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Analysis of purified wild type and mutant adenovirus particles by SILAC based quantitative proteomics.

Alqahtani A, Heesom K, Bramson JL, Curiel D, Ugai H, Matthews DA - J. Gen. Virol. (2014)

Bottom Line: In all the samples analysed we found no evidence of consistent packaging or contamination with cellular proteins.We believe this technique is a powerful method to analyse the protein composition of this important gene therapy vector and genetically engineered or synthetic virus-like particles.The raw data have been deposited at proteomexchange, identifer PXD001120.

View Article: PubMed Central - PubMed

Affiliation: School of Cellular and Molecular Medicine, University Walk, University of Bristol, Bristol BS8 1TD, UK College of Applied Medical Sciences, Najran University, Najran 1983, Saudi Arabia.

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Related in: MedlinePlus

Abundance ratios of viral proteins detected and quantified. Single batches of WT adenovirus labelled with R6K6 or R10K8 were compared quantitatively with 11 or six different virus batches, respectively. (a–c) Coomassie-stained gels of the viruses. The type of virus used, whether mature or immature particles and isotopic labelling are indicated. Samples marked with an asterisk were purified by H. U. at Washington State University at St Louis. (a) Mature WT particles and mature recombinant adenoviruses, (b) mature dV/TSB particles, and (c) immature particles. For each gel, a ladder with molecular mass standards is included and the virus proteins are labelled. (d–g) Mean ratio±sd (two or more values available). Those viral proteins where the ratio of abundance was ~1 : 1 in all experiments are shown in (d). Note that all subsequent charts use a log scale because of the wide variability of abundance. (e) Range of quantification using R6K6 virus as the comparator and (f) the same proteins where their abundance ratios were compared with the R10K8 virus preparation. (g) A similar analysis of immature particles where the relative abundance of viral proteins in immature particles was compared with the batch of R6K6-labelled mature particles used in (d, e).
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f1: Abundance ratios of viral proteins detected and quantified. Single batches of WT adenovirus labelled with R6K6 or R10K8 were compared quantitatively with 11 or six different virus batches, respectively. (a–c) Coomassie-stained gels of the viruses. The type of virus used, whether mature or immature particles and isotopic labelling are indicated. Samples marked with an asterisk were purified by H. U. at Washington State University at St Louis. (a) Mature WT particles and mature recombinant adenoviruses, (b) mature dV/TSB particles, and (c) immature particles. For each gel, a ladder with molecular mass standards is included and the virus proteins are labelled. (d–g) Mean ratio±sd (two or more values available). Those viral proteins where the ratio of abundance was ~1 : 1 in all experiments are shown in (d). Note that all subsequent charts use a log scale because of the wide variability of abundance. (e) Range of quantification using R6K6 virus as the comparator and (f) the same proteins where their abundance ratios were compared with the R10K8 virus preparation. (g) A similar analysis of immature particles where the relative abundance of viral proteins in immature particles was compared with the batch of R6K6-labelled mature particles used in (d, e).

Mentions: Each batch of virus contained only the major viral proteins normally detected by SDS-PAGE and Coomassie staining (Fig. 1a–c). We found that protein V was missing from all dV/TSB preparations (Fig. 1b), and proteins V and VII were missing from immature particles in WT Ad5 and dV/TSB, as expected (Fig. 1c).


Analysis of purified wild type and mutant adenovirus particles by SILAC based quantitative proteomics.

Alqahtani A, Heesom K, Bramson JL, Curiel D, Ugai H, Matthews DA - J. Gen. Virol. (2014)

Abundance ratios of viral proteins detected and quantified. Single batches of WT adenovirus labelled with R6K6 or R10K8 were compared quantitatively with 11 or six different virus batches, respectively. (a–c) Coomassie-stained gels of the viruses. The type of virus used, whether mature or immature particles and isotopic labelling are indicated. Samples marked with an asterisk were purified by H. U. at Washington State University at St Louis. (a) Mature WT particles and mature recombinant adenoviruses, (b) mature dV/TSB particles, and (c) immature particles. For each gel, a ladder with molecular mass standards is included and the virus proteins are labelled. (d–g) Mean ratio±sd (two or more values available). Those viral proteins where the ratio of abundance was ~1 : 1 in all experiments are shown in (d). Note that all subsequent charts use a log scale because of the wide variability of abundance. (e) Range of quantification using R6K6 virus as the comparator and (f) the same proteins where their abundance ratios were compared with the R10K8 virus preparation. (g) A similar analysis of immature particles where the relative abundance of viral proteins in immature particles was compared with the batch of R6K6-labelled mature particles used in (d, e).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202269&req=5

f1: Abundance ratios of viral proteins detected and quantified. Single batches of WT adenovirus labelled with R6K6 or R10K8 were compared quantitatively with 11 or six different virus batches, respectively. (a–c) Coomassie-stained gels of the viruses. The type of virus used, whether mature or immature particles and isotopic labelling are indicated. Samples marked with an asterisk were purified by H. U. at Washington State University at St Louis. (a) Mature WT particles and mature recombinant adenoviruses, (b) mature dV/TSB particles, and (c) immature particles. For each gel, a ladder with molecular mass standards is included and the virus proteins are labelled. (d–g) Mean ratio±sd (two or more values available). Those viral proteins where the ratio of abundance was ~1 : 1 in all experiments are shown in (d). Note that all subsequent charts use a log scale because of the wide variability of abundance. (e) Range of quantification using R6K6 virus as the comparator and (f) the same proteins where their abundance ratios were compared with the R10K8 virus preparation. (g) A similar analysis of immature particles where the relative abundance of viral proteins in immature particles was compared with the batch of R6K6-labelled mature particles used in (d, e).
Mentions: Each batch of virus contained only the major viral proteins normally detected by SDS-PAGE and Coomassie staining (Fig. 1a–c). We found that protein V was missing from all dV/TSB preparations (Fig. 1b), and proteins V and VII were missing from immature particles in WT Ad5 and dV/TSB, as expected (Fig. 1c).

Bottom Line: In all the samples analysed we found no evidence of consistent packaging or contamination with cellular proteins.We believe this technique is a powerful method to analyse the protein composition of this important gene therapy vector and genetically engineered or synthetic virus-like particles.The raw data have been deposited at proteomexchange, identifer PXD001120.

View Article: PubMed Central - PubMed

Affiliation: School of Cellular and Molecular Medicine, University Walk, University of Bristol, Bristol BS8 1TD, UK College of Applied Medical Sciences, Najran University, Najran 1983, Saudi Arabia.

Show MeSH
Related in: MedlinePlus