 4,000 × g at 4 °C. Supernatant containing the secreted recombinant NS1 protein was filtered through a 0.2-μm Millipore filter unit (Millipore, Australia) at room temperature. The flow-through was then subjected to ultrafiltration and buffer exchange through two Vivaflow200 (Sartorius, Australia) units at room temperature. Final concentration and buffer exchange reduced the original harvest volumes that were in excess of 2 liters to 50 ml in a buffer comprising 1 mm imidazole, 0.15 m NaCl, and 20 mm Tris, pH 8.0. This concentrate was applied to a 2-ml nickel-nitrilotriacetic acid (Qiagen) column at a flow rate of 0.5 ml/min. The column was washed with 20 mm imidazole, 0.15 m NaCl, 20 mm Tris, pH 8.0, and then eluted with 100 mm imidazole, 0.15 m NaCl, and 20 mm Tris, pH 8.0, at room temperature. Elution fractions were analyzed for purity by 15% SDS-PAGE, pooled, and concentrated through a Centricon spin column. The yield of purified AcHis.D2.NS1 was routinely ∼1 mg/liter. Pooled fractions of nickel-nitrilotriacetic acid-purified NS1 were concentrated by Vivaspin with 50-kDa molecular mass cut-off (Sartorius, Germany) at room temperature and passed through a 0.2-μm filter before loading onto a Superdex 200 HR 10/30 column (Amersham Biosciences) linked to an ÄKTA™ FPLC™ system (Amersham Biosciences). FPLC was performed in 20 mm Tris, 0.15 m NaCl, pH 8, with a flow rate of 0.6 ml/min at 4 °C. The column was calibrated in a separate run using High and Low FPLC™ standard molecular masses of 440, 232, 67, 43, 25, and 13.7 kDa (ferritin, catalase, bovine serum albumin, chymotrypsinogen A, and ribonuclease A, respectively, from Amersham Biosciences). The elution profile data were collected and analyzed by UNICORN™ version 2.31 (Amersham Biosciences). A standard plot was obtained by plotting the diffusion constant Kav, which was calculated from the equation Kav = (elution volume–void volume)/(column bed volume–void volume), versus log Mr (relative weight). The standard curve was computed using EXCEL™ (Microsoft) and a standard formula was generated as log Mr = –3.2991 × (Kav) + 3.2041. The elution volume for NS1 in this column was 11.53 ml, which was used to obtain a Kav = 0.2404 and an Mr of 257,600. The secreted recombinant NS1 was assayed against an extended panel of both linear sequence and conformational specific monoclonal antibodies (38Falconar A.K. Young P.R. J. Gen. Virol. 1991; 72: 961-965Crossref PubMed Scopus (62) Google Scholar). Each binding experiment was done in triplicate and the volume used per well was 50 μl. Immulon 4 microtiter plates were coated with 0.01 mg/ml Protein A (Pharmacia) in coating buffer overnight at 4 °C. After washing the plates with 0.05% Tween 20 in phosphate-buffered saline (PBST), 150 μl of blocking solution of 1% gelatin in phosphate-buffered saline (PBS) was added to each well and left at room temperature for 1 h. The plates were washed with PBST and then 0.018 mg/ml rabbit anti-mouse IgG (H&L) in PBST, 0.25% gelatin was allowed to bind to the Protein A for 1 h at 37 °C. Following four washes with PBST, relevant monoclonal antibodies (diluted 1/100 in PBST, 0.25% gelatin) were added and incubated at 37 °C for 1 h. After removing monoclonal antibodies and washing, immunoaffinity purified 35S-labeled NS1 (2,000 cpm/well) in PBST, 0.25% gelatin was added to the plate and incubated at 37 °C for 1 h. The plate was then washed four times with PBST, and disruption solution (2% SDS, 1% 2-mercaptoethanol in SDS-PAGE sample buffer) was added to each well. After 10 min incubation at room temperature the disruption solution was removed individually from each well, added to 1.0 ml of scintillation fluid (Optiphase HiSafe II, Wallac), and counted in a scintillation counter (Amersham Biosciences). Trypsin