Answer | Researchers who would like to dive deeper into their Phosphorylation Analysis results may be interested in viewing the effects of specific phosphosites upon a pathway or network. This goes beyond the normal means of analyzing phosphoproteomics datasets in IPA and is designed to track and visualize specific changes in specific phosphosites. It will involve making a special sort of dataset where phosphosites are numbered in a particular way. IPA’s Overlay feature allows you to add bar charts to your pathway or network, making it easier to visualize and investigate how phosphorylation differences in dataset proteins are contributing to the biology of your experiment.You can view and interpret changes in specific phosphosites in two ways in IPA:
- Examine the effects of multiple phosphorylation sites for a protein in one experimental condition.
- View the effects of a single phosphorylation site across multiple biological conditions.
First, we recommend that you upload a standard phosphoproteomics dataset containing information from all phosphopeptides and observations (i.e., experimental comparisons such as treatment vs control, knockout vs wildtype, etc.) and perform a Phosphorylation Analysis. Next, you should identify the most relevant pathways or networks from the analysis that you would like to further investigate. For example, you may be interested in a specific Canonical Pathway or want to further investigate an Upstream Analysis network.You can then create and upload additional special datasets that focus on specific phosphosites in the proteins in these pathways and networks and apply the Overlay tool to visualize your data.To illustrate how to format your data and use the Overlay tool, we will use a network that was generated from an IPA Phosphorylation Analysis of mouse adipocytes stimulated with insulin for 15 seconds (from Table S3 of PMC 3690479). Option 1: Examine how an experimental condition results in changes to a protein’s phosphorylation sites.Each peptide residue from proteins in a pathway or network generated from one observation will be displayed as a bar in an Overlay bar chart.This option allows you to answer questions such as: “What is the effect of a drug treatment on specific protein phosphorylation sites?” “What are the differences in phosphorylation of each peptide residue in a knockout compared to a wildtype organism?”How to create and upload the special dataset:
- Each experimental group comparison (i.e., observation) will be placed into one dataset.
- We recommend creating an index file. This file will not be uploaded into IPA but will help serve as a reference for the uploaded datasets.
- To create the index file, place the protein IDs in the first column and any peptide-specific information, such as peptide sequences, in the second column. Each row will contain one phosphosite. You will then assign a number for each phosphosite.
In the example below, the first protein in this abbreviated dataset (IPI00830670) has one phosphosite, with its peptide sequence listed in the second column. Because there is one phosphosite in this protein, it is given a value of 1 in the third column. The second protein (IPI00830670) has six phosphosites. Therefore, there are six rows of phosphosites for this protein, and each is given a value ranging from 1 to 6. The third protein (IPI00623570) has two phosphosites, which are given values of 1 and 2, respectively.
| ID | Phosphosite | Peptide number | | IPI00670928 | S_664 NPREALSPCPST | 1 | | IPI00830670 | T_85 WLKDCRTPLGASL | 1 | | IPI00830670 | S_268 PLQRIGSMSSVTS | 2 | | IPI00830670 | S_271 RIGSMMSVTSTKE | 3 | | IPI00830670 | T_273 GSMSSVTSTKEVA | 4 | | IPI00830670 | S_274 SMSSVTSTKEVAD | 5 | | IPI00830670 | S_441 KSAQASSSEKEPC | 6 | | IPI00623570 | T_1454 KRGRAQTAPTKTS | 1 | | IPI00623570 | S_1460 TAPTKTSPRNAKK | 2 |
- Next, add the information from the index file into a new file that you will upload into IPA. The phosphopeptide data should be formatted so that:
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- Each experimental condition (observation) is in a separate file.
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- Each phosphopeptide has its own column of data measurement values (e.g., log ratios or fold changes) and will be mapped as an “Observation” when uploading the dataset.
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- Each file can contain up to 20 phosphorylation sites.
The example below shows the three proteins from the index file in a new dataset that includes their log ratio values.
| ID | Peptide 1 | Peptide 2 | Peptide 3 | Peptide 4 | Peptide 5 | Peptide 6 | | IPI00670928 | -1.873 | | | | | | | IPI00830670 | -3.644 | -2.762 | -3.324 | -5.313 | -5.313 | -2.167 | | IPI00623570 | 3.674 | 2.281 | | | | |
After uploading your dataset, you can open the network/pathway of interest, navigate to the Overlay button in the toolbar, select the option Overlay Analyses, Datasets and Lists, then select the dataset file you uploaded. Note: If you have opened the network or pathway from a Phosphorylation Analysis, the nodes will automatically be colored with phosphorylation values from that analysis. If you only want to view bar charts from the phosphosite dataset, you will need to remove the Phosphorylation Analysis results from the Overlay datasets, analyses and lists section by using the Remove Selected button. If you do not remove the Phosphorylation Analysis data, the first bar in the bar charts will be from the phosphosite that had the greatest absolute phosphorylation value in the analyzed dataset.The example network below shows the results of the uploaded phosphosites dataset. Each bar in the bar chart represents the phosphorylation measurement values of each phosphopeptide in the proteins in this network after 15 seconds of stimulation of insulin. Clicking on the graphs in the network shows a larger version that includes the phosphorylation log ratio measurements. The coloring of the nodes represents the increase or decrease in phosphorylation for the index number selected in the left panel.  For example, when Peptide 1 (Index 1) is selected, UBE4B is colored red, indicating that after 15 seconds of insulin stimulation this phosphorylation site shows an increase in phosphorylation. If Peptide 2 (Index 2) or 3 (Index 3) is selected, this node’s color would change to green, reflecting the decrease in phosphorylation at each of those two phosphosites. Gray represents nodes that do not contain phosphosites for the selected peptide (index) number. Option 2: Examine phosphorylation levels of a peptide across multiple conditions
You can use the Overlay tool to display relative phosphorylation levels of each site in a protein on a pathway or network for more than one experimental condition. This option can answer questions such as: “How do different drug compounds affect the phosphorylation of a specific site in a protein?” “How does a phosphorylation site change over time after treatment with a compound?”This visualization can lead to a greater understanding of how different phosphorylation states correlate with differences in treatments, timepoints, or phenotypes.We can use the Overlay tool to view bar charts that will display phosphorylation values of one phosphopeptide from a single protein across all conditions (observations). These files are set up very much like a typical dataset that you would upload into IPA, except each phosphosite is in a separate file.The dataset will not require an index file as mentioned in Option 1 above. Instead, phosphorylation data is formatted so that:
- Each protein phosphopeptide is placed into a separate data file.
