In the list of experiments, click “quantCOMPARE” to list all existing quantitation comparisons.
Figure 10.3.1: Link for Quantitative Comparison.
Click “run new quantitative ©COMPARE” to build a new comparison. This section shows how to submit comparisons on ms1 based labeling analysis such as SILAC, 15N, Dimethyl, etc
Click “run new quantitative ©COMPARE” to build a new comparison. For different labeling types, the procedure and result formats are different. This section shows how to submit comparisons with both “ratio-to-ratio” and “compare directly” comparisons.
Figure 10.3.2 view of Quant Compare
“Ratio-to-Ratio” Comparison at Protein Level
The following are the steps to submit comparison with “MS1 labeling – ratio-to-ratio” label type, and protein with redundant compare level as an example. Click the next button to view the list of experiments to be compared.
Figure 10.3.3: Quant Compare Options.
In the page shown below, users need to assign a name for the group and select at least two experiments to add to the group. Click the “Add to group” button. Name this group “1” and select experiments for the quant comparison. In the next step, the user will build a second group.
Figure 10.3.4 Interface to add experiments to compare: group 1 create.
Name a second group and select the experiments to add to this group. The previous group will be listed at the top of the page.
Figure 10.3.5 Interface to add experiments to compare: group 2 create.
Repeat the previous step if you want to add more groups to compare. Once you have defined all desired groups, click the “Next” button to finish the comparison construction.
Figure 10.3.6 Interface to “add experiments to compare”.
All selected experiments will be displayed at the top of the page. Name this comparison and click the “Run it now” button to execute. IP2 will load the list of the quantitation comparisons. This table is sorted by date to easily locate the newly created comparison.
Figure 10.3.7 Specified groups for quant comparison.
To view the results, both “ANOVA_comparison” and “Singleton” links are available, as shown below:
Figure 10.3.8 View quant comparison results list
The first link will demonstrate the statistical analysis results in the table, see Figure 6.11.6. The second link will display the singleton result.
Figure 10.3.9 view of the quant ANOVA comparison, “ratio-to-ratio”.
Clicking the singleton link as previously shown in Figure 10-26 gives the following result:
Figure 10.3.10 view of Quant ANOVA comparison – singleton
“Compare Directly” on Protein Level
Returning to the selection of labeling type as shown previously, if the user selects the second radio button to compare the ratios directly, only one group will need to be built.
Figure 10.3.11 Select “comparison directly” for labeling type for quant comparison.
As shown previously to build groups for “ratio-to-ratio” comparison, the user needs to specify the comparison name. However, for direct comparison, the user needs only to assign a name for the group and the comparison will take that name for itself. There is no “add to group” function for the “compare directly” label type. Click the “run it now” button in Figure 10-30 below to submit the job:
Figure 10.3.12 Interface to “add experiments to compare” directly.
The browser will redirect to the list of comparisons as shown in Figure 10-31 below:
Figure 10.3.13 updated list of quant comparison.
By clicking the “ANOVA_comparison” link, the page will be navigated to the comparison result.
Figure 10.3.14 view of quant ANOVA comparison – compare directly.
“Ratio-to-Ratio” Comparison on Peptide Level
This section shows how to implement a “ratio-to-ratio” comparison on the peptide level. Similar to protein level comparison, click the “run new quantitative COMPARE” link and select the “peptide” radio button, then select “next” to select desired experiments.
Figure 10.3.15 Selecting “Peptide” as compare level for quantitative comparison.
After creating at least two groups in the same manner as with the previously described ratio to ratio comparison on the protein level, the job can be submitted to the server.
Figure 10.3.16 Submitting the quantitative comparison request to server.
The results of the comparison will be completed in approximately one minute. Check the updated list for comparisons and click the new one to view the result.
Figure 10.3.17 The updated comparison list with the new comparison.
The process for viewing ANOVA comparison or singleton results are similar to the previous protein level comparisons:
Figure 10.3.18 ANOVA comparison at peptide level.
“Compare Directly” on Peptide Level
As shown in the previous section, the only difference between quantitative comparison on the protein level and peptide level is the “compare level.” Select “compare directly” and “Peptide,” then click “next” as shown below:
Figure 10.3.19 Selecting “compare directly” and “peptide”.
The next step is the same as for comparing directly at the protein level. Select the experiments to compare and click the “run it now” button to submit the job to the server. The new comparison will be displayed in the updated comparison list.
Figure 10.3.20 Updated comparison list.
Click the “ANOVA comparison” link to view the result. For the “compare directly” option, the result will calculate the t-test p-value for all ratios and use “Benjamin and Hochberg Correction” algorithm to transfer the p-values more like a normal distribution.