FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W4306174941> ?p ?o ?g. }

• W4306174941 endingPage "336" @default.
• W4306174941 startingPage "327" @default.
• W4306174941 abstract "Charge detection mass spectrometry (CDMS) was used to analyze recombinant adeno-associated virus serotype 8 (rAAV8) vectors after incubation at elevated temperatures. rAAV8 vectors with a range of genomes of interest (GOIs) from 2.22 to 4.84 kb were investigated. For the shorter GOIs, GOI release occurred at surprisingly low temperatures (15 min at 45°C for cytomegalovirus [CMV]-GFP). The released DNA and intermediates with the GOI extruded from the capsid were detected. The temperature required to release the short GOIs is well below the 65°C incubation temperature required to disassemble the empty rAAV8 capsid. The temperature for GOI release increased with its GOI length. With the longer GOIs, the GOI stabilized the capsid so that it remained intact under conditions that would disassemble the empty particle. After incubation at 65°C, the main species in the CDMS mass distributions for the longer GOIs was the vector with the GOI. However, for GOIs longer than the wild-type genome (∼4.7 kb), the stability diminished, and genome release occurred at a lower temperature. Heterogeneous DNA fragments from the host cells or plasmids is released at a lower temperature than the longer GOIs, suggesting that the GOIs have a feature that resists early release. Charge detection mass spectrometry (CDMS) was used to analyze recombinant adeno-associated virus serotype 8 (rAAV8) vectors after incubation at elevated temperatures. rAAV8 vectors with a range of genomes of interest (GOIs) from 2.22 to 4.84 kb were investigated. For the shorter GOIs, GOI release occurred at surprisingly low temperatures (15 min at 45°C for cytomegalovirus [CMV]-GFP). The released DNA and intermediates with the GOI extruded from the capsid were detected. The temperature required to release the short GOIs is well below the 65°C incubation temperature required to disassemble the empty rAAV8 capsid. The temperature for GOI release increased with its GOI length. With the longer GOIs, the GOI stabilized the capsid so that it remained intact under conditions that would disassemble the empty particle. After incubation at 65°C, the main species in the CDMS mass distributions for the longer GOIs was the vector with the GOI. However, for GOIs longer than the wild-type genome (∼4.7 kb), the stability diminished, and genome release occurred at a lower temperature. Heterogeneous DNA fragments from the host cells or plasmids is released at a lower temperature than the longer GOIs, suggesting that the GOIs have a feature that resists early release. Recombinant adeno-associated virus (rAAV) is a leading gene therapy vector for monogenetic diseases. There are currently two FDA-approved therapies and hundreds more in clinical trials.1Wang D. Tai P.W.L. Gao G. Adeno-associated virus vector as a platform for gene therapy delivery.Nat. Rev. Drug Discov. 2019; 18: 358-378https://doi.org/10.1038/s41573-019-0012-9Crossref PubMed Scopus (708) Google Scholar,2Keeler A.M. Flotte T.R. Recombinant adeno-associated virus gene therapy in light of Luxterna (and Zolgensma and Glybera): where are we, and how did we get here?.Annu. Rev. Virol. 2019; 6: 601-621https://doi.org/10.1146/annurev-virology-092818-015530Crossref PubMed Scopus (126) Google Scholar Its favorable attributes include low immunotoxicity and wide cell trophism. AAV, a member of the parvovirus family, is one of the smallest and simplest viruses. It is around 25 nm in diameter and consists of a capsid surrounding a packaged genome. The capsid contains 60 proteins, a mixture of the viral proteins VP1, VP2, and VP3, arranged in pseudo icosahedral symmetry.3Kronenberg S. Kleinschmidt J.A. Böttcher B. Electron cryo-microscopy and image reconstruction of adeno-associated virus type 2 empty capsids.EMBO Rep. 2001; 2: 997-1002https://doi.org/10.1093/embo-reports/kve234Crossref PubMed Scopus (110) Google Scholar,4Kaludov N. Padron E. Govindasamy L. McKenna R. Chiorini J.A. Agbandje-McKenna M. Production, purification, and preliminary X-ray crystallographic studies of adeno-associated virus serotype 4.Virology. 