TPP Demo2009 - 6. Validation of Peptide-Spectrum assignments with PeptideProphet 使用 PetideProphet 檢驗 pepitde 與 Spectrum 對應的關係

PeptideProphet provides statistical validation of search engine results by assigning a probability to each peptide-spectrum match.

PeptideProphet 給予搜尋結果的每對 peptide 與 spectrum 一個機率，以統計模型來檢定是否為 false match。

• Click on the Analyze Peptides tab under the Analysis Pipeline section in Petunia to access the xinteract interface.

回到首頁並選擇"Tandem"為分析流程後，將滑鼠移至上方的"Analysis Pipeline(Tandem)" 選項，並從跳出的視窗中，選擇"Analyze Peptdies"選項進入 xinteract 程式的介面。

xinteract is a general utility that is able to launch several components of the TPP, including PeptideProphet.

xinteract 是通用的工具套件，可以執行 TPP 的數種功能，包含 PeptideProphet。

• Select the two OR2008*.pep.xml files in the directory demo2009\tandem. Make sure that there are only two files selected for analysis; you can edit the selections using the checkboxes and Remove button on the right-hand side.

將 demo2009\tandem 下的 *.pep.xml 加入。

• Under PeptideProphet Options, find and select the option to Use accurate mass binning since this is a high-accuracy data.

在 PeptideProphet Options 區段中，勾選"Use accurate mass binning"，因為 Demo 的數據為高精準度的數據。

• Leave all other options set to their defaults, and click on Run XInteract at the bottom of the page to run PeptideProphet.

保留其他選項為預設值，再按下方的"Run XInteract"按鈕以執行 PeptideProphet。

• Once the command finishes running, you can click on the view results link that appears in the Command Status box to view and analyze the results. IMG:PepProphet On this page, sort the list in descending order based on Probabilities. The identifications at the top of the resulting list are most likely to be correct. Click on the hypertext link for any probability. This brings up a details page IMG:PlotModel which shows graphically how successful the modeling was. In the upper pane, it is desirable for the red curve (sensitivity) to hug the upper right corner, and for the green curve (error) to hug the lower left corner. The lower pane shows how well the data (black line) follows the PeptideProphet modeling for each charge state. The blue curve describes the modeling of the negative results, and the purple one, the positive results. If these two curves are well separated and fit the black line well, then the analysis for that charge state was successful.

一旦完成後，可以點擊上方"Command Status"欄位中的的"here"連結，

再點擊"Output Files"區塊中的"View"以瀏覽分析結果。

在 results 結果中，可依照機率的降冪方式進行排列，意為最上面的項目是最可能正確。

若按下任何機率的超連結，則會跳出一個視窗以圖形的方式代表該 modeling 的結果。

上方的區塊中，紅色曲線代表 sensitivity ，綠色曲線代表 error。橫軸代表可接受的最小機率，該值越大則 Error 越小，但是 Sensitivity 也越小。

下方的區塊中，各個圖分別代表不同 charge state 下的 modeling 情況，黑線代表數據依 PeptideProphet 模式的走向，

藍色曲線與紫色曲線各表示 modeling 的 negative results 及 positive results，如果這2條曲線分很開且與黑色吻合度高，則代表該 charge state 是很成功的。

• You can now go back run this analysis on the SpectraST results. Again, make sure you are only analyzing two input files.

SpectraST 的結果檔亦可用 PeptideProphet 來分析。