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• W2163442590 abstract "Using high-resolution MS-based proteomics in combination with multiple protease digestion, we profiled, with on average 90% sequence coverage, all 13 viral proteins present in an human adenovirus (HAdV) vector. This in-depth profile provided multiple peptide-based evidence on intrinsic protease activity affecting several HAdV proteins. Next, the generated peptide library was used to develop a targeted proteomics method using selected reaction monitoring (SRM) aimed at quantitative profiling of the stoichiometry of all 13 proteins present in the HAdV. We also used this method to probe the release of specific virus proteins initiated by thermal stimulation, mimicking the early stage of HAdV disassembly during entry into host cells. We confirmed the copy numbers of the most well characterized viral capsid components and established the copy numbers for proteins whose stoichiometry has so far not been accurately defined. We also found that heating HAdV induces the complete release of the penton base and fiber proteins as well as a substantial release of protein VIII and VI. For these latter proteins, maturational proteolysis by the adenoviral protease leads to the differential release of fragments with certain peptides being fully released and others largely retained in the AdV particles. This information is likely to be beneficial for the ongoing interpretation of high resolution cryoEM and x-ray electron density maps.Background: Adenoviruses (AdV) are broadly employed as gene delivery vectors.Results: Copy numbers of all AdV proteins were measured, and the release of proteins upon heat stress investigated.Conclusion: The viral protease plays a distinct role in the segmented release of AdV proteins.Significance: Our characterization by mass spectrometry provides new insight in HAdV disassembly during entry into host cells. Using high-resolution MS-based proteomics in combination with multiple protease digestion, we profiled, with on average 90% sequence coverage, all 13 viral proteins present in an human adenovirus (HAdV) vector. This in-depth profile provided multiple peptide-based evidence on intrinsic protease activity affecting several HAdV proteins. Next, the generated peptide library was used to develop a targeted proteomics method using selected reaction monitoring (SRM) aimed at quantitative profiling of the stoichiometry of all 13 proteins present in the HAdV. We also used this method to probe the release of specific virus proteins initiated by thermal stimulation, mimicking the early stage of HAdV disassembly during entry into host cells. We confirmed the copy numbers of the most well characterized viral capsid components and established the copy numbers for proteins whose stoichiometry has so far not been accurately defined. We also found that heating HAdV induces the complete release of the penton base and fiber proteins as well as a substantial release of protein VIII and VI. For these latter proteins, maturational proteolysis by the adenoviral protease leads to the differential release of fragments with certain peptides being fully released and others largely retained in the AdV particles. This information is likely to be beneficial for the ongoing interpretation of high resolution cryoEM and x-ray electron density maps. Background: Adenoviruses (AdV) are broadly employed as gene delivery vectors. Results: Copy numbers of all AdV proteins were measured, and the release of proteins upon heat stress investigated. Conclusion: The viral protease plays a distinct role in the segmented release of AdV proteins. Significance: Our characterization by mass spectrometry provides new insight in HAdV disassembly during entry into host cells. Adenoviruses (AdVs) 7The abbreviations used are: AdVadenovirusHAdVhuman AdVTPterminal proteinAVPadenovirus proteinaseSRMselected reaction monitoring. are large (∼150 MDa protein content), double-stranded DNA viruses found in all vertebrates (1.Davison A.J. Benko M. Harrach B. et al.Genetic content and evolution of adenoviruses.J. Gen. Virol. 