2003; 306: 1-6https://doi.org/10.1016/S0042-6822(02)00037-5Crossref PubMed Scopus (20) Google Scholar,5Nam H.J. Lane M.D. Padron E. Gurda B. McKenna R. Kohlbrenner E. Aslanidi G. Byrne B. Muzyczka N. Zolotukhin S. Agbandje-McKenna M. Structure of adeno-associated virus serotype 8, a gene therapy vector.J. Virol. 2007; 81: 12260-12271https://doi.org/10.1128/JVI.01304-07Crossref PubMed Scopus (165) Google Scholar The three capsid proteins are generated from overlapping reading frames in the cap gene. They all contain a common VP3 sequence at the C terminus. VP2 is missing residues 1–137 of the VP1 sequence, and VP3 is missing an additional 66 residues (i.e., 1–203) of the VP1 sequence. The VP1 unique region contains a motif homologous to a phospholipase A2 domain. The VP1 unique region and VP1/VP2 common regions are initially internalized,6Kronenberg S. Böttcher B. von der Lieth C.W. Bleker S. Kleinschmidt J.A. A conformational change in the adeno-associated virus type 2 capsid leads to the exposure of hidden VP1 N termini.J. Virol. 2005; 79: 5296-5303https://doi.org/10.1128/JVI.79.9.5296-5303.2005Crossref PubMed Scopus (129) Google Scholar,7Bleker S. Sonntag F. Kleinschmidt J.A. Mutational analysis of narrow pores at the fivefold symmetry axes of adeno-associated virus type 2 capsids reveals a dual role in genome packaging and activation of phospholipase A2 activity.J. Virol. 2005; 79: 2528-2540https://doi.org/10.1128/JVI.79.4.2528-2540.2005Crossref PubMed Scopus (133) Google Scholar but at some point during infection, a conformational change promotes their exposure.6Kronenberg S. Böttcher B. von der Lieth C.W. Bleker S. Kleinschmidt J.A. A conformational change in the adeno-associated virus type 2 capsid leads to the exposure of hidden VP1 N termini.J. Virol. 2005; 79: 5296-5303https://doi.org/10.1128/JVI.79.9.5296-5303.2005Crossref PubMed Scopus (129) Google Scholar,8Sonntag F. Bleker S. Leuchs B. Fischer R. Kleinschmidt J.A. Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus.J. Virol. 2006; 80: 11040-11054https://doi.org/10.1128/JVI.01056-06Crossref PubMed Scopus (184) Google Scholar It is thought that each site on the icosahedral T = 1 capsid is populated randomly by a VP1, VP2, or VP3 in ratios that mainly reflect their expression stoichiometry. The overall ratio for rAAV from human embryonic kidney (HEK) cells is around 1:1:10.9Rose J.A. Maizel J.V. Inman J.K. Shatkin A.J. Structural proteins of adenovirus-associated viruses.J. Virol. 1971; 8: 766-770https://doi.org/10.1128/JVI.8.5.766-770.1971Crossref PubMed Google Scholar,10Johnson F.B. Ozer H.L. Hoggan M.D. Structural proteins of adenovirus associated virus type 3.J. Virol. 1971; 8: 860-863https://doi.org/10.1128/JVI.8.6.860-863.1971Crossref PubMed Google Scholar,11Buller R.M. Rose J.A. Characterization of adenovirus associated virus induced polypeptides in KB cells.J. Virol. 1978; 25: 331-338https://doi.org/10.1128/JVI.25.1.331-338.1978Crossref PubMed Google Scholar,12Samulski R.J. Muzyczka N. AAV-Mediated gene therapy for research and therapeutic purposes.Annu. Rev. Virol. 2014; 1: 427-451https://doi.org/10.1146/annurev-virology-031413-085355Crossref PubMed Scopus (270) Google Scholar Capsids from Spodoptera frugiperda (Sf9) cells using baculovirus expression appear to be less heterogeneous (i.e., they contain more VP3 and less VP1 and/or VP2).13Kohlbrenner E. Aslanidi G. Nash K. Shklyaev S. Campbell-Thompson M. Byrne B.J. Snyder R.O. Muzyczka N. Warrington K.H. Zolotukhin S. Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system.Mol. Ther. 2005; 12: 1217-1225https://doi.org/10.1016/j.ymthe.2005.08.018Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar The genome is single-stranded DNA, 4.7 kb in the wild-type (WT) virus, with identical T-shaped inverted terminal repeats at the ends. Both + and – strands are packaged with equal frequency.14Berns K.I. Rose J.A. Evidence for a single-stranded adenovirus-associated virus genome: isolation and separation of complementary single strands.J. Virol. 1970; 5: 693-699https://doi.org/10.1128/JVI.5.6.693-699.1970Crossref PubMed Scopus (0) Google Scholar Incomplete genomes can be packaged by both WT-AAV particles and rAAV vectors.15Hauswirth W.W. Berns K.I. Adeno-associated virus DNA replication: non-unit length molecules.Virology. 