1. After protein identification is done, click the ‘run now’ link underneath the quantitative analysis column on the experiment page.
2. Select isobaric labeling. If you have less than or equal to 6-plex, select “Isobaric labeling”. If you have more than 6-plex labeling, select “isobaric N-plex labeling”.
If you select isobaric N-plex labeling, you will see a box to type the number of reporter ions. Up to 11-plex, IP2 will automatically fill in default TMT values. If you specify more than 11-plex or iTRAQ, you will need to manually type in values (e.g. reporter ion masses, purity values, etc.).
3. Fill in parameters for isobaric data analysis.
- Tandem scan shift: If you want to perform a quantitative analysis on a different scan (e.g. for CID/HCD alternation), you can shift the targeted scan for quantitative analysis with this parameter. e.g., 1 is for the scan right after an identified scan. Leave it at 0 if you do not have mixed scan types.
- MS3-based TMT: Check the box if your data is multi-notch or synchronous precursor selection (SPS) ms3 to minimize contaminant peaks. Refer to Nature Methods 8, 937-940 (2011)
- Mass tolerance: Reporter ion mass tolerance.
- Intensity threshold: Filter out low signal scans by the intensity threshold. When the sum of all reporter ions is lower than the threshold, the scan will be discarded.
- Reporter ion: Reporter ion m/z values. Any blank fields will be ignored in the data analysis.
- Reporter ion isotope: Reporter ion purity correct value in percentage.
- Isobaric purity filter: Negative value (e.g. -1) means there is no filter to apply.
Determination of isolation specificity
In an attempt to estimate the intensity of contamination in precursor isolation window for each peptide, we calculated a precursor purity value. This was done by first recording all ion intensities in survey MS data within the isolation width m/z range (+/- x m/z) around target precursor ion m/z values. Using given tolerance, the summed intensity of ions assigned to the target ion isotopic envelope (including 13C ions) was then divided by the total ion intensity. The calculated isolation specificity was graded on a scale from 0 to 1, where 0 represented 100% interference (none of the signal belonged to the precursor); and 1 represented 0% interference (the entire signal belonged to the precursor).
Ting, et al, Nat Methods. 2011
If it is lower than the threshold, peptides will be discarded. After analysis, users can view purity distribution in a graph to get a better idea of the data (see result page figure below). It can then be re-filtered in the results page instead of re-running the whole quantitative analysis (see Figure 188.8.131.52.2 below).
In static modification fields (Figure 184.108.40.206), TMT static masses are already considered.
If you have any additional static mass changes in the amino acids, change atomic information
4. Click Submit button to run data analysis.
5. After analysis, in the experiment page, there are two links to click. One is ‘View data’ in the quantitative analysis column to view data in the table, and the other is an image link in the graph view column to view data in graphs.
When you click ‘graph view’ image, you can see the default result page shown in Figure 220.127.116.11.1.
There are six (6) top menus in the current version:
- Protein table view: display quantitative results in table
- Protein graph view: display results in graph view in protein level(Figure 18.104.22.168.1)
- Peptide graph view: display results in graph view in peptide level
- Reporter ion delta mass: display all peaks in reporter ion range
- Intensity distribution: distribution of all reporter ion intensities
- precursor purity and S/N distribution: graphs to show precursor purity distribution and S/N distribution. This information can give idea of what values can be applied in purity and S/N filters. Users can run re-filter in the result page.
At first, the user can click “precursor purity and S/N distribution” button to evaluate purity and S/N values.
Precursor purity graph shows the distribution of a number of peaks in the ms1 isolation window. Users can review the S/N distribution graph, too.
Regarding S/N calculation, for each MS spectrum, the peaks were ranked by intensity, and the bottom 10% of peaks were considered chemical noise. The average intensity of these peaks is defined as the background noise level.
The signal to noise of a given ion is defined as the ratio of that peaks intensity the background noise level. (J Am Soc Mass Spectrom. 2009 Aug).
Let us call S/N value from the paper, S/Ns. For TMT S/N, the IP2 calculates like below:
- Find the maximum intensity for the reporters for a given peptide
- Divide this value by S/Ns
- Calculate S/N value for each reporter within a peptide by dividing the intensity by the calculated noise level for that peptide.
- Then, average the signal to noise values for each reporter within a peptide and discard any peptides that do not have an average signal to noise of a given threshold. Threshold 10 is recommended.
To re-filter data, click ‘Protein table view’ button on the top of the page.
In the protein table view page, users can see protein (or peptide level by changing radio buttons above table) quantitative results in the table view. There are three boxes to run filters.
- intensity threshold: Intensity is the sum of all reporter ions on each PSM. If it is smaller than the threshold, peptides will be discarded
- isobaric_purity_filter: Calculate purity value in MS1 isolation window (See below for algorithm in detail). If it is lower than the threshold, peptides will be discarded
- isobaric_signal_to_noise: The biggest intensity among reporter ions is less than background noise, peptides will be discarded
Click Refilter button to apply new filters
User can come back to graph view page to review reporter ion intensities. User can find reporter ion intensities in stacked view. This is protein level, but similar graphs can be displayed in each PSM level, or a peptide for multiple PSMs
This graph displays all reporter ions
Raw spectrum can be displayed. This example is from TMT 10-plex. 2nd and 3rd reporter ions are very close as expected in 10-plex TMT, but they can be easily separated in high-resolution data
6. User can check the global view of ion distribution based on their m/z values by clicking “Reporter ion delta mass tab”. Also, the next tab “intensity distribution” displays reporter ion intensity distributions
7. After clicking the view data link, you can see isobaric results on the web. You can find both raw and normalized reporter ion intensity values on the protein level.
Click ‘census-out.txt view’ link on the top to view detailed information for the data analysis including peptide/spectrum level values.
8. Next, you can statically compare samples by grouping reporter ions. In this case, we assume some reporter ions are biological replicates.
To compare sample, click QuantationCompare link in the project page.
9. In the quantCompare page, click ‘run new quantitative Compare’ link.
10. Select Isobaric labeling (single experiment).
11. Select an experiment to run the analysis.
12. Type a group name and select reporter ions (biological replicates), and click ‘add to group’. Repeat this for samples (e.g., control, biological sample, etc).
13. Click next, and type user-defined comparison name. Click ‘run it now’ button
14. After analysis, click ‘View Results’ link in the quant compare page. There is p-value based on t-test. Also, corresponding q-value is in the next column. You can find normalized protein-level intensities. Outlier Number column displays how many peptides are discarded as outliers per protein. ‘peptide ratio average’ column is an average value from all peptide ratios for each protein.
‘peptide ratio StDev’ is peptide ratio standard deviation. ‘peptide ratio RSD’ is a relative standard deviation.