A volcano plot is a type of scatter plot used to quickly identify protein or peptide changes in large data sets composed of sample pairs. The left upper quadrant (highlighted in green) displays those proteins or peptide groups that are significantly downregulated. The upper right quadrant (highlighted in red) displays those proteins or peptide groups that are significantly upregulated. You can choose where the green and red highlighted areas are displayed by using the P-Value and Log2Fold Change sliders. Use these areas to choose proteins or peptides that might be biologically relevant to your study.

The x axis displays the abundance ratio in log2, and the y axis displays p-values in log10.

For information on p-values, see Calculate p-values and adjusted p-values for quantification results.

The result report must contain replicate quantification data in order for you to display this plot or to run a new analysis.

Procedure

  1. Open the result file of interest.
  2. Select View > Distribution Charts, or select Distribution Charts in the toolbar.
  3. Select the Volcano Plots tab.
  4. From the Data Source list, select the type of data to plot, either Proteins or Peptide Groups, peptide isoforms.
  5. From the Comparison list, select the items to be compared.
  6. Drag the P-Value slider to set the range of p-values (shaded areas) along the y axis, or enter the appropriate number in the box to the right of the slider and press the ENTER key.
  7. Drag the Log2 Fold Change slider to set the range of abundance values (shaded areas) along the x axis, or enter the appropriate number in the box to the right of the slider and press the ENTER key.
  8. The following figure gives an example of a volcano plot that compares two tissues, kidney and liver.
  9. The data points are color-coded as defined by the legend.
  10. Volcano plot
  11. If the result file contains p-values and ratios, the volcano plot opens with the differential analysis from the data processing. The ratios in the Comparison list match the ratios on the Ratios and Ratio Groups page of the Quantification page in the Result Summaries view. The initial p value setting is 0.05 (–log10 0.05 = 1.3), and the initial Log2 Fold Change setting is 1 (a ratio of 2 to 1). Depending on the setting of the Log10 Transform parameter in the Reporter Ions Quantifier node or the Precursor Ions Quantifier node, the y-axis scale spans the p-value range (0–1) or the –log10 p-value range (–log10 0 = infinity, –log10 1 = 0).
  12. Identical protein ratios in the Comparison list and on the Ratios and Ratio Groups summary page
  13. The volcano plot highlights selected items in blue. You can select and clear items of interest. The corresponding items are then selected or cleared in the result file page.

Task

Procedure

Display the legend

Right-click the plot and select Show Legend.

Change the analysis displayed in the volcano plot

Select a different ratio from the Comparison list.

This step changes the data points in the plot.

Change the p-value setting

Drag the P-Value slider to the left or right, or enter the p-value in the box to the right of the slider and press the ENTER key.

This step changes the y-axis range of the shaded areas.

Change the log2 fold change setting

Drag the Log2 Fold Change slider to the left or right, or enter the fold change value in the box to the right of the slider and press the ENTER key.

This step changes the x-axis range of the shaded areas.

Select a single data point in the volcano plot

Right-click the data point in the volcano plot, and select Check Point.

The selected point turns blue in the plot, and in the Checked column of the result report, the application selects the checkbox of the corresponding protein or peptide group.

Clear a single data point in the volcano plot

Right-click the data point in the volcano plot, and select Uncheck Point.

The selected point resumes its original color in the plot, and in the Checked column of the result report, the application clears the checkbox of the corresponding protein or peptide group.

Select all visible data points

Right-click the volcano plot, and select Check All Visible Points.

All visible data points turn blue in the plot, and the legend disappears. In the Checked column of the result report, the application selects the checkboxes of the corresponding proteins or peptide groups.

Clear all visible data points

Right-click the volcano plot, and select Uncheck All Visible Points.

All visible data points resume their original colors in the plot, and the legend reappears. In the Checked column of the result report, the application clears the checkboxes of the corresponding proteins or peptide groups.

Display tooltips

Right-click the view and select Show Position Tooltips.

The tooltips display the location of the cursor by time and peak height.

Enlarge the view

Drag your cursor to the right and select the area to enlarge in size.

Reduce the view

  • Drag your cursor to the left and select the area to reduce in size.

–or–

  • Right-click the view and select Zoom Out.

Return to the default view

Right-click the chart and select Undo All Zoom/Pan.

Copy the data points in a volcano plot in an image format

Procedure

  1. Right-click the chart and select Copy > Image.
  2. Paste the contents of the chart into another document.
  3. The contents appear as an image of the scatter plot in the document.

Copy the data points in a volcano plot as text

Procedure

  1. Right-click the chart and select Copy > Points.
  2. Paste the contents of the chart into another document.
  3. The contents appear as two textual columns in the document, Log2 Ratio and -Log10 P-value, which correspond to each point’s location coordinates in the plot.

Copy the Proteins or Peptide Groups page as text in CSV format

Procedure

  1. Right-click the chart and select Copy > Point Details.
  2. Paste the contents of the chart into another document.
  3. All items on the selected page (Proteins or Peptide Groups) appear in a CSV-format text file.

Export the data points from the volcano plot as an image

Procedure

  1. Right-click the volcano plot and select Export > Image As.
  2. In the Save As dialog box, enter the name of the file to save the image in.
  3. Select Save.
  4. The application saves the volcano plot as an Enhanced Metafile (EMF) file.

Export the data points from the volcano plot as text

Procedure

  1. Right-click the volcano plot and select Export > Points As.
  2. In the Save As dialog box, enter the name of the file to save the image in.
  3. Select Save.
  4. The application saves the volcano plot as a text file that contains log2 ratios and Log10 p-values.

Export the Proteins or Peptide Groups page as text in CSV format

Procedure

  1. Right-click the volcano plot and select Export > Point Details As.
  2. In the Save As dialog box, enter the name of the file to save the image in.
  3. Select Save.
  4. All items on the selected page (Proteins or Peptide Groups) appear in a CSV-format text file.