1979; 93: 57-68https://doi.org/10.1016/0042-6822(79)90275-7Crossref PubMed Scopus (45) Google Scholar,16Laughlin C.A. Myers M.W. Risin D.L. Carter B.J. Defective-interfering particles of the human parvovirus adeno-associated virus.Virology. 1979; 94: 162-174https://doi.org/10.1016/0042-6822(79)90446-xCrossref PubMed Scopus (0) Google Scholar,17Pierson E.E. Keifer D.Z. Asokan A. Jarrold M.F. Resolving adeno-associated viral particle diversity with charge detection mass spectrometry.Anal. Chem. 2016; 88: 6718-6725https://doi.org/10.1021/acs.analchem.6b00883Crossref PubMed Scopus (76) Google Scholar Small DNA fragments can also be packaged as well as heterogeneous DNA.18Smith P.H. Wright J.F. Qu G. Patarroyo-White S. Parker A. Sommer J.M. Packaging of host cell and plasmid DNA into recombinant adeno-associated virus particles produced by triple transfection.Mol. Ther. 2003; 7: S348https://doi.org/10.1016/S1525-0016(16)41342-0Abstract Full Text Full Text PDF Google Scholar,19Chadeuf G. Ciron C. Moullier P. Salvetti A. Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery.Mol. Ther. 2005; 12: 744-753https://doi.org/10.1016/j.ymthe.2005.06.003Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar,20Barnes L.F. Draper B.E. Chen Y.-T. Powers T.W. Jarrold M.F. Quantitative analysis of genome packaging in recombinant AAV vectors by charge detection mass spectrometry.Mol. Ther. Methods Clin. Dev. 2021; 23: 87-97https://doi.org/10.1016/j.omtm.2021.08.002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar,21Tran N.T. Lecomte E. Saleun S. Namkung S. Robin C. Weber K. Devine E. Blouin V. Adjali O. Ayuso E. et al.Human and insect cell produced recombinant adeno-associated viruses show differences in genome heterogeneity.Hum. Gene Ther. 2022; 33: 371-388https://doi.org/10.1089/hum.2022.050Crossref PubMed Scopus (8) Google Scholar Like all viruses, AAV must perform a thermodynamic balancing act.22Katen S. Zlotnick A. The thermodynamics of virus capsid assembly.Methods Enzymol. 2009; 455: 395-417https://doi.org/10.1016/S0076-6879(08)04214-6Crossref PubMed Scopus (120) Google Scholar The capsid must be stable enough to protect the genome from the environment but also must be able to release its genetic payload at the right time and place for replication. The thermal stability of AAV particles and the structural changes induced by heating have been investigated in a number of studies.23Kronenberg S. Böttcher B. von der Lieth C.W. Bleker S. Kleinschmidt J.A. A conformational change in the adeno-associated virus type 2 capsid leads to exposure of hidden VP1 N-termini.J. Virol. 2005; 79: 5296-5303https://doi.org/10.1128/JVI.79.95296-5303.2005Crossref PubMed Scopus (0) Google Scholar,24Johnson J.S. Li C. DiPrimio N. Weinberg M.S. McCown T.J. Samulski R.J. Mutagenesis of adeno-associated virus type 2 Capsid protein VP1 uncovers new roles for basic amino acids in trafficking and cell specific transduction.J. Virol. 2010; 84: 8888-8902https://doi.org/10.1128/JVI.00687-10Crossref PubMed Scopus (68) Google Scholar,25Horowitz E.D. Finn M.G. Asokan A. Tyrosine cross-linking reveals interfacial dynamics in Adeno-associated viral capsids during infection.ACS Chem. Biol. 2012; 7: 1059-1066https://doi.org/10.1021/cb3000265Crossref PubMed Scopus (19) Google Scholar,26Horowitz E.D. Rahman K.S. Bower B.D. Dismuke D.J. Falvo M.R. Griffith J.D. Harvey S.C. Asokan A. Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release.J. Virol. 2013; 87: 2994-3002https://doi.org/10.1128/JVI.03017-12Crossref PubMed Scopus (65) Google Scholar,27Venkatakrishnan B. Yarbrough J. Domsic J. Bennett A. Bothner B. Kozyreva O.G. Samulski R.J. Muzyczka N. McKenna R. Agbandje-McKenna M. Structure and dynamics of adeno-associated virus serotype 1 VP1-unique N-terminal domain and its role in capsid trafficking.J. Virol. 2013; 87: 4974-4984https://doi.org/10.1128/JVI.02524-12Crossref PubMed Scopus (101) Google Scholar,28Rayaprolu V. Kruse S. Kant R. Venkatakrishnan B. Movahed N. Brooke D. Lins B. Bennett A. Potter T. McKenna R. et al.Comparative analysis of adeno-associated virus capsid stability and dynamics.J. Virol. 