- Each data file contains values for all experimental observations.
The example below shows two simple datasets with three timepoints.Peptide 1 dataset file
| ID | Phospho Site | 15 sec | 30 sec | 1 min | | IPI00623249 | S_121 DSSPPDSPASSPC | -2.152 | | | | IPI00670928 | S_664 NPREALSPCPSTI | -1.873 | -1.405 | | | IPI00225761 | S_334 PDGHAVSPRNTET | 1.933 | | | | IPI00115900 | S_2282 SSGLPSSPSSPRL | 2.652 | -0.872 | -0.666 | | IPI00221566 | S_392 MDSIAGSERPGPS | -2.353 | | -0.887 | | IPI00133608 | S_122 SGELRRSSPPGHY | 1.802 | | |
Peptide 2 dataset file
| ID | Phospho Site | 15 sec | 30 sec | 1 min | | IPI00623249 | S_97 AGPLDMSLPSTPD | 0.413 | 0.413 | 0.397 | | IPI00670928 | S_1137 EKLDGESDKEQFD | -1.405 | 0.093 | 1.405 | | IPI00225761 | S_431 VLLRSVSSDSLGP | -0.266 | -0.266 | | | IPI00115900 | S_620 VSKGLASPSLKEK | | 0.711 | 0.913 | | IPI00221566 | S_408 HFSEHPSMSNMKA | 0.826 | 0.826 | 0.471 | | IPI00133608 | S_123 GELRRSSPPGHYT | 0.301 | 0.301 | 0.329 |
The dataset is structured so that the first column contains the protein ID, the second column contains the phosphosite information (which can be any combination of up to 256 characters), and the remaining columns contain the data measurement values for each observation. When uploading the dataset into IPA, the Phospho Site column will be mapped as Observation 1. The next column that contains the data measurement value for the first observation will also be mapped as Observation 1. The remaining columns will contain the data measurement values for the other observations, and will be mapped as Observation 2, 3, etc. The Phospho Site information will be applied to all Observations even though it is assigned as Observation 1. You can rename the Observations using the Edit Observation Names button. After uploading your dataset, you can open the network/pathway of interest, navigate to the Overlay button in the toolbar, select the option Overlay Analyses, Datasets and Lists, then select the dataset file(s) you uploaded. Note: If you have opened the network or pathway from a Phosphorylation Analysis, the nodes will be automatically colored with phosphorylation values from that analysis. If you only want to view bar charts that contain information about each observation, you will need to remove the Phosphorylation Analysis information from the Overlay datasets, analyses and lists section by using the Remove Selected button. If you do not remove the analysis, the first bar in the bar charts will be from the phosphosite that was used in the Phosphorylation Analysis.We will compare the effects of phosphorylation on specific phosphosites across three timepoints: 15 seconds, 30 seconds, and one minute after the adipocytes were stimulated with insulin. The network below shows the information uploaded with data measurement values from multiple observations for one phosphosite in each protein. Each bar in the bar chart represents timepoint. Clicking on a graph in the network shows a larger version that includes the phosphorylation log ratio values. This option makes it easier to view trends across observations. For example, the first phosphosite for UBE4B shows an increase of phosphorylation after 15 seconds of insulin treatment that decreases over time, while the TRIP12 phosphosite shows a consistent increase in phosphorylation after stimulation among all timepoints. The coloring of the nodes represents the increase or decrease in phosphorylation for the index number selected. The index can be changed in the Overlay datasets, analyses and lists section of the left panel. For example, selecting Index 1 (the 15 seconds timepoint observation shown in the networks above) shows UBE4B’s node colored in red, indicating an increase in phosphorylation at that site 15 seconds after insulin stimulation. Selecting Index 3 (the 1 minute timepoint observation) changes the coloration of the node to green, representing a decrease in phosphorylation after one minute of stimulation of insulin. Grey nodes display timepoints that do not have measurable differences in phosphorylation between treated and untreated adipocytes at the phosphorylated site, such as TNC1D15, which shows an increase in phosphorylation at this site at only 15 seconds after treatment. More information about using the Overlay tool can be found in this help document:
Overlay Analyses, Datasets and Lists
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