Site-mapping of PTM- how to know whether it is real

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Pippuri
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Site-mapping of PTM- how to know whether it is real

Postby Pippuri » Mon Mar 19, 2012 7:25 am

Hi All,

I am wondering whether anyone can share their experience/tips with manual data interpretation for PTM assignment. I have to confess (once again) that I am a amateur in mass-spec, so, I will not be offended if you feel like explaining things to a kindergarten kid.

Here is the thing- After optimizing everything for sample prep, I was able to obtain ~80% coverage (based on sequest search) on my protein of interested. I was told that, since my protein is fairly big (>180kDa), this is a good thing. But, the sad thing is, I don't get any modified peptides that have decent Xcorr. However, I was also told not to expect any high Xcorr from the modified peptides, because they don't, and that I should go through my data manually.

So, the next thing is to go through my data manually. I have run my samples many times with CID/ETD (decision-tree), HCD/ETD. Based on what I read in the literature, looking for the oxonium ions specific to the PTM under HCD fragmentation is the most reliable way to locate a modified peptide.-- But how to do that, exactly?

I have started with looking at all the spectra pulled out by sequest, says, an ETD spectrum that the software determined there is a PTM on it, and looked the the HCD spectrum from the same precursor ion for the oxonium ions. So far, every attempt come out fruitless!!

So, for all the experts out there, can you please share your experience with me?

Million THANKS!!!

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Doug
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Postby Doug » Mon Mar 19, 2012 10:01 am

In my experience, finding PTMs not identified in database searches is difficult and validating them is even harder. But for a first step you might want to look for neutral losses. In the QualBrowser software you can make a filtered chromatogram that shows the relative intensities of specific neutral losses (you have to tell it what neutral loss you are looking for). You can look at some of the scans indicated by that chromatogram and they will likely have your modified peptides in them (if they are known to give a neutral loss). I have found glycosylated peptides this way.

Having both HCD and ETD should help in the identification process.

Are there specific modifications you are looking for?

Pippuri
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Postby Pippuri » Mon Mar 19, 2012 1:11 pm

Hi Doug,

I am looking for O-GlcNAc.

Unfortunately, I wasn't able to enrich at the peptide level very effectively- I tried the others have published without any luck so far. Since I am focusing on just one protein, I am hoping to use a combination of MS-spec workflows to dig out the sites.

I did include neutral loss setting in my CID method, but Sequest search turned out nothing. Do you think it is still possible for me to dig out spectra with neutral loss in my raw file? Will I see the neutral loss in my HCD run too? I am a little bit confuse at this moment.

Thanks.

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Doug
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Postby Doug » Mon Mar 19, 2012 1:31 pm

I see. Yes, you can still dig out spectra with neutral losses in your RAW file. I think you should probably be able to see the neutral losses in both CID and HCD spectra.

In QualBrowser open your RAW file, right click on your chromatogram, select "ranges", from the "plot type" menu select "neutral fragment", and enter the mass of the neutral loss you are looking for. Hit Ok. This should result in a chromatogram showing where the most intense neutral losses were. You can look through the most abundant peaks in this chromatogram for putative modified peptides. It won't necessarily find only modified peptides but it might be a good place to start.


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