2013; 87: 13150-13160https://doi.org/10.1128/JVI.01415-13Crossref PubMed Scopus (80) Google Scholar,29Bennett A. Patel S. Mietzsch M. Jose A. Lins-Austin B. Yu J.C. Bothner B. McKenna R. Agbandje-McKenna M. Thermal stability as a determinant of AAV serotype identity.Mol. Ther. Methods Clin. Dev. 2017; 6: 171-182https://doi.org/10.1016/j.omtm.2017.07.003Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar,30Bernaud J. Rossi A. Fis A. Gardette L. Aillot L. Büning H. Castelnovo M. Salvetti A. Faivre-Moskalenko C. Characterization of AAV vector particle stability at the single-capsid level.J. Biol. Phys. 2018; 44: 181-194https://doi.org/10.1007/s10867-018-9488-5Crossref PubMed Scopus (31) Google Scholar,31Xu Y. Guo P. Zhang J. Chrzanowski M. Chew H. Firrman J.A. Sang N. Diao Y. Xiao W. Effects of thermally induced configuration changes on rAAV genome’s enzymatic accessibility.Mol. Ther. Methods Clin. Dev. 2020; 18: 328-334https://doi.org/10.1016/j.omtm.2020.06.005Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Several studies indicate that heating leads to externalization of the VP1 N termini.23Kronenberg S. Böttcher B. von der Lieth C.W. Bleker S. Kleinschmidt J.A. A conformational change in the adeno-associated virus type 2 capsid leads to exposure of hidden VP1 N-termini.J. Virol. 2005; 79: 5296-5303https://doi.org/10.1128/JVI.79.95296-5303.2005Crossref PubMed Scopus (0) Google Scholar,24Johnson J.S. Li C. DiPrimio N. Weinberg M.S. McCown T.J. Samulski R.J. Mutagenesis of adeno-associated virus type 2 Capsid protein VP1 uncovers new roles for basic amino acids in trafficking and cell specific transduction.J. Virol. 2010; 84: 8888-8902https://doi.org/10.1128/JVI.00687-10Crossref PubMed Scopus (68) Google Scholar,28Rayaprolu V. Kruse S. Kant R. Venkatakrishnan B. Movahed N. Brooke D. Lins B. Bennett A. Potter T. McKenna R. et al.Comparative analysis of adeno-associated virus capsid stability and dynamics.J. Virol. 2013; 87: 13150-13160https://doi.org/10.1128/JVI.01415-13Crossref PubMed Scopus (80) Google Scholar It is also well established that heating leads to genome release. Atomic force microscopy (AFM) has been used to probe structural changes and genome release in several studies where images show single-stranded DNA (ssDNA) extruding from the capsid.26Horowitz E.D. Rahman K.S. Bower B.D. Dismuke D.J. Falvo M.R. Griffith J.D. Harvey S.C. Asokan A. Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release.J. Virol. 2013; 87: 2994-3002https://doi.org/10.1128/JVI.03017-12Crossref PubMed Scopus (65) Google Scholar,30Bernaud J. Rossi A. Fis A. Gardette L. Aillot L. Büning H. Castelnovo M. Salvetti A. Faivre-Moskalenko C. Characterization of AAV vector particle stability at the single-capsid level.J. Biol. Phys. 2018; 44: 181-194https://doi.org/10.1007/s10867-018-9488-5Crossref PubMed Scopus (31) Google Scholar An inverse correlation between the packaged genome length and the temperature needed to induce uncoating was noted.26Horowitz E.D. Rahman K.S. Bower B.D. Dismuke D.J. Falvo M.R. Griffith J.D. Harvey S.C. Asokan A. Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release.J. Virol. 2013; 87: 2994-3002https://doi.org/10.1128/JVI.03017-12Crossref PubMed Scopus (65) Google Scholar Measurements using differential scanning fluorimetry (DSF) and differential scanning calorimetry (DSC) show serotype melting temperatures that vary by more than 20°C, with rAAV2 being the least stable and rAAV5 being the most.28Rayaprolu V. Kruse S. Kant R. Venkatakrishnan B. Movahed N. Brooke D. Lins B. Bennett A. Potter T. McKenna R. et al.Comparative analysis of adeno-associated virus capsid stability and dynamics.J. Virol. 2013; 87: 13150-13160https://doi.org/10.1128/JVI.01415-13Crossref PubMed Scopus (80) Google Scholar The transgene (luciferase) had only a minor effect, with the melting temperatures of empty and full particles differing by 1°C to 2°C at most.29Bennett A. Patel S. Mietzsch M. Jose A. Lins-Austin B. Yu J.C. Bothner B. McKenna R. Agbandje-McKenna M. Thermal stability as a determinant of AAV serotype identity.Mol. Ther. Methods Clin. Dev. 2017; 6: 171-182https://doi.org/10.1016/j.omtm.2017.07.003Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar The nature of the buffer had a significant effect on the melting temperatures of AAV2 and AAV3, but for other serotypes studied, the effect was modest (a few degrees). It has been suggested that the thermal stability measured by DSF could be used as a determinant of AAV