Steve Gygi talks about next generation of MS3 for quantitation of isobaric tags

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Doug
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Steve Gygi talks about next generation of MS3 for quantitation of isobaric tags

Postby Doug » Fri Feb 17, 2012 1:37 pm

I saw a talk by Steve Gygi today called "Solving the mysteries of MS multiplxing". It was focused on application of MS3 in order to get more accurate quantitation for isobaric tagging experiments. This was a method he published in Nature methods last year. (http://www.nature.com/nmeth/journal/v8/n11/full/nmeth.1714.html). His group has applied it to compare cancer cell lines with some interesting results.

He spoke of some of the drawbacks to the current implementation of the method as well as some ways to fix them. He mentioned that 20% of peptides don't give rise to enough ions in MS3 scans to accurately quantify peptides and described a new method that reduces this problem. In the new method they employ "multi notch waveforms" in the isolation of fragment ions after MS2. Basically, instead of isolating a single MS2 fragment for MS3 they can now isolate multiple at once. He said this results in an 8-fold increase in MS3 signal. That should definitely improve quantitation. He mentioned that of course you can't isolate too many "notches" or else you will just get back the interference that you are trying to filter out.

Are any of you using this method? How is it working in your hands?

arledvina
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Postby arledvina » Fri Feb 17, 2012 2:06 pm

So the question I would have is this: how much of a hit to isolation efficiency does one take when using the isolation waveforms? By this I mean that isolation efficiency in a 2-D trap is dependent upon the reduced mathieu parameter (q-value); the higher the q-value, the deeper the pseudo-potential well, and the more efficient the isolation.

If he is using multiple isolation notches, not every species can be placed at a high q-value; only the lowest m/z ion is. I wonder how the efficiency of isolating a precursor at a q-value of, for example, 0.4 compares to isolation at a q-value of 0.8.

Finally, I am curious how many notches were used. I assume it must be at least 8 to gain 8-fold?

Craig
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Postby Craig » Fri Feb 17, 2012 3:24 pm

It sounds like a great idea. Sensitivity (along with throughput) appears to be the key weakness of the MS[SUP]3[/SUP] method, although its accuracy should be very good. Being able to select the number of precursors to isolate for MS[SUP]3[/SUP] is a great way to tune the balance between sensitivity and accuracy. The more precursors you select for MS[SUP]3[/SUP], the more signal you should get for quantitation but at the same time more interference and therefore less accuracy, and vice versa.


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