TIQAM-Digestor – Tutorial

Mbrusnia

Informations

• Publié par: Onfue
• Nombre de lectures: 34
• Langue: English

Extrait

TIQAM-Digestor – Tutorial
Mi-Youn Brusniak

1. Double Click Short cut
of TIQAM-Digestor
2. File >> New >> Create
new PD project
3. Type PD Project Name
and Description as
shown in the screen
then click “Finish”






4. Click “Select Fasta file
holding proteins in
Protein Loader” Select
StreptococcusPyogene.
fasta in the “My
Document/TIQAM-
ClassDemoData”







TIQAM-Digestor – Tutorial



5. Fructose-bisphosphate aldolase will be
loaded as it is shown in the left figure.
Click ClassDemo in “Select PD Project”
tab, then click “Load Proteins” button






6. Change “pep mass >” query to be 600 as
shown in the left figure and then click
“Make Petides” button

7. Click “Annotate Peptides” tab and type
in Name filed and select Type field and
select File
StreptococcusPyogene_pepXML.xml
using “Brower” button shown in the left
bottom figure. The clock “Import
Annotations” button to see the right bottom figure.


TIQAM-Digestor – Tutorial

8. Click “Proceed to Make
Transitions” button it will show
Generate Transitions page as
you see in the left figure. Select
+2 and +3 in z1 column while
holding down shift key then
click “Make Transitions”




9. It will show Transition Table,
click “Save to File” tab then it
will show “save” button (shown
in the left figure) and save as
StreptococcusPyogene_Digestor
in “My Document/TIQAM-
ClassDemoData”.




10. Exercise
Open the exported file StreptococcusPyogene.txt in Excel and order by CE
(Collision Energy). Is there any correlation with Mass?
Could you think of way to get better Retention time for schedule SRM?

TIQAM-PeptideAtlas – Tutorial
Mi-Youn Brusniak

1. Double Click short cut of TIQAM-
PeptideAtlas
2. Click “PeptideAtlas MRM WS” in the
left panel shown in the left panel
3. Follow instruction in “Get Web
Service ID” using a any web brower.
4. Cut and paste ID obtained from the
to the “Web Service ID” http://db.systemsbiology.net/webapps/mrmWSID
then Click “Save ID” button.
5. Click back to “Web Service Query” tab on the top shown in the left figure.
6. Select Sp in “org” drop down menu and SpectraST_Sp in “lib” drop down
menu for Streptococcus Pyogenes library. Currently, it shows Hs (Human)
and Sc (yeast) consensus library is available.
7. Fill 0.9 in “pepLowLimP” text box for lower limit of peptide prophet and 10
in “pepLowLimNObs” text box for lower limit of number of observation and
1 in”pepNumGenLocs” text box to get unique protein mapping by selecting
one for genome
chromosomal
locations. Then
select getYIons ,
getTrans,
getLibSpecURL
as shown in the
left figure. Click
“Send Query”

TIQAM-PeptideAtlas – Tutorial



8. You will see the resulted
Transition Table. Click “Save
To File” tab and click
“Export To File” and give file
name
“Streptococcu_TIQAM_PA_
out.csv” and click “save”
button as shown in the left.




9. Scroll the table to the right to see spectrum url. Click spectrum url shown in
the below left figure (any url belong to TYDAQHLLDESK will be ok). Then
select “Brower” tab at the top to exam the spectra shown in the below
right figure.


10. Exercise
What Q3 would you pick for peptide TYDAQHLLDESK transition?
Open the exported file using Excel. How many transitions did you get?
TIQAM-Viewer – Tutorial
Mi-Youn Brusniak
1. Double Click short cut of TIQAM-
Viewer.
2. File >> Import
Select “A new project”
Click “Next”
3. Fill out the Create Project wizard as
shown in the left figure
4. Click “Next”
5. Fill out the Experiment wizard as
shown in the left figure





6. Click “Manually select files” and
select the following files located in
the “My Document/TIQAM-
ClassDemoData” : Transition File (StreptococcusPyogene.csv), mzXML
(0223House4.mzXML, 0223House5.mzXML), pepXML
(StreptococcusPyogene_pep.xml). Select Peptides only before click “Next” . TIQAM-Viewer – Tutorial


7. When it’s imported, it will show the
summary dataset shown in the right
figure
8. Click “refresh” button to show your
imported project and experiment.

9. Select “all proteins” in Queries
section and you will have the screen
shown below












TIQAM-Viewer – Tutorial

10. Exercise.
Go through the transitions and you can decide the level of validation
whether the transition sets for a given peptide validate the peptide
identification.
For example, the peptide AVQGAVEER of ALF_STRP1 Protein generates
strong peptide (shown below figure). You can annotate this as strong
category in Peptide Validation.
Which three transitions would you like to use for quantifying AVQGAVEER
peptide? Please find your top three candidates from transitions table and
write.
You may go through more peptides and proteins to see how you would like
to annotate peptide validation and select best transitions.