Sunday, July 15, 2012

SWATH: The worst idea in proteomics?

I've been reserving my opinion on the new MS/MS technique called SWATH for several weeks now. I like to think about things for a long time before I commit to them, sometimes.  And I felt like I really needed to learn more about this procedure, particularly when my initial reaction was so overwhelmingly negative.  After weeks of thinking about it, I'm convinced that I don't think SWATH is a good idea at all.

The SWATH technique is a data independent fragmentation method.  Every ion is fragmented.  The good, the bad, the intense, the weak -- every ion.  In order to narrow this down, small mass ranges (or swaths) are chosen, often in 25 amu windows.  Every ion coming through with a m/z of 350-375 is then fragmented and MS/MS spectra collected.  The machine then goes to fragmenting all ions from 375-400.  To be clear, the MS1 mass is never recorded.  You simply do not know the mass of your parent ion.

In order to get over this minor obstacle of not actually knowing what you fragmented (other than its m/z, plus or minus 25 amu!), the processing program compares the MS/MS spectra generated to a pre-generated spectral library.

Okay, so maybe I'm being old fashioned, and I have been doing this a while.  But not that long ago, we looked at the parent mass of every ion we fragmented and made a hypothesis of what it might be.  Then we examined each ion generated in MS/MS individually and matched them to see if our hypothesis was correct. I know, mass spectrometry has been growing at an insane rate.  It is almost impossible to keep up with the advances in hardware, software, and processing technologies.  But at the heart of it, I personally believe that it still breaks down to:  can we make a structural hypothesis based on the MS1 and support it with the MS2?

Besides these perceived shortcomings, there are some other concerns.  Moving sequentially through mass ranges takes a long time.  A method that requires such intense scan times can not possibly be compatible with rapid chromatography systems.  The real limitation of most proteomics labs is the amount of time it takes to get data from a sample.  This is most often limited by the amount of time required to get a good chromatographic separation.  Ideally, these would be getting shorter all the time.  I know I was thrilled when I found a superior column manufacturer that could trim 15 minutes off of my gradient time  -- and here we are looking at a slower method?

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