02 Differential Abundance
These small examples have been produced with the provided example data set, an interaction proteomics study focusing on PGRMC1, a protein from the MAPR family with a range of cellular functions. In this study, MIA PaCa-2 cells were stably transfected with a PGRMC1-HA plasmid and Co-IPs of PGRMC1 interacting proteins were isolated from cells expressing PGRMC1-HA, as well as from non-transfected parental MIA PaCa-2 cells as a negative control, with or without AG-205 treatment (a PGRMC1-specific inhibitor). As the example data contains AP-MS data, we set the selection parameter to ”enriched” to retrieve differentially abundant proteins compared against the control.
Then we select a single group comparison (PRGMC1 bait vs MIA PaCa-2 cell background) and press "Submit Analysis".
When you hover over the volcano - or MA - plot you can see features to manipulate the plot. When we utilize the select box or lasso tool we can annotate the highest enriched proteins, as seen in the figure above.
Further information on most plots can be acquired when you press the "info" icon. Plot parameters can be changed when pressing the "wrench" icon and can be saved upon hovering over the plot and clicking on the "camera" icon.
The example data contains Co-IPs of PGRMC1 in untreated MIA PaCa-2 cells and MIA PaCa-2 cells treated with AG-205. In this example, we are interested in whether some prey proteins of PGRMC1 are sensitive to AG-205.
When we select the ”Analyze multiple comparisons” tab pill we can select the two comparisons
of the bait versus the negative controls (PGRMC1__vs__MIAPACA and PGRMC1_AG205__vs__MIAPACA_AG205):
Scrolling down we can figure out the quantitive changes of prey proteins with and without AG-205 treatment in a fold change plot:
This small examples have been produced with the provided example data set (using the same global paramters as in the previous tutorial). An over-representation analysis was conducted utilizing the 46 enriched proteins from the comparison PGRMC1__vs__MIAPACA. The "Show genes in functional enrichment?" button was selected.
Sorting the output table from most significant p-value to least significant we find the term "actin binding" on top of the list. 15 proteins from the enriched proteins are annotated with this term.
All visualizations (heatmap, fold change plot and PPI network) work only on the proteins selected in the above output table we can filter that table to only show proteins annotated with our term of interest. The output table can parse "regular expressions", so all we need to do is to copy paste the comma-delimited gene names into a text editor (or text processing tool like MS Word) and replace all commas with viertical line symbols ("|" which is the logical "or" operator) with the "Find and Replace" tool:
We can now paste the the vertical line delimited gene names into the "Gene.names" search bar in the output table and we could successfully subset the data table to only show proteins of our interest:
Below the table there is now a text message telling us that the original table has been filtered and that only the remaining proteins are used in subsequent visualizations. As an example you can now observe how the selected proteins compare across different group comparisons in a heatmap or fold change plot.