Saturday, March 16, 2013

How to set up a binning experiment on an Orbitrap

Recently, we've started doing quantitative experiments by looking in smaller MS1 ranges.  But what if you could do discovery based proteomics in the smaller ranges?  Would this help your results?  Absolutely.

This idea has been touched on before by other groups, but this new paper from CE Vincent et. al., out of the University of Wisconsin really knocks it out of the park.  The paper focuses on two concepts, one a little older and one brand new for doing this.  They call the first one a binning experiment, and their new approach a tiling experiment.  For now, I'm going to skip the tiling experiment which is the real star of this paper.  This requires subtle alterations of the code controlling Xcalibur on the Orbitrap computer.  The lesser note is the binning experiment, which everyone can do on every Orbitrap or LTQ system.

Above is a quick schematic I made of a binning workflow.  In this you would run the same sample multiple times, but during each run you can only perform your search for your TopN to fragment from within a narrow mass range.  It increases your dynamic range within that area and lets you dig a whole lot deeper.  The more narrow the bin, the deeper you'll dig into your sample.

In a tiling experiment, this is taken one step further, where multiple narrow ranges are selected in one run.  For example, if this is a Top20 experiment, the first ten MS/MS events can only occur from mass range 400-500 and the next ten can only occur on precursors from 500-600.  Without hacking Xcalibur using the developer's kit this option simply isn't available.  I would have to think though that the binning experiment would have to do a better job in obtaining sample depth than the tiling experiment, since you are concentrating more time on a more narrow range.  The benefit in the tiling approach is that, if utilized well, you would get depth without re-running the sample multiple times, something most of us can't afford.

The real gold in this experiment is that this group used these approaches quantitatively.  In each run, even when you were binning a different mass range, you still have the MS1 scans that you could use to for XICs for quan.  So you were able to dig much deeper and still have replicates for accurate quantification.

I encourage you to take a look at this paper.  And if you have questions on how to set up a binning experiment, I have a method that I successfully utilized on an Orbitrap Velos that I can make available.  As always, feel free to email me at:

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