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Quantitative temporal viromics: an approach to investigate host-pathogen interaction.

Weekes MP, Tomasec P, Huttlin EL, Fielding CA, Nusinow D, Stanton RJ, Wang EC, Aicheler R, Murrell I, Wilkinson GW, Lehner PJ, Gygi SP - Cell (2014)

Bottom Line: QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets.Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined.QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA; Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK. Electronic address: mpw1001@cam.ac.uk.

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Temporal Plasma Membrane Profiling of HCMV-Infected Fibroblasts, Related to Figure 1(A) Gene ontology annotation of proteins quantified in experiment PM1. Peptides were analyzed by mass spectrometry either unfractionated, or after division into 6 fractions using offline strong cation exchange. ‘'Short GO” refers to a subset of proteins annotated by GO as integral to the membrane, but with no subcellular assignment and a short 4- or 5-part GO cellular compartment term (Weekes et al., 2012).(B) Quantitation of all cell surface proteins that exhibit previously reported changes during productive HCMV infection in HFFFs. Protein temporal profiles correlated well between repeat time courses PM1 (upper panels) and PM2 (lower panels). MICB and ULBP1 were not quantified in experiment PM1. Because of sequence similarities it was not possible to distinguish individual isoforms of HLA-B, and these data are not included. Red diamonds – irradiated HCMV infection at 12h. One-way ANOVA with multiple hypothesis correction: ∗p < 0.005, ∗∗p < 0.0001. Red diamonds – 12h after infection with irradiated HCMV.(C) Hierarchical clustering of proteins quantified in whole-cell lysate experiment WCL1. Fold change was limited to a maximum of 50.(D) Workflow of 10-plex TMT experiments. ∗12h after infection with irradiated HCMV.(E) Hierarchical clustering of proteins quantified in plasma membrane experiment PM2. Fold change was limited to a maximum of 50.(F) Correlation between proteins quantified in both experiments WCL1 and WCL2.
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figs1: Temporal Plasma Membrane Profiling of HCMV-Infected Fibroblasts, Related to Figure 1(A) Gene ontology annotation of proteins quantified in experiment PM1. Peptides were analyzed by mass spectrometry either unfractionated, or after division into 6 fractions using offline strong cation exchange. ‘'Short GO” refers to a subset of proteins annotated by GO as integral to the membrane, but with no subcellular assignment and a short 4- or 5-part GO cellular compartment term (Weekes et al., 2012).(B) Quantitation of all cell surface proteins that exhibit previously reported changes during productive HCMV infection in HFFFs. Protein temporal profiles correlated well between repeat time courses PM1 (upper panels) and PM2 (lower panels). MICB and ULBP1 were not quantified in experiment PM1. Because of sequence similarities it was not possible to distinguish individual isoforms of HLA-B, and these data are not included. Red diamonds – irradiated HCMV infection at 12h. One-way ANOVA with multiple hypothesis correction: ∗p < 0.005, ∗∗p < 0.0001. Red diamonds – 12h after infection with irradiated HCMV.(C) Hierarchical clustering of proteins quantified in whole-cell lysate experiment WCL1. Fold change was limited to a maximum of 50.(D) Workflow of 10-plex TMT experiments. ∗12h after infection with irradiated HCMV.(E) Hierarchical clustering of proteins quantified in plasma membrane experiment PM2. Fold change was limited to a maximum of 50.(F) Correlation between proteins quantified in both experiments WCL1 and WCL2.

Mentions: We infected primary human fetal foreskin fibroblasts (HFFFs) with HCMV strain Merlin and initially used 8-plex TMT to quantify changes in PM protein expression. We assessed in biological duplicate three of the reference time points in productive HCMV infection and mock infection (experiment “PM1”, Figure 1A). We quantified 927 PM proteins (Figure S1A available online). Surprisingly, 56% of proteins changed >2-fold and 33% >3-fold by 72 hr of infection. Replicates clustered tightly (Figure 1B).


