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Role of cytoskeletal proteins in cerebral cavernous malformation signaling pathways: a proteomic analysis.

Baxter SS, Dibble CF, Byrd WC, Carlson J, Mack CR, Saldarriaga I, Bencharit S - Mol Biosyst (2014)

Bottom Line: Loss of expression of each CCM gene results in loss of in vitro endothelial tube formation.Principal component analysis and cluster analysis show the effects of individual knockdown.While all CCM mutations result in similar pathology, different CCM mutations have their own distinct pathogenesis in cell signaling.

View Article: PubMed Central - PubMed

Affiliation: David H. Murdock Research Institute, North Carolina Research Campus, Kannapolis, NC 28081, USA.

ABSTRACT
Three genetic mutations were found to cause cerebral cavernous malformation (CCM), a vascular anomaly predisposing affected individuals to hemorrhagic stroke. These CCM proteins function together as a protein complex in the cell. Loss of expression of each CCM gene results in loss of in vitro endothelial tube formation. Label-free differential protein expression analysis using multidimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) was applied to explore the proteomic profile for loss of each CCM gene expression in mouse endothelial stem cells (MEES) compared to mock shRNA and no shRNA control cell-lines. Differentially expressed proteins were identified (p < 0.05). 120 proteins were differentially expressed among the cell-lines. Principal component analysis and cluster analysis show the effects of individual knockdown. In all knockdown cell-lines, altered expression of cytoskeletal proteins is the most common. While all CCM mutations result in similar pathology, different CCM mutations have their own distinct pathogenesis in cell signaling.

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Mass Spectrometry Analysis (A–B). Aligned Mass Data. An example of aligned peptide signals for the data set is presented. Sample labels: figures are color coded according to cell line. Selection (red) references an option to manually select a single sample to be highlighted. This option was not selected. (A) Extracted ion chromatogram for m/z 633.0408–633.7484 from retention time 548 391–556 203 demonstrates retention time alignment across all samples for this signal. (B) Aligned masses in the mass range of 633.0408–633.7484 for all samples in the study set. (C). PCA plot for the study set. An ANOVA test was performed using all mass signals detected to compare the expression results from the five cell lines. Results demonstrate an unsupervised separation of the five cell lines based on the mass signal patterns. (D). PCA for cell line replicates. An ANOVA test was performed at the protein level to compare the protein expression results from the five cell lines. Candidate differentially expressed proteins were determined and are presented in the ESI,† Table S1. Results demonstrate separation of the five cell lines based on the protein patterns determined based on an ANOVA test. (E). Cell line cluster based on differentially expressed proteins determined by ANOVA.
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fig2: Mass Spectrometry Analysis (A–B). Aligned Mass Data. An example of aligned peptide signals for the data set is presented. Sample labels: figures are color coded according to cell line. Selection (red) references an option to manually select a single sample to be highlighted. This option was not selected. (A) Extracted ion chromatogram for m/z 633.0408–633.7484 from retention time 548 391–556 203 demonstrates retention time alignment across all samples for this signal. (B) Aligned masses in the mass range of 633.0408–633.7484 for all samples in the study set. (C). PCA plot for the study set. An ANOVA test was performed using all mass signals detected to compare the expression results from the five cell lines. Results demonstrate an unsupervised separation of the five cell lines based on the mass signal patterns. (D). PCA for cell line replicates. An ANOVA test was performed at the protein level to compare the protein expression results from the five cell lines. Candidate differentially expressed proteins were determined and are presented in the ESI,† Table S1. Results demonstrate separation of the five cell lines based on the protein patterns determined based on an ANOVA test. (E). Cell line cluster based on differentially expressed proteins determined by ANOVA.

Mentions: To examine the global changes of each CCM knockdown, a label-free differential expression LC/MS/MS method was used to quantitatively compare the protein expression among all five cell lines; three CCM knockdown cell lines and two control cell lines (Fig. 1A–E). The total protein in each sample was calibrated (Fig. 2A and B). MS data from all five cell lines were analyzed in triplicate (15 replicates). Data processing in Elucidator was performed in multiple steps that included mass correction and mass signal alignment of detected signals, retention time alignment of detected signals, and feature selection, with features representing mass signals detected within the study set. QC review of the aligned data indicated the mass signals were correctly aligned in the Elucidator software (Fig. 2A and B). For retention time alignment, mathematical adjustments are performed to time-align the same detected signals across all samples. QC assessment of this retention time shift indicates that all adjustments were <3 min. Principal component analysis (PCA) was performed on the collected data set to evaluate the data for outliers (Fig. 2C and D) and sample trends. Results from the unsupervised analysis suggest that the samples differentiate by cell line based on the mass spectrometric patterns detected.


