Difference between Rapid and Thorough Searches in ProteinPilot™ Software


日付: 11/02/2018
カテゴリー: Academia Omics , Pharma CRO , ProteinPilot Software

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For research use only. Not for use in diagnostic procedures.


Answer

When setting up a search in ProteinPilot™ software, either a Rapid Search or a Thorough Search is selected in the Search Effort section. This setting determines which parts of the algorithm will be invoked and effectively how deep the sample will be searched to find the right answers.

When a Rapid Search is selected, a Fraglet search type will be performed. This type of search is similar to most search engines where the fragment ion spectrum is matched against the possible matches from the database and then scored (matches being pre-selected based on precursor mass). 

When a Thorough search is selected, both a Fraglet search followed by a Taglet search will be performed. This type of search is novel and allows for a much more rich definition of the proteome and a much deeper search to be performed. 

Note: when a no enzyme search is selected, only a Taglet search is performed. Please watch this video on the Paragon™ Algorithm to learn more details on how this algorithm works.

After peptide identification is complete, the Pro Group™ algorithm assembles the results into a minimal list of detected proteins.

Selection of the search effort also changes the way things like digestion definition and modifications are handled. In rapid mode, the Paragon Algorithm uses simple canonical definitions of proteases, much like other search engines. Trypsin cleaves at Lys and Arg but nowhere else (left side of picture). Here, up to 2 missed cleavages are allowed.


In Thorough mode, the algorithm uses feature probability sets that allow a more refined description of what a given protease does. For the trypsin example, there is a high probability that trypsin will cleave at Lys and Arg, but also we know that we will see semi-tryptic cleavages at other residues at much lower frequencies, and these are described using empirically determined probabilities. With Thorough, up to 5 missed cleavages allowed as well as semi-tryptics (right of figure).
User-added image

The same approach is used for post-translational modifications (PTMs); a much more sophisticated and biologically relevant view of the sample is used when the Thorough search effort is selected. To see which PTMs are searched when doing a Rapid search, refer to the Standard workup modification set in column V of  the "Unified Modification Catalog.xlsx" found in the Help folder.  In ProteinPilot software 5.0, 63 PTMs are searched when Rapid is selected.  For a typical search where Thorough is selected with biological modifications, a much bigger set of modifications is invoked (see the Standard biological mod set in column W: additional 337 mods). By using the Taglet search in combination with feature probabilities, this much expanded set of PTMs can be searched with only a small impact on search time and minimal impact on false discovery rates. The Feature probabilities used are the numbers shown in these columns in the Mod Catalogue.

More details on the Paragon Algorithm can be found in: Mol. Cell. Proteomics, 6: 1638-1655, (2007).
ProteinPilot™ Software Overview - High Quality, In-Depth Protein Identification and Protein Expression Analysis, RUO-MKT-02-1777-A.

Note: Blue text includes links to videos in the Sciex Community under "Proteomics" and "ProteinPilot".