Digestion—100 μg of NS1 was co-precipitated with 2 μg of trypsin (Roche modified sequencing grade) by addition of 10 volumes of methanol at –20 °C, and incubation at –20 °C overnight. The precipitate was pelleted by centrifugation at 12,000 × g for 10 min at 4 °C in a microcentrifuge, air dried, resuspended in 20 μl of freshly prepared 100 mm ammonium bicarbonate, pH 8.0, in H 162O, and incubated for 2 h at 37 °C. An additional 3 μg of trypsin was then added, to give a final trypsin:NS1 ratio of 1:20 (w/w), and the reaction incubated for a further 3 h at 37 °C. Digestion was terminated by storage at –20 °C. Tryptic subdigestions of isolated peptides were performed by resuspending dried peptides in 20 μl of 100 mm ammonium bicarbonate in H 162O containing trypsin at 5 ng/μl, and incubating at 37 °C for 3 h. Pepsin Digestion—100 μg of NS1 was precipitated by addition of 10 volumes of methanol at –20 °C, and incubation was at –20 °C overnight. The precipitate was pelleted and air dried as described above, and resuspended in 20 μl of buffer containing 100 mm formic acid, 100 mm acetic acid, 1 μg of pepsin (Sigma number P6887) and H 182O (97%, Enritech, diluted to give a final concentration of 50% (v/v)). Digestion was conducted at 37 °C for 3 h and terminated by storage at –20 °C or immediate secHPLC as described below. Thermolysin Digestion—100 μg of NS1 was precipitated, pelleted, and air dried as described above, and resuspended in 20 μl of buffer containing 100 mm ammonium bicarbonate, pH 8, 10 mm CaCl2, 5 μg of thermolysin (Roche number 161586) and H 182O at a final concentration of 50% (v/v). Digestion was conducted at 65 °C for 2 h, and terminated by storage at –20 °C. All peptide chromatography was performed on Agilent 1100 liquid chromatography systems running Chemstation software, with peptide absorbance monitored at 214 nm. SecHPLC was performed using a 3.2 mm × 30-cm Superdex Peptide column (Amersham Biosciences). Peptides were separated over 90 min in 0.05% (v/v) aqueous trifluoroacetic acid in 6% (v/v) aqueous acetonitrile at 30 μl/min. Peptides were separated by rpHPLC using a 90-min gradient from 0.05% (v/v) aqueous trifluoroacetic acid to 0.045% (v/v) trifluoroacetic acid in 80% (v/v) aqueous acetonitrile. Flow rates of 40 and 5 μl/min were used where rpHPLC was performed using a 1 mm × 25-cm C18 column (Vydac) and 0.5 mm × 15-cm C18 capHPLC column (Zorbax), respectively. A constant temperature of 25 °C was used for capHPLC. To prevent unwanted exchange of 18O from labeled peptides with aqueous buffers, collected peptides were immediately freeze dried following chromatography using a centrifugal evaporator (Heto) and stored dried at –20 °C. MALDI-TOF-MS—Reduced and non-reduced samples were analyzed using a Bruker Reflex MALDI-TOF mass spectrometer in positive ion reflector mode. Data were acquired and analyzed using the Bruker XMass suite of software as described previously (39Gorman J.J. Ferguson B.L. Nguyen T.B. Rapid Commun. Mass. Spectrom. 1996; 10: 529-536Crossref PubMed Scopus (69) Google Scholar). 2,6-Dihydroxyacetophenone (DHAP, Fluka)/diammonium hydrogen citrate (Fluka) matrix was prepared as described previously (39Gorman J.J. Ferguson B.L. Nguyen T.B. Rapid Commun. Mass. Spectrom. 1996; 10: 529-536Crossref PubMed Scopus (69) Google Scholar). Samples were diluted 1:5 in 33% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid prior to analysis. 1–2 μl of diluted sample was mixed with an equivalent volume of matrix, 1 μl deposited on a Bruker Scout 26 MALDI target, and allowed to air dry for 10 min before analysis. Peptides were reduced with Tris(2-carboxyethyl)phosphine (TCEP, Pierce) and analyzed using a previously described modification of the DHAP/diammonium hydrogen citrate protocol (34Pitt J.J. Da Silva E. Gorman J.J. J. Biol. Chem. 2000; 275: 6469-6478Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 39Gorman J.J. Ferguson B.L. Nguyen T.B. Rapid Commun. Mass. Spectrom. 