help with data analysis for iTRAQ on LTQ Orbitrap with CID for peptide identification and HCD for reporter ion

[parimal samir]

I am using LTQ Orbitrap XL for iTRAQ or TMT based quantitation. I
fragment the peptide using CID in ion trap to get the sequence
information. I use HCD fragmentation with high fragmentation energy to
get the reporter ion spectra. Since, the reporter ion spectra and
sequence spectra are in different scan events, I would assume that
once converted to .dta, these spectra will be in different .dta files.
So my question is there a way that SEQUEST can be used to identify the
peptides in such a way that downstream quantitation tool can use the
corresponding reporter ions information for quantitation in this case?
If not, can I somehow merge the two spectra so that I will have both
the information in the same .dta file? Then I can use Scaffold or
Libra for quantitation.

[dctrud]

There is an ExPASy tool to merge CID/HCD spectra from a Mascot MGF
format peaklist for this purpose. Can you go from RAW/MzXML/MzML into
MGF, use this tool, and then convert to dta for SEQUEST? We've done
iTRAQ on an XL, but only using PQD not mixed CID/HCD, so it's just a
suggestion to try, can't say I've tried it.

http://expasy.org/tools/HCD_CID_merger.html

Cheers,

DT

http://expasy.org/tools/HCD_CID_merger.html

[tiannan guo]

We have worked on merging PQD and CID spectra for iTRAQ before. Raw could be converted to mgf using extract_msn.exe packed in Sequest/Bioworks. I can provide a script to merge spectra with reporter peaks if you need. You may find some description of the method in JPR.7:4831.
Now I have also developed some script to extract spectra with reporters from mzXML directly, which I think is more convenient. I have not tried HCD with my script, but I think it won't be too difficult to take a try if you provide some example data.

cheers,
Tiannan

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