2003; 84: 2895-2908Crossref PubMed Scopus (424) Google Scholar). Human AdVs (HAdVs) are normally associated with mild infections but can cause potentially severe disease in immunocompromised individuals. First isolated in the early 1950s, HAdV has served as a model virus to elucidate the molecular basis of viral structure, replication, and pathogenesis (2.Reddy V.S. Natchiar S.K. Stewart P.L. Nemerow G.R. et al.Crystal structure of human adenovirus at 3.5 Å resolution.Science. 2010; 329: 1071-1075Crossref PubMed Scopus (191) Google Scholar, 3.Liu H. Jin L. Koh S.B. Atanasov I. Schein S. Wu L. Zhou Z.H. et al.Atomic structure of human adenovirus by cryo-EM reveals interactions among protein networks.Science. 2010; 329: 1038-1043Crossref PubMed Scopus (281) Google Scholar4.Nemerow G.R. et al.Cell receptors involved in adenovirus entry.Virology. 2000; 274: 1-4Crossref PubMed Scopus (160) Google Scholar), with notable contributions to the field of eukaryotic molecular biology (5.Chow L.T. Gelinas R.E. Broker T.R. Roberts R.J. et al.An amazing sequence arrangement at the 5′ ends of adenovirus 2 messenger RNA.Cell. 1977; 12: 1-8Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 6.Berget S.M. Moore C. Sharp P.A. et al.Spliced segments at the 5′ terminus of adenovirus 2 late mRNA.Proc. Natl. Acad. Sci. U.S.A. 1977; 74: 3171-3175Crossref PubMed Scopus (848) Google Scholar). Most importantly, HAdVs are broadly employed in molecular biotechnology as gene delivery vectors and DNA-based vaccine vehicles (7.Xin K.Q. Sekimoto Y. Takahashi T. Mizuguchi H. Ichino M. Yoshida A. Okuda K. et al.Chimeric adenovirus 5/35 vector containing the clade C HIV gag gene induces a cross-reactive immune response against HIV.Vaccine. 2007; 25: 3809-3815Crossref PubMed Scopus (13) Google Scholar, 8.Sharma A. Tandon M. Bangari D.S. Mittal S.K. et al.Adenoviral vector-based strategies for cancer therapy.Curr. Drug Ther. 2009; 4: 117-138Crossref PubMed Scopus (54) Google Scholar). Nevertheless, these applications are partly hampered by the immune response elicited by the capsid proteins, as well as the difficulty in targeting specific cell types. Structure-based redesign for vector cell targeting and alteration of antigenic properties is therefore needed, but this requires a detailed knowledge of the HAdV structure. The recently reported crystal structure (2.Reddy V.S. Natchiar S.K. Stewart P.L. Nemerow G.R. et al.Crystal structure of human adenovirus at 3.5 Å resolution.Science. 2010; 329: 1071-1075Crossref PubMed Scopus (191) Google Scholar) and high-resolution cryoelectron microscopy reconstruction (3.Liu H. Jin L. Koh S.B. Atanasov I. Schein S. Wu L. Zhou Z.H. et al.Atomic structure of human adenovirus by cryo-EM reveals interactions among protein networks.Science. 2010; 329: 1038-1043Crossref PubMed Scopus (281) Google Scholar) of intact HAdV have provided a more detailed molecular model of the structure and assembly of the HAdV particle (Fig. 1); however, a complete and accurate assignment of all capsid proteins is still lacking. The structure of the outer capsid is mostly well defined, but the stoichiometry and structure of the inner capsid and core proteins, many of them interacting with the DNA, remains elusive. HAdV assembles from 13 distinct proteins to form an icosahedral capsid (9.Burnett R.M. et al.The structure of the adenovirus capsid. II. The packing symmetry of hexon and its implications for viral architecture.J. Mol. Biol. 1985; 185: 125-143Crossref PubMed Scopus (73) Google Scholar). The main components are the 240 trimers of protein II (hexon) that comprise the faces and edges of the capsid structure. Polypeptide III (penton) is present in 60 copies and forms 12-penton base complexes centered on the 12 vertices of the icosahedron. Extending outward from each penton base are 12 polypeptide IV (fiber) trimers (10.Liu H. Wu L. Zhou Z.H. et al.Model of the trimeric fiber and its interactions with the pentameric penton base of human adenovirus by cryo-electron microscopy.J. Mol. Biol. 2011; 406: 764-774Crossref PubMed Scopus (34) Google Scholar). The locations, interactions, and stoichiometries of these three