serotype identity.29Bennett A. Patel S. Mietzsch M. Jose A. Lins-Austin B. Yu J.C. Bothner B. McKenna R. Agbandje-McKenna M. Thermal stability as a determinant of AAV serotype identity.Mol. Ther. Methods Clin. Dev. 2017; 6: 171-182https://doi.org/10.1016/j.omtm.2017.07.003Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar In this study, we have used charge detection mass spectrometry (CDMS) to investigate thermally induced transitions for rAAV8 particles with a variety of genome of interest (GOI) lengths from 2,219 to 4,844 nt. CDMS is a single-particle technique where the masses of individual ions are measured directly. It allows accurate mass distributions to be recorded for highly heterogeneous samples that are beyond the capabilities of conventional MS.32Fuerstenau S.D. Benner W.H. Molecular weight determination of megadalton DNA electrospray ions using charge detection time-of-flight mass spectrometry.Rapid Commun. Mass Spectrom. 1995; 9: 1528-1538https://doi.org/10.1002/rcm.1290091513Crossref PubMed Scopus (164) Google Scholar,33Pierson E.E. Keifer D.Z. Selzer L. Lee L.S. Contino N.C. Wang J.C.Y. Zlotnick A. Jarrold M.F. Detection of late intermediates in virus capsid assembly by charge detection mass spectrometry.J. Am. Chem. Soc. 2014; 136: 3536-3541https://doi.org/10.1021/ja411460wCrossref PubMed Scopus (103) Google Scholar,34Doussineau T. Désert A. Lambert O. Taveau J.-C. Lansalot M. Dugourd P. Bourgeat-Lami E. Ravaine S. Duguet E. Antoine R. Charge detection mass spectrometry for the characterization of mass and surface area of composite nanoparticles.J. Phys. Chem. C. 2015; 119: 10844-10849https://doi.org/10.1021/jp510081vCrossref Scopus (44) Google Scholar,35Harper C.C. Elliott A.G. Oltrogge L.M. Savage D.F. Williams E.R. Multiplexed charge detection mass spectrometry for high throughput single ion analysis of large molecules.Anal. Chem. 2019; 91: 7458-7465https://doi.org/10.1021/acs.analchem.9b01669Crossref PubMed Scopus (23) Google Scholar,36Keifer D.Z. Pierson E.E. Jarrold M.F. Charge detection mass spectrometry: weighing heavier things.Analyst. 2017; 142: 1654-1671https://doi.org/10.1039/C7AN00277GCrossref PubMed Google Scholar,37Jarrold M.F. Applications of charge detection mass spectrometry in molecular biology and biotechnology.Chem. Rev. 2022; 122: 7415-7441https://doi.org/10.1021/acs.chemrev.1c00377Crossref PubMed Scopus (11) Google Scholar In CDMS, the m/z ratio and charge are determined simultaneously for each ion and then multiplied to give their masses. Masses measured for thousands of ions are binned into a mass distribution. In conventional MS, where just the m/z ratio is measured, the ion’s charge must be deduced from the m/z spectrum. However, when the sample has a heterogeneous mass distribution, there is a large number of overlapping m/z peaks in the m/z spectrum, and the charge cannot be determined. Heterogeneity increases with size, and conventional MS usually cannot determine masses for ions larger than around a 1 MDa without prior knowledge of the mass. CDMS has been used to measure molecular weights for species well over 100 MDa, such as adenovirus.38Barnes L.F. Draper B.E. Jarrold M.F. Analysis of recombinant adenovirus vectors by ion trap charge detection mass spectrometry: accurate molecular weight measurements beyond 150 MDa.Anal. Chem. 2022; 94: 1543-1551https://doi.org/10.1021/acs.analchem.1c02439Crossref PubMed Scopus (2) Google Scholar CDMS has previously been used to investigate changes induced by incubation of AAV8 vectors at elevated temperatures, and it was found that incubation significantly narrowed the peaks attributed to both empty and full particles.20Barnes L.F. Draper B.E. Chen Y.-T. Powers T.W. Jarrold M.F. Quantitative analysis of genome packaging in recombinant AAV vectors by charge detection mass spectrometry.Mol. Ther. Methods Clin. Dev. 2021; 23: 87-97https://doi.org/10.1016/j.omtm.2021.08.002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar This observation was attributed to the release of small DNA fragments. Marty and coworkers39Kostelic M.M. Ryan J.P. Brown L.S. Jackson T.W. Hsieh C.