Role of cytoskeletal proteins in cerebral cavernous malformation signaling pathways: a proteomic analysis.

Baxter SS, Dibble CF, Byrd WC, Carlson J, Mack CR, Saldarriaga I, Bencharit S - Mol Biosyst (2014)

Mass Spectrometry Analysis (A–B). Aligned Mass Data. An example of aligned peptide signals for the data set is presented. Sample labels: figures are color coded according to cell line. Selection (red) references an option to manually select a single sample to be highlighted. This option was not selected. (A) Extracted ion chromatogram for m/z 633.0408–633.7484 from retention time 548 391–556 203 demonstrates retention time alignment across all samples for this signal. (B) Aligned masses in the mass range of 633.0408–633.7484 for all samples in the study set. (C). PCA plot for the study set. An ANOVA test was performed using all mass signals detected to compare the expression results from the five cell lines. Results demonstrate an unsupervised separation of the five cell lines based on the mass signal patterns. (D). PCA for cell line replicates. An ANOVA test was performed at the protein level to compare the protein expression results from the five cell lines. Candidate differentially expressed proteins were determined and are presented in the ESI,† Table S1. Results demonstrate separation of the five cell lines based on the protein patterns determined based on an ANOVA test. (E). Cell line cluster based on differentially expressed proteins determined by ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4043921&req=5

fig2: Mass Spectrometry Analysis (A–B). Aligned Mass Data. An example of aligned peptide signals for the data set is presented. Sample labels: figures are color coded according to cell line. Selection (red) references an option to manually select a single sample to be highlighted. This option was not selected. (A) Extracted ion chromatogram for m/z 633.0408–633.7484 from retention time 548 391–556 203 demonstrates retention time alignment across all samples for this signal. (B) Aligned masses in the mass range of 633.0408–633.7484 for all samples in the study set. (C). PCA plot for the study set. An ANOVA test was performed using all mass signals detected to compare the expression results from the five cell lines. Results demonstrate an unsupervised separation of the five cell lines based on the mass signal patterns. (D). PCA for cell line replicates. An ANOVA test was performed at the protein level to compare the protein expression results from the five cell lines. Candidate differentially expressed proteins were determined and are presented in the ESI,† Table S1. Results demonstrate separation of the five cell lines based on the protein patterns determined based on an ANOVA test. (E). Cell line cluster based on differentially expressed proteins determined by ANOVA.
Mentions: To examine the global changes of each CCM knockdown, a label-free differential expression LC/MS/MS method was used to quantitatively compare the protein expression among all five cell lines; three CCM knockdown cell lines and two control cell lines (Fig. 1A–E). The total protein in each sample was calibrated (Fig. 2A and B). MS data from all five cell lines were analyzed in triplicate (15 replicates). Data processing in Elucidator was performed in multiple steps that included mass correction and mass signal alignment of detected signals, retention time alignment of detected signals, and feature selection, with features representing mass signals detected within the study set. QC review of the aligned data indicated the mass signals were correctly aligned in the Elucidator software (Fig. 2A and B). For retention time alignment, mathematical adjustments are performed to time-align the same detected signals across all samples. QC assessment of this retention time shift indicates that all adjustments were <3 min. Principal component analysis (PCA) was performed on the collected data set to evaluate the data for outliers (Fig. 2C and D) and sample trends. Results from the unsupervised analysis suggest that the samples differentiate by cell line based on the mass spectrometric patterns detected.

Bottom Line: Loss of expression of each CCM gene results in loss of in vitro endothelial tube formation.Principal component analysis and cluster analysis show the effects of individual knockdown.While all CCM mutations result in similar pathology, different CCM mutations have their own distinct pathogenesis in cell signaling.

View Article: PubMed Central - PubMed

Affiliation: David H. Murdock Research Institute, North Carolina Research Campus, Kannapolis, NC 28081, USA.

ABSTRACT
Three genetic mutations were found to cause cerebral cavernous malformation (CCM), a vascular anomaly predisposing affected individuals to hemorrhagic stroke. These CCM proteins function together as a protein complex in the cell. Loss of expression of each CCM gene results in loss of in vitro endothelial tube formation. Label-free differential protein expression analysis using multidimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) was applied to explore the proteomic profile for loss of each CCM gene expression in mouse endothelial stem cells (MEES) compared to mock shRNA and no shRNA control cell-lines. Differentially expressed proteins were identified (p < 0.05). 120 proteins were differentially expressed among the cell-lines. Principal component analysis and cluster analysis show the effects of individual knockdown. In all knockdown cell-lines, altered expression of cytoskeletal proteins is the most common. While all CCM mutations result in similar pathology, different CCM mutations have their own distinct pathogenesis in cell signaling.

Show MeSH
Related in: MedlinePlus