proteins are revealed in the crystal structure and EM models. However, HAdV particles contain 10 additional proteins, for which precise structural information remains unclear. Four minor cement proteins are primarily associated with the capsid shell proteins IIIa, VI, VIII, and IX. The genomic core of the virion is associated with six additional proteins, i.e. V, VII, Mu, IVa2, terminal protein, and the 23K maturational protease. An amazing attribute of one of these cement proteins, VI, is that it serves several other crucial functions in the virus life cycle (11.Wiethoff C.M. Wodrich H. Gerace L. Nemerow G.R. et al.Adenovirus protein VI mediates membrane disruption following capsid disassembly.J. Virol. 2005; 79: 1992-2000Crossref PubMed Scopus (328) Google Scholar, 12.McGrath W.J. Ding J. Didwania A. Sweet R.M. Mangel W.F. et al.Crystallographic structure at 1.6-A resolution of the human adenovirus proteinase in a covalent complex with its 11-amino-acid peptide cofactor: insights on a new fold.Biochim. Biophys. Acta. 2003; 1648: 1-11Crossref PubMed Scopus (28) Google Scholar13.Wodrich H. Guan T. Cingolani G. Von Seggern D. Nemerow G. Gerace L. et al.Switch from capsid protein import to adenovirus assembly by cleavage of nuclear transport signals.EMBO J. 2003; 22: 6245-6255Crossref PubMed Scopus (75) Google Scholar). Table 1 summarizes key characteristics of the proteins known to be present in HAdV particles. Developing a method to determine the exact stoichiometry for all of the HAdV proteins is likely to be beneficial to improving the x-ray and EM models of HAdV. MS-based proteomics techniques have emerged as the method of choice to characterize the protein content of a given biological sample (14.Altelaar A.F. Munoz J. Heck A.J. et al.Next-generation proteomics: towards an integrative view of proteome dynamics.Nat. Rev. Genet. 2013; 14: 35-48Crossref PubMed Scopus (521) Google Scholar, 15.Mann M. Kulak N.A. Nagaraj N. Cox J. et al.The coming age of complete, accurate, and ubiquitous proteomes.Mol. Cell. 2013; 49: 583-590Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar16.Bensimon A. Heck A.J. Aebersold R. et al.Mass spectrometry-based proteomics and network biology.Annu. Rev. Biochem. 2012; 81: 379-405Crossref PubMed Scopus (317) Google Scholar). Recent developments in new analyzers (17.Olsen J.V. Schwartz J.C. Griep-Raming J. Nielsen M.L. Damoc E. Denisov E. Lange O. Remes P. Taylor D. Splendore M. Wouters E.R. Senko M. Makarov A. Mann M. Horning S. et al.A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed.Mol. Cell Proteomics. 2009; 8: 2759-2769Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 18.Second T.P. Blethrow J.D. Schwartz J.C. Merrihew G.E. MacCoss M.J. Swaney D.L. Russell J.D. Coon J.J. Zabrouskov V. et al.Dual-pressure linear ion trap mass spectrometer improving the analysis of complex protein mixtures.Anal. Chem. 2009; 81: 7757-7765Crossref PubMed Scopus (122) Google Scholar19.Andrews G.L. Simons B.L. Young J.B. Hawkridge A.M. Muddiman D.C. et al.Performance characteristics of a new hybrid quadrupole time-of-flight tandem mass spectrometer (TripleTOF 5600).Anal. Chem. 2011; 83: 5442-5446Crossref PubMed Scopus (223) Google Scholar) and fragmentation methods (20.Syka J.E. Coon J.J. Schroeder M.J. Shabanowitz J. Hunt D.F. et al.Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9528-9533Crossref PubMed Scopus (1999) Google Scholar, 21.Olsen J.V. Macek B. Lange O. Makarov A. Horning S. Mann M. et al.Higher-energy C-trap dissociation for peptide modification analysis.Nat. Methods. 2007; 4: 709-712Crossref PubMed Scopus (719) Google Scholar) dramatically increased MS sensitivity, accuracy, and sequencing speed. Consequently, the characterization of several thousands of proteins expressed in complex biological samples (e.g. mammalian cells or tissues) is achievable employing two-dimensional chromatography strategies or even in a single shotgun LC-MS analysis (22.Cristobal A. Hennrich M.L. Giansanti P. Goerdayal S.S. Heck A.J. Mohammed S. et al.In-house construction of a UHPLC system enabling the identification of over 4000 protein groups in a single analysis.Analyst. 