-C. Zak C.K. Sanders H.M. Liu Y. Chen V.S. Byrne M. et al.Stability and dissociation of adeno-associated viral capsids by variable temperature-charge detection-mass spectrometry.Anal. Chem. 2022; 94: 11723-11727https://doi.org/10.1021/acs.analchem.2c02378Crossref PubMed Scopus (3) Google Scholar have also recently reported some preliminary results on heating of AAV vectors using a related approach, Orbitrap individual ion MS (I2MS).40Makarov A. Denisov E. Dynamics of ions of intact proteins in the Orbitrap mass analyzer.J. Am. Soc. Mass Spectrom. 2009; 20: 1486-1495https://doi.org/10.1021/jasms.8b03533Crossref PubMed Scopus (0) Google Scholar,41Kafader J.O. Melani R.D. Senko M.W. Makarov A.A. Kelleher N.L. Compton P.D. Measurement of individual ions sharply increases the resolution of Orbitrap mass spectra of proteins.Anal. Chem. 2019; 91: 2776-2783https://doi.org/10.1021/acs.analchem.8b04519Crossref PubMed Scopus (34) Google Scholar,42McGee J.P. Melani R.D. Yip P.F. Senko M.W. Compton P.D. Kafader J.O. Kelleher N.L. Isotopic resolution of protein complexes up to 466 kDa using individual ion mass spectrometry.Anal. Chem. 2021; 93: 2723-2727https://doi.org/10.1021/acs.analchem.0c03282Crossref PubMed Scopus (15) Google Scholar,43Kafader J.O. Melani R.D. Durbin K.R. Ikwuagwu B. Early B.P. Fellers R.T. Beu S.C. Zabrouskov V. Makarov A.A. Maze J.T. et al.Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes.Nat. Methods. 2020; 17: 391-394https://doi.org/10.1038/s41592-020-0764-5Crossref PubMed Scopus (63) Google Scholar,44Wörner T.P. Snijder J. Bennett A. Agbandje-McKenna M. Makarov A.A. Heck A.J.R. Resolving heterogeneous macromolecular assemblies by Orbitrap-based single-particle charge detection mass spectrometry.Nat. Methods. 2020; 17: 395-398https://doi.org/10.1038/s41592-020-0770-7Crossref PubMed Scopus (69) Google Scholar Previously, CDMS has been used to characterize empty-full ratios and the mass distributions for the packaged DNA, including partially filled and overpackaged particles.17Pierson E.E. Keifer D.Z. Asokan A. Jarrold M.F. Resolving adeno-associated viral particle diversity with charge detection mass spectrometry.Anal. Chem. 2016; 88: 6718-6725https://doi.org/10.1021/acs.analchem.6b00883Crossref PubMed Scopus (76) Google Scholar,20Barnes L.F. Draper B.E. Chen Y.-T. Powers T.W. Jarrold M.F. Quantitative analysis of genome packaging in recombinant AAV vectors by charge detection mass spectrometry.Mol. Ther. Methods Clin. Dev. 2021; 23: 87-97https://doi.org/10.1016/j.omtm.2021.08.002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar CDMS only requires 10–20 uL of material at >5 × 1011 particles/mL, and a spectrum can typically be collected in 25–60 min (depending on the signal intensity, which, in turn, depends on the sample concentration). Measurements have been performed for a wide variety of serotypes. As a demonstration, Figure 1 shows CDMS mass distributions recorded for AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, AAV9, AAVDJ, AAVphp.eb, and AAVshH10 with a cytomegalovirus (CMV)-GFP genome prepared in Sf9 cells and for AAV3-CAG-EGFP and AAV8-CMV-EGFP prepared in HEK cells. Dashed gray lines show the expected location of the empty and full particles for each serotype and genome. Except for AAV3-CAG-EGFP, the dominant peak is due to the full particle, and the intensity at the expected mass of the empty is small. In addition to the dominant peak located at the expected mass of the full particle with the GOI, there is another smaller peak at a higher mass (around 5 MDa) for all spectra except AAV3-CAG-EGFP. The CMV-GFP and CMV-EGFP genomes are much smaller than the AAV packaging capacity. AAV particles that have packaged DNA to the packaging capacity are expected to have a mass around of 5 MDa, which corresponds to the mass of the smaller peak mentioned above. This peak has been attributed mainly to the packaging of a single strand of heterogeneous DNA from the host or plasmid.20Barnes L.F. Draper B.E. Chen Y.-T. Powers T.W. Jarrold M.F. Quantitative analysis of genome packaging in recombinant AAV vectors by charge detection mass spectrometry.Mol. Ther. Methods Clin. Dev. 2021; 23: 87-97https://doi.org/10.1016/j.omtm.2021.08.002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar These results suggest that packaging of heterogeneous DNA occurs for samples prepared in HEK cells with roughly the same frequency as those prepared in Sf9 cells.20Barnes L.F. Draper B.E. Chen Y.