1996; 10: 529-536Crossref PubMed Scopus (69) Google Scholar). MALDI-PSD—Analysis was performed using the Bruker Reflex mass spectrometer and samples were prepared in α-cyano-4-hydroxycinnamic acid (CHCA) matrix essentially as described previously (40Gorman J.J. Ferguson B.L. Speelman D. Mills J. Protein Sci. 1997; 6: 1308-1315Crossref PubMed Scopus (63) Google Scholar, 41Lopaticki S. Morrow C.J. Gorman J.J. J. Mass. Spectrom. 1998; 33: 950-960Crossref PubMed Google Scholar). Variation to the previously described procedures involved use of a 100-ns delay before extraction of ions from the source, and data acquisition using a 2 GHz digitizer. MALDI-MS/MS—Analysis was performed using a Q-STAR XL hybrid Quadrupole quadrupole (Qq)-TOF-MS equipped with an O-MALDI 2 source (Applied Biosystems MDS Sciex). All samples were dissolved in 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid and crystallized with CHCA matrix (Agilent) by spotting 0.5 μl of matrix + 0.5 μl of sample and allowing air drying at ambient temperature. TOF-MS data were acquired in the mass range of 1000–4000 atomic mass units. The mass spectrometer was calibrated using Glu-fibrinopeptide B in MS/MS mode. Subsequent MS and MS/MS data on the samples were acquired using external calibration. In this external calibration mode ∼30 ppm or better mass accuracy was obtained with mass spectrometer resolution of ∼10,000. The O-MALDI 2 source is equipped with a N2 laser that was operated at 7–8 μJ. The same laser power (energy) was used for MS and MS/MS experiments. MS/MS data were acquired with slightly open resolution (about 4-atomic mass unit wide open window for precursor selection in Quadrupole 1) to allow transmission of all the isotopes of the high mass peptide (2858 atomic mass units) for fragmentation. The collision energy (Q0–RO2) used varied depending on peptide mass and was 90 eV for the peptide at 2858 atomic mass units, 75 eV at 1700 atomic mass units, and around 70 eV at 1500 atomic mass units. Acquisition time (specified as accumulation time in the software) for one spectrum was 1 s for MS and MS/MS, and all the data were acquired in MCA mode (multiple channel addition or summed spectra) over a period of 30 s to 2 min. Analyst QS software was used for all the data acquisition software. Under the normal operating conditions of the O-MALDI 2 source, very little in-source fragmentation of tryptic peptides is observed. In this case of disulfide-linked peptides some insource fragmentation did occur and can be seen in MS spectra. Most of the disulfide-linked peptide remained intact for MS/MS analysis. For MS/MS/MS experiments slightly higher laser power (8–9 μJ) was used. Because of the orthogonal nature of this TOF instrument use of higher laser power has no effect on instrument resolution or mass accuracy. Peptide and fragment ion masses were localized to the NS1 sequence using PAWS freeware version 8.4 (Proteometrics), and Bioanalyst (Applied Biosystems) software. The 12 half-cystines of NS1 are indicated as C1 to C12 (starting from the amino proximal half-cystine), with interchain disulfide linkages indicated with a forward slash, C3/C4, C5/C6, and so on (34Pitt J.J. Da Silva E. Gorman J.J. J. Biol. Chem. 2000; 275: 6469-6478Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Brackets are used to indicate that alternative disulfide linkages were possible. C8/[C9,C10]/C11 thus indicates a three-chain peptide where separate peptides containing C8 and C11 are linked to a peptide containing C9 and C10, although the exact disulfide linkage pa
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