2012; 137: 3541-3548Crossref PubMed Scopus (42) Google Scholar, 23.Nagaraj N. Kulak N.A. Cox J. Neuhauser N. Mayr K. Hoerning O. Vorm O. Mann M. et al.System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap.Mol. Cell Proteomics. 2012; (10.1074/mcp.M111.013722)Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). A variety of methods have focused on the relative quantification of protein expression, defining protein level changes in two (or more) samples or conditions. Although typical MS-based methods can accurately detect expression changes of thousands of proteins, the estimation of their absolute abundance (e.g. essential to define the stoichiometry of a protein in a protein complex) remains a much more challenging task. The most commonly used approach relies on reference peptides that contain heavy isotope labels and are further chemically identical to the original peptides (so-called AQUA peptides (24.Gerber S.A. Rush J. Stemman O. Kirschner M.W. Gygi S.P. et al.Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6940-6945Crossref PubMed Scopus (1542) Google Scholar)). Relying on a subset of specific peptides, absolute protein quantification can be best approached by targeted proteomics strategies. Selected reaction monitoring (SRM) has emerged as a method to quantitatively assess the abundance of not only small molecules but also a targeted set of proteins. SRM assays are typically performed by LC-MS/MS employing triple quadrupole mass spectrometers. In this approach, the selectivity of two mass filters is applied to target a specific peptide via the combination of its precursor ion m/z and multiple diagnostic collision-induced dissociation fragment ions (named peptide transitions), along with the specific peptide retention time. This setup allows targeting of specific peptides even in complex samples and removes most of the background chemical noise, increasing both sensitivity and dynamic range (25.Picotti P. Aebersold R. et al.Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions.Nat. Methods. 2012; 9: 555-566Crossref PubMed Scopus (991) Google Scholar). To design an SRM assay, the transitions specific for each quantified peptide needs to be extracted from shotgun MS/MS spectra or, if available, from public repositories (26.Craig R. Cortens J.P. Beavis R.C. et al.Open source system for analyzing, validating, and storing protein identification data.J. Proteome Res. 2004; 3: 1234-1242Crossref PubMed Scopus (576) Google Scholar, 27.Desiere F. Deutsch E.W. King N.L. Nesvizhskii A.I. Mallick P. Eng J. Chen S. Eddes J. Loevenich S.N. Aebersold R. et al.The PeptideAtlas project.Nucleic Acids Res. 2006; 34: D655-658Crossref PubMed Scopus (590) Google Scholar28.Prince J.T. Carlson M.W. Wang R. Lu P. Marcotte E.M. et al.The need for a public proteomics repository.Nat. Biotechnol. 2004; 22: 471-472Crossref PubMed Scopus (137) Google Scholar). Here, we employed a combination of shotgun and targeted proteomics to characterize the HAdV (Ad5F35) proteome (Fig. 2). We first used high resolution LC-MS/MS analysis, employing three different proteases (trypsin, chymotrypsin, and Lys-N (29.Taouatas N. Heck A.J. Mohammed S. et al.Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions.J. Proteome Res. 2010; 9: 4282-4288Crossref PubMed Scopus (33) Google Scholar)) to improve coverage of all proteins present in the HAdV particles, with the aim to evaluate intrinsic adenovirus proteinase activity and to select the best peptide candidates for downstream SRM assays. Next, we developed a SRM assay to define the copy number of each of the 13 detected HAdV proteins and further applied this method to monitor the changes occurring in HAdV upon heat-induced virion disassembly that mimics a key step in host infection (11.Wiethoff C.M. Wodrich H. Gerace L. Nemerow G.R. et al.Adenovirus protein VI mediates membrane disruption following capsid disassembly.J. Virol. 2005; 79: 1992-2000Crossref PubMed Scopus (328) Google Scholar, 30.Russell W.C. Valentine R.C. Pereira H.G. et al.The effect of heat on the anatomy of the adenovirus.J. Gen. Virol. 