10.4 Quantification Compare Isobaric (TMT, iTRAQ, etc)

In the list of experiments, click “quantitativeCOMPARE” to list all existing quantitation comparisons.

Figure 10.4.1: Link for Quantitative Comparison.

Click “run new quantitative ©COMPARE” to build a new comparison.

Figure 10.4.2 view of Quant COMPARE

This section shows how to submit comparisons on isobaric labeling analysis such as TMT and iTRAQ
In Figure 10.4.3, there are four (4) options for isobaric labeling analysis.

Isobaric labeling (Single experiment. This option will be replaced by version 2 below)
Isobaric labeling comparing multiple experiments version 1 (this option will be replaced by version 2 below)
Isobaric labeling data comparison version 2 (New version. Flexible way to compare multiple experiments. Beta version)
Isobaric labeling multiple experiments. Simply concatenate multiple quantitative outputs (census-out.txt) files

1st and 2nd options are deprecated and replaced by a 3rd option. Users can select compare report ions from single or multiple experiments. It also provides a tool to normalize intensities by using internal standards. If there is no internal standards channel, users can ignore the internal standard option.

Figure 10.4.3: Quant Compare Options.

Please note that in the project page, users can tag experiments active or archive. In the QuantCompare analysis, only active experiments are used for analysis. If you want to analyze any experiments in your archive, you need to tag them as active before running QuantCompare tool.
This is the internal standard option. If internal standards are used in the experiments, select ‘Use Internal Standard’ option. If not, select ‘No Internal Standard’. If the ‘Use Internal Standard’ option is selected, users can select internal standard report ions in the next page. Otherwise, it will skip this step and move to the next page.

Figure 10.4.4 Internal standard option

Select the internal standard for each experiment and click next. If the internal standard option in the previous page is not selected, IP2 skips this step.

Figure 10.4.5 Select internal standards

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 10.4.6 Group report ions

Type in your sample name in the “group name” box and select multiple reporter ions in checkboxes. Sample can be a control sample or a biological sample with treatments, for example. Multiple reporter ions, typically, represent replicates of the sample. Then, click the “Add to group” button. Repeat this step to build samples with replicates to compare. See Figure 10.4.7 for example. Figure 10.4.7 shows “control” and “sample A” to compare. Users can build any number of samples to compare. When it is done, click the “Next” button.

Figure 10.4.7 Build samples with reporter ions to compare

In the final step before running a comparison tool, users can review samples to compare an internal standard (if any) to double-check. Once it is ready, type in “Give a name to save” on the bottom of the page with a name for this analysis. Later, users can find the results by using this name.

Figure 10.4.8 Review samples and internal standards before running analysis

In the QuantCompare page, users can find the new entry with the analysis name that the users defined in the previous step. Click the “View Results” link.

Figure 10.4.9 QuantCompare page

Figure 10.4.10 is comparison result page. User can click ‘quant_isobaric_output_xxx.txt’ link on the top of the page to open and click ‘save as…’ in their browser. This txt file is standard tab-delimited format, so users can open in excel or input to another script to process further. Result page basically parses same txt file and display on IP2 web page.
User can find “column description”.

Column description

ACCESSION: Protein accession from fasta database
GENE: Gene name
TOTAL_INTENSITY: Total intensity from census-out.txt file. Simple sum of peptide intensities without any normalization or correction
NORM_INTENSITY: Protein total intensity on each channel is normalized by the intensity sum of all proteins in the same channel.
INTERNAL_STANDARD_NORM_INTENSITY: When an internal standard is used, NORM_INTENSITY value is divided by internal standard intensity selected by the user and then multiplied by the average intensity of all channels. 2. When the internal standard is not used, the value is NA.
PVALUE: We used total intensities for calculating the p-value. The p-value is calculated by comparing 1st and 2nd group. If there is a 3rd group, 2nd p-value is from a comparison of 1st and 3rd groups. And so on.
QVALUE: We used total intensities for calculating q-value. We used the Benjamini-Hochberg posthoc analysis for adjusted p-value correction (q-value). 1st q-value is from a comparison of 1st and 2nd group. If there is a 3rd group, 2nd q-value is from a comparison of 1st and 3rd groups. And so on.
AVERAGE_INTENSITY: Average intensity of each group total intensity. When the value is zero or X, we exclude them for average intensity calculation
NORM_AVERAGE_INTENSITY: Average intensity of each group normalized intensity. When the value is zero or X, we exclude them for average intensity calculation
RATIO_2_1: 2nd group average intensity divided by 1st group average intensity. When there is a 3rd group, 2nd ratio value is from the average intensity of the 3rd group divided by the 1st group average intensity.
NORM_RATIO_2_1: 2nd group normalized average intensity divided by 1st group normalized average intensity. When there is a 3rd group, 2nd ratio value is from the normalized average intensity of 3rd group divided by 1st group normalized average intensity.
DESCRIPTION: protein description line from the protein database

Note. If users can give us suggestions, such as additional columns, we will greatly appreciate it.

Figure 10.4.10 Isobaric QuantCompare result page

Users can click “Show and Hide columns” button to organize columns. Users can apply filters using the ‘Add Filter’ tool. For example, users can keep proteins that have a p-value less than 0.05.