Is ETD necessary or can a QTOF in CID mode work well for phosphoproteomics?

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congriver
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Is ETD necessary or can a QTOF in CID mode work well for phosphoproteomics?

Postby congriver » Tue Mar 05, 2013 1:22 am

Hi
I currently have to start some phosphoproteomics work soon. We have an agilent QTOF in the lab with chip cube interface and I could try a phospho chip workflow to see how this works but would CID be an ineffective tool to use in this case due to phospholoss during CID? We have an ETD enabled Bruker ion trap in the lab but it is switched off for the last year as we just needed the QTOF for straightford protein id workflows.I could start up the ion trap but I thought I'd see what you thought. Most CID phosphoproteomics talks about the phospholoss but are usually referring to CID in an ion trap. Does the phospholoss also occur to the same extent in a CID QTOF experiment.many people seem to use HCD in orbitraps for phosphoproteomics and I beleive HCD is pretty identical to CID in a QTOF
thanks
Brendan

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Doug
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Postby Doug » Tue Mar 05, 2013 8:18 am

Hi Brendan,

I think you'll find different opinions on this subject but I will summarize my experiences for you. I have no experience with a Qtof but I do have experience using Thermo Orbitrap instruments which are capable of low energy ion trap CAD, high energy beam-type CID that is similar to Qtof fragmentation (HCD), and ETD. In my experience, HCD provides by far the most phosphopeptide identifications from complex mixtures (whole cell lysates enriched for phosphopeptides using IMAC). Ion trap CAD, in my hands, identified far fewer phosphopeptides. This is true even if fragments were detected in the Orbitrap indicating that the difference is in the fragmentation patterns. This suggests the a Qtof should be well equipped for phosphopeptide analysis. You should keep in mind that several expert groups still use ion trap CAD for phosphopeptides (even on Orbitrap instruments) so they presumably get better results using ion trap CAD. ETD gives far fewer phosphopeptide identifications. This is not because it isn't good for phosphopeptides but because it works best for higher charged peptides (and peptides with an mz < ~900 Th). Trypsin generates a lot of +2 peptides which are not ideally suited for ETD. This results in lower overall numbers for ETD. However, if you use different enzymes ETD can give you access to regions of proteins not observed using trypsin and CAD. A common example is the use of alternative enzymes and ETD to characterize highly charged, highly modified, histone proteins. ETD can be very helpful is when you care more about specific proteins that shear number of phosphopeptides. Significant portions of many proteins are not easily observed using trypsin and CAD. So if you want to thoroughly map modifications on a protein you will almost definitely need to use multiple enzymes and multiple fragmentation methods. I can attach papers later today that highlight some of these differences both for large scale studies and for studies mapping PO4 sites on a single protein.

In summary, if the Qtof is running well and the bruker's condition is unknown just go with the Qtof. It should be fine.

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Postby achimt » Wed Mar 06, 2013 12:58 am

Hi Doug,

forgive me the really naive question: how do you perform ion trap CAD with fragment detection in the orbitrap? Are there situations where this is a useful thing to do (rather than performing HCD?).

best wishes,
Achim

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Doug
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Postby Doug » Wed Mar 06, 2013 8:23 am

For resonant-excitation CAD (ion trap CAD), ETD, and PQD, the actual fragmentation takes place in the ion trap. Once you have generated the fragments they can be detected in the ion trap (faster but with lower resolution and lower mass accuracy) or in the Orbitrap (slower but with higher resolution and mass accuracy). It's basically a balance of speed vs quality. Once the Velos Orbis first came out I generally got more IDs detecting fragments in the Orbitrap. But good friends (now in other labs) tell me that they get more IDs with ion trap detection. And since they still do mass spec and I don't I am inclined to trust them, though I would recommend testing for yourself if you are interested.

The reason I did CAD with Orbitrap detection in the past was so that I could directly compare the fragmentation method to HCD. HCD is a form of beam-type CAD that takes place in a dedicated collision cell that is (approximately) in between the ion trap and the orbitrap. The Thermo software only allows for detection of HCD fragments in the Orbitrap.

That was a bit long winded. So your question...

Are there situations where this is a useful thing to do (rather than performing HCD)?

For general identification purposes I can 't think of any reason why you might want to do this. But for studies of fragmentation you might want to use it. If you want to increase confidence in an ID you might want to acquire data with multiple fragmentation patterns followed by Orbitrap detection.

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Postby karthikskamath » Wed Mar 06, 2013 11:24 pm

Hi Doug,

Just a question in relation with detecting in ion-trap and orbi. Have you observed any difference in sensitivity between IT and Orbi while running whole cell lysate? Through my experience sensitivity is better in IT than orbi (yes on the cost of specificity).

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Postby Doug » Thu Mar 07, 2013 7:21 am

It's hard to say exactly. I think the IT is more sensitive but that doesn't necessarily translate int more IDs. MS2 scans of low abundant peptides look much better in the IT. But the high mass accuracy and better fragmentation offered by HCD/Orbi detection often result in more IDs (even if the MS2 spectra look much more sparse). I should mention that it also took quite a bit of care to maintain the instrument so that the MS2 Orbi scans kept their sensitivity. A lot of calibrations and cleaning.

I need to correct a comment I made in a previous post. For unmodified peptides, CAD with Orbi detection gives similar numbers of IDs compared with HCD with Orbi detection. For phosphopeptides HCD with Orbi detection works much better. See figure one in this paper.

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Postby neil777 » Thu Mar 07, 2013 2:09 pm

Hi
I have used bruker tofs for phospo work. You do get some good ids, but you do get quite alot of neutral loss too. I have also used bruker etd ion trap and in my opinion, for analysis of complex samples is about as much use as a chocolate tea pot


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