1967; 1: 509-522Crossref PubMed Scopus (74) Google Scholar, 31.Pérez-Berná A.J. Ortega-Esteban A. Menéndez-Conejero R. Winkler D.C. Menéndez M. Steven A.C. Flint S.J. de Pablo P.J. San Martín C. et al.The role of capsid maturation on adenovirus priming for sequential uncoating.J. Biol. Chem. 2012; 287: 31582-31595Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar32.Russell W.C. et al.Adenoviruses: update on structure and function.J. Gen. Virol. 2009; 90: 1-20Crossref PubMed Scopus (257) Google Scholar).TABLE 1Detailed features on each of the 13 HAdV (Ad5F35) proteinsPolypeptideUniprot IDResiduesMW (kDa)LocationProtein copy number (2, 3, 42)Measured protein copy numberHexonP04133952107.9Coat720821 ± 40PentonP1253857163.3Vertex6060FiberQ6773332335.3Vertex3636 ± 1IIIaP1253758565.2Cement68 ± 257 ± 6VIIIP2493622724.7Cement127 ± 3102 ± 11VIP2493725027Cement342 ± 4359 ± 24IXP0328114014.4Cement247 ± 2676 ± 122VIIP6895119822Core833 ± 19527 ± 44VP2493836641.3Core157 ± 1148 ± 15TPP0449965374.6Core25 ± 1μP14269808.8Core∼104290 ± 18AVPP0325320423.1Core15 ± 57 ± 1IVa2P0327144950.97 ± 15 ± 1 Open table in a new tab FIGURE 2.Schematic of the MS-based workflow combining shotgun and targeted approaches to characterize HAdV (Ad5F35). HAdV from intact and heat-stressed particles were purified by HistoDenz density gradients. A first analysis was performed by combining shotgun LC-MS/MS analysis with multiple proteases for protein digestion. This analysis was used to identify all HAdV proteins with high sequence coverage, identify AVP cleavage sites, and build a spectral library from which we selected the peptides employed in the SRM assay. Using these assays, we quantitatively profiled the stoichiometry of all 13 proteins present, as well as their (partial) release upon heat stress.View Large Image Figure ViewerDownload Hi-res image Download (PPT) adenovirus human AdV terminal protein adenovirus proteinase selected reaction monitoring. Replication-defective HAdV type 5 pseudotyped with the HAdV type 35 fiber (Ad5F35) was purified as described previously (33.Reddy V.S. Natchiar S.K. Gritton L. Mullen T.M. Stewart P.L. Nemerow G.R. et al.Crystallization and preliminary X-ray diffraction analysis of human adenovirus.Virology. 2010; 402: 209-214Crossref PubMed Scopus (19) Google Scholar) using cesium chloride density gradients. The virus was dialyzed into DX10/10 buffer (40 mm Tris, pH 8.1, 500 mm NaCl, 10% glycerol, 10% ethylene glycol, 2% sucrose, and 1% mannitol). Partial capsid disassembly was achieved using thermal perturbation (34.Smith J.G. Nemerow G.R. et al.Mechanism of adenovirus neutralization by human α-defensins.Cell Host Microbe. 2008; 3: 11-19Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 35.Moyer C.L. Nemerow G.R. et al.Disulfide-bond formation by a single cysteine mutation in adenovirus protein VI impairs capsid release and membrane lysis.Virology. 2012; 428: 41-47Crossref PubMed Scopus (18) Google Scholar). Briefly, Ad5F35 (5 mg) was diluted in 7.5 mm HEPES, 50 mm NaCl, pH 7.4, divided into 6 × 500 μl aliquots, and incubated at 55 °C for 15 min. The released proteins were isolated from the intact particles and viral cores by density sedimentation on discontinuous histodenz gradients (30–80%). For DNA exposure experiments, 5 μg of Ad5F5 (HAdV5) or Ad5F35, in 10 μl A195 buffer, was mixed with 5 μl of Pico Green (Invitrogen), diluted 1:20 in A195 buffer and transferred to a 96-well RT-PCR plate (Bio-Rad). Triplicate samples were heated for 1 min in a Bio-Rad CFX96 real-time PCR system from 37–55 °C in 0.5 °C increments. Data collection was done with Channel 1/FAM settings (450–490 nm excitation and 510–530 nm emission). Measurements of capsid disassembly via immunoblot with antibodies to protein VI or fiber was done as described previously (34.Smith J.G. Nemerow G.R. et al.Mechanism of adenovirus neutralization by human α-defensins.Cell Host Microbe. 2008; 3: 11-19Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 35.Moyer C.L. Nemerow G.R. et al.Disulfide-bond formation by a single cysteine mutation in adenovirus protein VI impairs capsid release and membrane lysis.Virology. 