Quantitative temporal viromics: an approach to investigate host-pathogen interaction.

Weekes MP, Tomasec P, Huttlin EL, Fielding CA, Nusinow D, Stanton RJ, Wang EC, Aicheler R, Murrell I, Wilkinson GW, Lehner PJ, Gygi SP - Cell (2014)

Temporal Plasma Membrane Profiling of HCMV-Infected Fibroblasts, Related to Figure 1(A) Gene ontology annotation of proteins quantified in experiment PM1. Peptides were analyzed by mass spectrometry either unfractionated, or after division into 6 fractions using offline strong cation exchange. ‘'Short GO” refers to a subset of proteins annotated by GO as integral to the membrane, but with no subcellular assignment and a short 4- or 5-part GO cellular compartment term (Weekes et al., 2012).(B) Quantitation of all cell surface proteins that exhibit previously reported changes during productive HCMV infection in HFFFs. Protein temporal profiles correlated well between repeat time courses PM1 (upper panels) and PM2 (lower panels). MICB and ULBP1 were not quantified in experiment PM1. Because of sequence similarities it was not possible to distinguish individual isoforms of HLA-B, and these data are not included. Red diamonds – irradiated HCMV infection at 12h. One-way ANOVA with multiple hypothesis correction: ∗p < 0.005, ∗∗p < 0.0001. Red diamonds – 12h after infection with irradiated HCMV.(C) Hierarchical clustering of proteins quantified in whole-cell lysate experiment WCL1. Fold change was limited to a maximum of 50.(D) Workflow of 10-plex TMT experiments. ∗12h after infection with irradiated HCMV.(E) Hierarchical clustering of proteins quantified in plasma membrane experiment PM2. Fold change was limited to a maximum of 50.(F) Correlation between proteins quantified in both experiments WCL1 and WCL2.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048463&req=5

figs1: Temporal Plasma Membrane Profiling of HCMV-Infected Fibroblasts, Related to Figure 1(A) Gene ontology annotation of proteins quantified in experiment PM1. Peptides were analyzed by mass spectrometry either unfractionated, or after division into 6 fractions using offline strong cation exchange. ‘'Short GO” refers to a subset of proteins annotated by GO as integral to the membrane, but with no subcellular assignment and a short 4- or 5-part GO cellular compartment term (Weekes et al., 2012).(B) Quantitation of all cell surface proteins that exhibit previously reported changes during productive HCMV infection in HFFFs. Protein temporal profiles correlated well between repeat time courses PM1 (upper panels) and PM2 (lower panels). MICB and ULBP1 were not quantified in experiment PM1. Because of sequence similarities it was not possible to distinguish individual isoforms of HLA-B, and these data are not included. Red diamonds – irradiated HCMV infection at 12h. One-way ANOVA with multiple hypothesis correction: ∗p < 0.005, ∗∗p < 0.0001. Red diamonds – 12h after infection with irradiated HCMV.(C) Hierarchical clustering of proteins quantified in whole-cell lysate experiment WCL1. Fold change was limited to a maximum of 50.(D) Workflow of 10-plex TMT experiments. ∗12h after infection with irradiated HCMV.(E) Hierarchical clustering of proteins quantified in plasma membrane experiment PM2. Fold change was limited to a maximum of 50.(F) Correlation between proteins quantified in both experiments WCL1 and WCL2.
Mentions: We infected primary human fetal foreskin fibroblasts (HFFFs) with HCMV strain Merlin and initially used 8-plex TMT to quantify changes in PM protein expression. We assessed in biological duplicate three of the reference time points in productive HCMV infection and mock infection (experiment “PM1”, Figure 1A). We quantified 927 PM proteins (Figure S1A available online). Surprisingly, 56% of proteins changed >2-fold and 33% >3-fold by 72 hr of infection. Replicates clustered tightly (Figure 1B).

Bottom Line: QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets.Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined.QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA; Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK. Electronic address: mpw1001@cam.ac.uk.

Show MeSH
Related in: MedlinePlus