8.5 Site Specific tag Analysis

This option is for site-specific chemical probe tagging (e.g TEV) or metabolomic labeling (e.g. AHA) analysis.

1. Protein Identification

In the ProLuCID search page, differential modification search options are recommended as below:

Please note that if you consider “57.02146 C for Carbamidomethylation” and your tag is on same Cystine, you need to consider mass difference for differential modification search (see below).

Figure 8.5.1

2. Quantitative Analysis

Before running Quantitative Analysis, we recommend you run Delta Mass Plotter to figure out what can be a good Mass Tolerance to use as Quantitative Parameters.
We suggest you make Mass Tolerance a bit wider than the Delta Mass Plotter shows. Click the ‘run now’ link in the search result page.

Figure 8.5.2

Next Page: click “Site-specific tag labeling”

Figure 8.5.3

In the site-specific tag labeling box of quant parameter page: Type in residue you labeled (in this example, C) together with light and heavy mass. As the IP2 displays differential residue and masses used in the search step, you can copy and paste proper residue and masses. In the extraction method box, click smooth XIC curve (recommended) and type in mass tolerance. Once finished, click the “Submit” button.
Please note that in the amino acids table below, Carbamidomethylation in C was already considered as default.

Figure 8.5.4

3. Results

After analysis, check the table view page.

Figure 8.5.5

In the table view page, users can view results either protein or peptide level by selecting radio button options.

Figure 8.5.6

Click show/hide filter parameters to display default parameters. The “view ratio distribution” link displays the overall peptide ratio distribution. Sample mixing error correction or normalization can be done in the “correction value” field, then click the re-filter button below.

Figure 8.5.7

Click the “Protein graph view” or “Peptide graph view” buttons to navigate proteins or peptides visually.

Figure 8.5.8

4. Compare Multiple Samples
The next step is to run QuantCompare to compare multiple experiments/samples to find statistically significant proteins or peptides. See more for Quantification Compare.