-T. Powers T.W. Jarrold M.F. Quantitative analysis of genome packaging in recombinant AAV vectors by charge detection mass spectrometry.Mol. Ther. Methods Clin. Dev. 2021; 23: 87-97https://doi.org/10.1016/j.omtm.2021.08.002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar The peak in the AAV4 spectrum at 7.2 MDa is at a mass slightly lower than expected for a dimer of the empty capsids, so it probably results from another impurity. The spectrum for AAV3-CAG-EGFP shows a sequence of peaks at 1.03, 2.06, and 3.09 MDa. These probably result from aggregates of an impurity with a molecular weight (MW) around 1.03 MDa. Finally, the AAV6 spectrum in Figure 1 shows a significant background signal at all masses. This probably results from disassembly and non-specific aggregation of the disassembly products. It appears that this AAV6 sample is particularly sensitive to freeze-thaw cycles, and after a second freeze-thaw cycle, peaks due to the full and overpackaged particles were no longer apparent in the spectrum. Figures 2A and 2B show CDMS mass distributions measured for an empty rAAV8 capsids after being incubated for 15 min at 60°C and 65°C, respectively. The main peak in the spectrum at around 3.7 MDa is attributed to the empty capsid. Measurements were also made for incubation temperatures between 45°C and 55°C in 5°C increments (data not shown). The spectra measured between 45°C and 60°C are not significantly different from those measured for unheated samples. As the incubation temperature is raised to 60°C, the 3.7 MDa peak becomes slightly narrower and shifts to a slightly lower mass. These changes are probably due to the loss of small heterogeneous DNA fragments as the samples are heated and have been discussed elsewhere.20Barnes L.F. Draper B.E. Chen Y.-T. Powers T.W. Jarrold M.F. Quantitative analysis of genome packaging in recombinant AAV vectors by charge detection mass spectrometry.Mol. Ther. Methods Clin. Dev. 2021; 23: 87-97https://doi.org/10.1016/j.omtm.2021.08.002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Incubation also appears to diminish the small amount of multimers present at masses between 6 and 8 MDa to the point where they are barely visible in the 60°C mass spectrum in Figure 2A. While incubation to 60°C causes only minor charges to the mass spectrum, dramatic changes occur for incubation at 65°C (see Figure 2B). The main peak at around 3.7 MDa is substantially diminished, and there is a broad distribution below the mass of the main peak and a high-mass tail that extends to beyond 50 MDa. The ions with masses below the main peak are attributed to capsid disassembly products, and the high-mass tail presumably results from their aggregation. Figures 2C and 2D show charge versus mass scatter plots measured after incubation at 60°C and 65°C. Each point in the plots represents the measurement for a single ion. The 60°C scatter plot is like those measured at room temperature and lower incubation temperatures (data not shown). The tight cluster of ions centered on a mass of around 3.7 MDa and a charge of around 160 elementary charges (e) corresponds to the main peak in the mass distribution. There are also clusters with a few ions at higher mass (6–8 MDa) and charge that result from multimers. Correlating mass and charge can provide information about the structure.45Keifer D.Z. Motwani T. Teschke C.M. Jarrold M.F. Acquiring structural information on virus particles via charge detection mass spectrometry.J. Am. Soc. Mass Spectrom. 2016; 27: 1028-1036https://doi.org/10.1007/s13361-016-1362-8Crossref PubMed Scopus (35) Google Scholar Large ions generated by electrospray are thought to be produced by the charge residue mechanism,46Fernandez de la" @default.
• W4306174941 created "2022-10-14" @default.
• W4306174941 creator A5061503158 @default.
• W4306174941 creator A5063887646 @default.
• W4306174941 creator A5064962658 @default.
• W4306174941 date "2022-12-01" @default.
• W4306174941 modified "2023-09-30" @default.
• W4306174941 title "Analysis of thermally driven structural changes, genome release, disassembly, and aggregation of recombinant AAV by CDMS" @default.
• W4306174941 cites W1517847098 @default.
• W4306174941 cites W1553716043 @default.
• W4306174941 cites W1601268295 @default.