2012; 428: 41-47Crossref PubMed Scopus (18) Google Scholar). For LC-MS analysis, purified HAdV particles were resuspended in 50 mm ammonium bicarbonate, 5% (w/v) sodium deoxycholate (Sigma Aldrich) and heated at 90 °C for 5 min to disrupt the virus particles. Proteins (∼200 μg) for each enzymatic digestion were first reduced using dithiothreitol (DTT, Sigma Aldrich) for 30 min at 56 °C and then alkylated by iodoacetamide (Sigma Aldrich) for 30 min in the dark. Samples were diluted such that the final concentration of sodium deoxycholate was 0.5%, followed by enzymatic digestion overnight at 37 °C employing three different proteases in parallel. Trypsin (Promega) and chymotrypsin (Roche Applied Science) were added in a substrate/enzyme ratio of 50:1 (w/w), whereas LysN (U-Protein Express, The Netherlands) was added in a substrate/enzyme ratio of 75:1. Digestion was quenched by acidification with trifluoroacetic acid (TFA, Sigma Aldrich) to a final concentration of 0.5%. Sodium deoxycholate removal was performed by liquid-liquid extraction. Briefly, samples were diluted two times with ethyl acetate, vortexed for 30 s, and then centrifuged at 20,000 × g for 1 min. The peptide-containing aqueous phase at the bottom of the Eppendorf tubes was recovered and desalted by solid phase extraction (Sep-pack Vac C18 cartridges, Waters). Desalted peptides were eluted in 80% acetonitrile, dried in a SpeedVac, and then resuspended in 10% formic acid solution. Sample digests from all three proteases were used for a shotgun experiment, whereas solely tryptic samples were analyzed by SRM and shotgun label-free quantification analyses. Sample digests from the three proteases were analyzed separately by a single LC-MS/MS run. We performed nanoflow LC-MS/MS using either an LTQ-Orbitrap XL coupled to an Agilent 1200 HPLC system (Agilent Technologies), an LTQ-Orbitrap Elite, and Orbitrap Q-exactive mass spectrometers (Thermo Electron, Bremen, Germany) coupled to a EASY nLC 1000 system (Thermo Fisher Scientific). Approximately 40 ng of digested HAdV was delivered to a trap column (ReproSil C18, (Dr. Maisch, GmbH, Ammerbuch, Germany); 20 mm × 100 μm inner diameter, packed in-house) at 5 μl/min in 100% solvent A (0.1 m acetic acid in water). Next, peptides were eluted from the trap column onto an analytical column (ReproSil-Pur C18-AQ (Dr. Maisch, GmbH, Ammerbuch, Germany); 40 cm in length, 50-μm inner diameter, packed in-house) at ∼100 nl/min in a 60 min or 3 h gradient from 0 to 40% solvent B (0.1 m acetic acid in 8:2 (v/v) acetonitrile/water). The eluent was sprayed via distal coated emitter tips. The mass spectrometers were operated in data-dependent mode, automatically switching between MS and MS/MS. In the LTQ-Orbitrap XL and LQT-Orbitrap Elite, the full-scan MS spectra (from m/z 350 to 1500) were acquired in the Orbitrap analyzer with a full width at half maximum resolution of 30,000 and 60,000 at 400 m/z. After the survey scans, the 10 most intense precursor ions at a threshold above 5000 were selected for MS/MS. Peptide fragmentation was carried out using a decision tree performed by collision-induced dissociation or electron transfer dissociation, depending on their charge state and mass. In both of the fragmentation methods employed, the fragment ions readout is in the ion trap analyzer. In the Q-exactive orbitrap full-scan MS spectra (from m/z 350 to 1500) were acquired with a full width at half maximum resolution of 35,000 at 400 m/z. After the survey scans, the 10 most intense precursor ions at an intensity threshold >4200 were selected for MS/MS. Peptide fragmentation was carried out by higher collisional dissociation with a normalized collision energy of 25. MS raw data from the shotgun LC-MS/MS analyses was processed by Proteome Discoverer (version 1.3, Thermo Electron). Peptide identification was performed with Mascot 2.3 (Matrix Science) against a concatenated forward-decoy UniPROT database, including the HAdV protein sequences and supplemented with all of the frequently observed contaminants in MS. The reversed sequences (decoy) were created for each protein entry present in the forward database and were employed to calculate the false discovery rate (36.Elias J.E. Gygi S.P. et al.Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.Nat. Methods. 2007; 4: 207-214Crossref PubMed Scopus (2827) Google Scholar) and therefore to adjust the filtering criteria. The following parameters were used: 50 ppm precursor mass tolerance, 0.6 Da (for collision-induced dissociation and electron transfer dissociation spectra) and 0.05 Da (for higher collisional dissociation spectra) fragment ion tolerance. To evaluate the adenovirus proteinase activity, we allowed the identification of peptides containing one nonspecific cleavage and a maximum of two missed cleavages. Carbamidomethyl cysteine was allowed as fixed modification, whereas oxidized methionine and protein N-terminal acetylation were set as variable modifications. Finally, results were filtered using the following criteria: (i) mass deviations of ±6 p.p.m., (ii) Mascot ion score of at least 20, (iii) a minimum of six amino acid residues per peptide, and (iv) position rank 1 in Mascot search. As a result, we obtained peptide false discovery rates below 1% for each of the three peptide mixtures analyzed. Label-free quantification of protein abundances was performed by calculating the area under the curve for each precursor ion identified as a peptide mapping to one of the pVI or pVIII proteolytic products. The area under the curves for these specific peptides were calculated by Proteome Discoverer (version 1.3, Thermo Electron) and used to compare the peptides abundance in the heated versus intact HAdV to calculate the ratios. For each of the HAdV proteins, we aimed to select up to three peptides based on the following criteria: (i) absence of nonspecific or missed cleavages, (ii) absence of methionine residues, and (iii) fragmentation efficiency. SRM transitions were created using Skyline software (37.MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. et al.Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (2963) Google Scholar) from the Orbitrap Q-exactive higher collisional dissociation spectral library. Synthetic stable isotope-labeled peptides with a C-terminal 15N-, and 13C-labeled arginine or lysine residue (>99 atom % isotopic enrichment) were purchased from Thermo Fisher Scientific (Ulm, Germany). For each synthetic peptide, 100 pmol were spiked into 50 μg of HAdV protein digests before LC-MS quantification. A total of 33 peptides and 210 transitions were employed for the whole assay. The LC-MS/MS was performed using a TSQ Vantage triple quadrupole (Thermo, San Jose, CA) coupled to an EASY nLC 1000 system (Thermo Fisher Scientific). Approximately 40 ng of digested HAdV are delivered to a trap column (ReproSil C18, (Dr. Maisch, GmbH, Ammerbuch, Germany); 20 mm × 100 μm inner diameter, packed in-house) at 5 μl/min in 100% solvent A (0.1 m acetic acid in water). Next, peptides eluted from the trap column onto an analytical column (ReproSil-Pur C18-AQ (Dr. Maisch, GmbH, Ammerbuch, Germany); 40-cm length, 50-μm inner diameter, packed in-house) at ∼100 nl/min in a 60-min gradient from 0 to 40% solvent B (0.1 m acetic acid in 8:2 (v/v) acetonitrile/water). The triple quadrupole mass analyzer setup was configured to perform MS/MS scans triggered on each target peptide according to their scheduled retention time window (3 min). Q1 and Q3 peak width set at 0.7 Da, and the cycle time corresponds to 1.8 s. The triple quadrupole collision energy was calculated according to the following equations: CE = 0.03× (m/z) + 2.905 and CE = 0.038× (m/z) + 2.281 (CE indicates collision energy and m/z indicates mass to charge ratio) for doubly and triply charged precursor ions, respectively. The area for each peptide peak was calculated by using Skyline software (37.MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. et al.Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (2963) Google Scholar). The quantification was then performed by comparing the area for each of the endogenous peptides, in comparison with the area of the corresponding heavy standard peptide added to the HAdV digest in known amount. The fi" @default.
