TPP Demo2009 - 9. Peptide Quantitation with ASAPRatio 使用 ASAPRatio 定量 Peptide

ASAPRatio is a tool for measuring relative expression levels of peptides and proteins from isotopically-labeled samples (e.g. ICAT, SILAC, etc).

ASAPRatio 是一套用來測量以同位素標定樣本的工具，可計算出該 peptides 與 proteins 層級的相對表現量。

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

將滑鼠移至上方的"Analysis Pipeline (Tandem)" 選項，並從跳出的視窗中，選擇"Analyze Peptides"選項以進入 xinteract 介面。

• Select the interact.iproph.pep.xml file in the directory demo2009. Make sure that there is only one file selected for analysis; you can edit the selections using the checkboxes and Remove button on the right-hand side.

加入 demo2009 目錄下的 interact.iproph.pep.xml 檔案。

• Important: Make sure that you uncheck the option to RUN PeptideProphet under PeptideProphet Options, as this file already contains results from PeptideProphet.

確認"PeptideProphet Options"區塊中的"RUN PeptideProphet"選項未被勾選。

因為我們選擇的檔案已經被 PeptideProphet 處理過。

• Under ASAPRatio Options, select to RUN ASAPRatio, change Labeled Residues to K and R, set m/z range to include in summation of peak to 0.05, set Specified masses to M 147.035, K 136.10916, and R 166.10941.

在"ASAPRatio"的區段中，勾選"RUN ASAPRatio"並更改以下的欄位值:

"Change labeled residues to": K,R

"m/z range to include in summation of peak: 0.05

"Specified mass 1": M, 147.035

"Specified mass 2": K, 136.10916

"Specified mass 3": R, 166.10941

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

保留其他預設值，再按下"Run XInteract"按鈕以執行 ASAPRatio。

• Once the command finishes running (about 4 hrs), you can click on the view results link that appears in the Command Status box to view and analyze the results. The "asapratio" column contains quantitation results with a link to the ASAPRatio ion trace. The number listed in the "asapratio" column is the light to heavy ratio. IMG:ASAPRatioProfiles

一旦執行完畢(約4個小時)，按下"Command Status"區塊下的"here"連結，

再按下"Output Files"區塊下的"View"連結瀏覽結果。

可以看到原本的 interact.pep.xml 多了"asapratio" 欄位，該欄位包含了定量的結果，

數字表示 light 與 heavy 之間的比例。

若點擊"asapratio"欄位中的超連結後，可以打開 ASAPRatio 的 ion trace。