• W4306174941 cites W1706435611 @default.
• W4306174941 cites W1787138288 @default.
• W4306174941 cites W1965108754 @default.
• W4306174941 cites W1967200408 @default.
• W4306174941 cites W1969538192 @default.
• W4306174941 cites W1993726835 @default.
• W4306174941 cites W2009455163 @default.
• W4306174941 cites W2012715792 @default.
• W4306174941 cites W2044008548 @default.
• W4306174941 cites W2050197623 @default.
• W4306174941 cites W2050285820 @default.
• W4306174941 cites W2059470894 @default.
• W4306174941 cites W2069800211 @default.
• W4306174941 cites W2078574809 @default.
• W4306174941 cites W2083392652 @default.
• W4306174941 cites W2101428586 @default.
• W4306174941 cites W2117514532 @default.
• W4306174941 cites W2127071500 @default.
• W4306174941 cites W2139166113 @default.
• W4306174941 cites W2141236766 @default.
• W4306174941 cites W2153140538 @default.
• W4306174941 cites W2154370603 @default.
• W4306174941 cites W2157464327 @default.
• W4306174941 cites W2164625869 @default.
• W4306174941 cites W2165192566 @default.
• W4306174941 cites W2312488225 @default.
• W4306174941 cites W2318089142 @default.
• W4306174941 cites W2321634589 @default.
• W4306174941 cites W2333112136 @default.
• W4306174941 cites W2439361170 @default.
• W4306174941 cites W2610101478 @default.
• W4306174941 cites W2739319013 @default.
• W4306174941 cites W2796959653 @default.
• W4306174941 cites W2887478002 @default.
• W4306174941 cites W2908399125 @default.
• W4306174941 cites W2913212173 @default.
• W4306174941 cites W2938981698 @default.
• W4306174941 cites W2945879121 @default.
• W4306174941 cites W2959256911 @default.
• W4306174941 cites W3009899209 @default.
• W4306174941 cites W3009934971 @default.
• W4306174941 cites W3034789387 @default.
• W4306174941 cites W3110774789 @default.
• W4306174941 cites W3197934364 @default.
• W4306174941 cites W3207464661 @default.
• W4306174941 cites W4206631757 @default.
• W4306174941 cites W4220909840 @default.
• W4306174941 cites W4242264039 @default.
• W4306174941 cites W4293678328 @default.
• W4306174941 doi "https://doi.org/10.1016/j.omtm.2022.10.008" @default.
• W4306174941 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/36381304" @default.
• W4306174941 hasPublicationYear "2022" @default.
• W4306174941 type Work @default.
• W4306174941 citedByCount "9" @default.
• W4306174941 countsByYear W43061749412023 @default.
• W4306174941 crossrefType "journal-article" @default.
• W4306174941 hasAuthorship W4306174941A5061503158 @default.
• W4306174941 hasAuthorship W4306174941A5063887646 @default.
• W4306174941 hasAuthorship W4306174941A5064962658 @default.
• W4306174941 hasBestOaLocation W43061749412 @default.
• W4306174941 hasConcept C104317684 @default.
• W4306174941 hasConcept C12554922 @default.
• W4306174941 hasConcept C141231307 @default.
• W4306174941 hasConcept C185592680 @default.
• W4306174941 hasConcept C40767141 @default.
• W4306174941 hasConcept C55493867 @default.
• W4306174941 hasConcept C70721500 @default.
• W4306174941 hasConcept C86803240 @default.
• W4306174941 hasConcept C95444343 @default.
• W4306174941 hasConceptScore W4306174941C104317684 @default.
• W4306174941 hasConceptScore W4306174941C12554922 @default.
• W4306174941 hasConceptScore W4306174941C141231307 @default.
• W4306174941 hasConceptScore W4306174941C185592680 @default.
• W4306174941 hasConceptScore W4306174941C40767141 @default.
• W4306174941 hasConceptScore W4306174941C55493867 @default.
• W4306174941 hasConceptScore W4306174941C70721500 @default.
• W4306174941 hasConceptScore W4306174941C86803240 @default.
• W4306174941 hasConceptScore W4306174941C95444343 @default.
• W4306174941 hasFunder F4320307765 @default.
• W4306174941 hasFunder F4320309291 @default.
• W4306174941 hasFunder F4320309577 @default.
• W4306174941 hasLocation W43061749411 @default.
• W4306174941 hasLocation W43061749412 @default.
• W4306174941 hasLocation W43061749413 @default.
• W4306174941 hasOpenAccess W4306174941 @default.
• W4306174941 hasPrimaryLocation W43061749411 @default.
• W4306174941 hasRelatedWork W1980578041 @default.

• next