User Manual POM
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Phenyx Extended PTM Search
What is the Phenyx Extended PTM Search?
Phenyx allows to search a sequence database with parameters set serially in two rounds. Classically, the set of parameters defined in the first round are used to search the entire selected databank(s) or a predefined taxonomy subset or even a predefined list of protein accession numbers. The list of the proteins identified after this first step is submitted to a second round, where typically the search space is open wider. For instance, one explicitly choose to search for semi-specific cleavage events, or/and a greater number of modifications, or/and to lower some score thresholds, or to widen the m/z tolerances, etc. To further open the search space and allow for a much larger number of amino acid modifications, there is a "extended PTM search" check box in the submission page.
This check box activates an extension to the second round, in that it allows a search for up to one amino acid modification more than all those already defined in the second round. The list of the considered modifications is the complete set of the modifications defined in the Phenyx default configuration file plus those defined by the user himself. It does correspond to the entire list of modifications visible in the AA modif field in the submission page. The list can include standard PTMs, artefactual modifications as well as mutations.
A z-score and a p-value are calculated for all peptide matches. The corresponding p-value is calculated according to the actual search space, which might vary a lot, depending on the number of proteins to be queried in the second round, the search parameters of the second round, and the list of modifications added by the extended PTM search mode. The results of such a search are to be considered with care in terms of confidence, as the FDR is usually increasing a lot. It is also to be noted that the number of conflicting identifications is also drastically increasing.
The peptides identified by the expended PTM search mode are annotated with a POM flag in a source column of peptide identification tables (in the Proteins Overview, in the Protein Match Details, in the Compounds Overview).
(here example from a Proteins Overview page, where a peptide was identified with a carbamidomethylated Cysteine (Cys_CAM) as searched in the second round, whereas the same peptide, carrying an acrylamide adduct (Cys_PAM) was detected in the Extended PTM search. a couple of scans later.)
Unvalidate the peptides identified via the Extended PTM mode
The number of possible candidate peptides increases drastically in an Extended PTM mode compared to a standard search. As a number of modifications share the same (or a very similar) mass deviation, the report includes a high number of conflicts. It is therefore up to the end user to select the relevant possible hits from the proposed crowd. As a consequence also, this mode increases drastically the FDR. If you wish to run a FDR calculation, it might be desirable to exclude all hits from the Extended PTM search, from both forward and decoy dearches, in the FDR calculation. To do so, one can automatically invalidate the peptide matches issued from the Extended PTM search using one export function.
Select the job of interest in the Desktop, click on the Export link on the left-hand menu. In the appearing management console, select "unselect peptide matched found with the Extended PTM Search" export.
This will invalidate the corresponding peptides. You will also get a small file summarising the number of selected peptides before and after the process.
If you wish to do this with more than one jobs, go to the management console, select the "unselect peptide matched found with the Extended PTM Search" export, and choose the jobs to process from the list (keep pressing on the SHIFT or CTRL key to select more than one jobs).
You can always revert the action by clicking on the "Revert to auto validation" button in the Compounds overview.
Use of the Extended PTM DB
If you wish to have access to a larger list of PTMs on the Phenyx public server or on the Phenyx online secured server, we have created a list based primarily on the entries available on the Unimod resource. These AA modifications are defined in a separate user. You can register to access this list in addition to the default one. Please send us a mail at phenyx@genebio.com.
If you have a local installation of Phenyx, here are the instructions to install the list.
