Thursday, December 10, 2015
Find unidentifed differentially regulated reporter biomarkers in reporter ion datasets!
I feel kind of smart for this one, though I'm afraid I'm getting to the point where I really really should get an indoor hobby of some kind since this is most of what I did last weekend. What do you guys do when its too cold to rock climb but you can't snowboard yet?
Anyway. I have access to an amazingly cool set of TMT/iTRAQ samples. I have access because there is a distinct and observable phenotype. Not a little one, either. The hundreds of samples in group 1 and group 2 are extremely different. Proteomics, so far, has shown just about nothing different between the two. Weird, right? For years we've been suspecting a novel mutational system or PTM that we've just never seen before, but we've not been able to find a way to hunt it down.
So, here was the thought that killed this last weekend: What if I completely ignored the IDs? What if I only looked at the spectra that showed a significant difference at the reporter ion level? And then I tried to figure out what they were later?
In PD 2.1 + Quan you can do this. There is a tab in your report that is your "Quan spectra".
You can actually go to that and look at every MS/MS spectra. You can see the RAW reporter values and you can even see your quantification spectra zoomed in.
So, you can actually go through and see all the stuff that is different. See the reporter ions above? This is exactly the trend I should be seeing in this sample set based on the phenotype. Exactly. And this MS/MS spectra is the most differentially regulated observation in this entire sample set of 1M or so MS/MS spectra. And this PSM shows up just like this three times in different, overlapping fractions. I think the precursor intensity for this is 1e6-5e6. More importantly, since in PD 2.1 we can plot our reporter ion intensities by their SIGNAL TO NOISE (yay!!!!!!), the S/N of these reporter ions are >500!!!
In sum, this is the perfect biomarker for this experiment and maybe the thing we've been trying to find in one form or another for 5 years (Holy cow, I don't think I'm exaggerating. Its 2015?!?!). Not to get my hopes up to high or anything....
Where it gets difficult, however, is linking that back to the full fragmentation spectra.
For example, check this out, and I'd LOVE it if you guys had advice. I'm putting in a feature request and will be bugging the great people at PD.Support but I'll take any ideas I can get.
Anything from the Protein/Peptide/PSM and MS/MS spectrum can be checked and exported to .DTA, mgf, or whatever. Then I can do big DeltaM searches in Byonic or DeNovo GUI it or PEAKS it.
But I've got to go through one at a time and find the MS/MS spectrum info to export. Kinda looks like next weekends gonna be a wash if I can't find a shortcut (cause I have about 200 interesting things to look at now that I have NO idea what the fudge they are!)
I suspect I'm looking at a PTM but I don't have anything to match any of our normal suspects. Or...I'm looking at unique class-switch sequences in the variable regions of antibodies! Either way, there are biomarkers in this dataset that traditional peptide searching can not identify and the dataset is just too big for Byonic WildCard, but here I've vastly reduced (computationally, at least...) the complexity of this problem! Will I find my biomarkers this way? Who knows, but on some of these hard datasets we need every lead we can get, right?
Again, if you have any advice or thoughts on how I might simplify this, I'd love to hear it!!!
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you can use peptide shaker to export the unidentified spectra as MGFReplyDelete
Okay. Thats a good idea! I bet PeptideShaker can find more stuff than Sequest can alone, as well. I'll check it out! Thanks!ReplyDelete