• W2163442590 created "2016-06-24" @default.
• W2163442590 creator A5017734471 @default.
• W2163442590 creator A5026834279 @default.
• W2163442590 creator A5027581149 @default.
• W2163442590 creator A5043090747 @default.
• W2163442590 creator A5044522325 @default.
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• W2163442590 creator A5053603496 @default.
• W2163442590 date "2014-04-01" @default.
• W2163442590 modified "2023-10-18" @default.
• W2163442590 title "Adenovirus Composition, Proteolysis, and Disassembly Studied by In-depth Qualitative and Quantitative Proteomics" @default.
• W2163442590 cites W1556304300 @default.
• W2163442590 cites W1734478427 @default.
• W2163442590 cites W1940378630 @default.
• W2163442590 cites W1968616393 @default.
• W2163442590 cites W1973829155 @default.
• W2163442590 cites W1979853788 @default.
• W2163442590 cites W1982844673 @default.
• W2163442590 cites W1990110703 @default.
• W2163442590 cites W1990822218 @default.
• W2163442590 cites W1995638700 @default.
• W2163442590 cites W1996948250 @default.
• W2163442590 cites W2005123048 @default.
• W2163442590 cites W2010316656 @default.
• W2163442590 cites W2027980197 @default.
• W2163442590 cites W2031095666 @default.
• W2163442590 cites W2047151196 @default.
• W2163442590 cites W2054116069 @default.
• W2163442590 cites W2060518210 @default.
• W2163442590 cites W2061204975 @default.
• W2163442590 cites W2063066512 @default.
• W2163442590 cites W2064631610 @default.
• W2163442590 cites W2065262709 @default.
• W2163442590 cites W2065881418 @default.
• W2163442590 cites W2068481474 @default.
• W2163442590 cites W2073641373 @default.
• W2163442590 cites W2078598194 @default.
• W2163442590 cites W2086190383 @default.
• W2163442590 cites W2087287669 @default.
• W2163442590 cites W2094752726 @default.
• W2163442590 cites W2096057003 @default.
• W2163442590 cites W2097780295 @default.
• W2163442590 cites W2105375220 @default.
• W2163442590 cites W2106376891 @default.
• W2163442590 cites W2106618212 @default.
• W2163442590 cites W2109055236 @default.
• W2163442590 cites W2110926538 @default.
• W2163442590 cites W2111496511 @default.
• W2163442590 cites W2112419844 @default.
• W2163442590 cites W2114216165 @default.
• W2163442590 cites W2122805769 @default.
• W2163442590 cites W2125214575 @default.
• W2163442590 cites W2130904964 @default.
• W2163442590 cites W2131741443 @default.
• W2163442590 cites W2141091417 @default.
• W2163442590 cites W2146968336 @default.
• W2163442590 cites W2150364154 @default.
• W2163442590 cites W2152687832 @default.
• W2163442590 cites W2154309992 @default.
• W2163442590 cites W2157918066 @default.
• W2163442590 cites W2161811833 @default.
• W2163442590 cites W2168887693 @default.
• W2163442590 cites W2434179572 @default.
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