Tuesday, April 14, 2020

FAIMSPro -- More proteins, but where did all the peptides go??

Mystery with a short answer (I think) that took me a while to figure out. However, since it isn't detailed precisely in the study above, I feel a little less unsmart. (1,2,3 negatives in that sentence? Meh.)

When you fire up your FAIMS Pro, you're going to be IMpressed. The background noise is great, the nitrogen consumption....less great..... and you're going to see WAY more protein IDs!

However, there is a cost to this. You're going to get less coverage of those proteins. The authors made this pretty green chart, so I don't have to (borrowed without any permission whatsoever)

Regardless of what FAIMS compensation voltage (CV) you use, you're going to end up with fewer peptides identified. But more proteins!

I'll even go one better with another stolen green plot -- the proteins you'll find will be lower abundance! (Deep HeLa is fractionated).

If you spend less time on albumin, titin and keratin, we all win (unless you're a keratin researcher, and you have your own challenges).

I scratched my head about this and plotted stuff a bunch of different ways. Surprisingly the MS1 isolation interference doesn't seem all that different (but -- keep in mind that this is essentially a normalized measurement)

<---No FAIMS left
FAIMS -75 right -->

However, if you take out all the z=1 peptides and your Signal (S) goes up, you're also raising low abundance peptides that are now contributing to your Noise (N), so probably this is all good stuff.

Okay -- so what is actually different between the files?

The stupid charge state distributions!

 No FAIMS left -- FAIMS -75 right. All the sudden you've got a bunch more +3 peptides (relatively)

Okay -- this gets better, I think, because you know what a lot of search engines assume? They assume that your MS/MS fragments will be +1 charged.

Sure, they'll try to look at more, but as awesome as a 1980s TransAm looks and sounds, it's only got 205 horsepower. With age, it's probably closer to 170 at the wheels without a full rebuild by someone good. You can get faster used hybrids on Craigslist.

Is that a long and unnecessary metaphor/analogy/something or other? Absolutely.

Nearly all of the newer search engines that I'm always going on about start by deconvoluting the MS/MS spectra prior to searching.

As a more controlled experiment (n=1! I'm winning science today) -- let's take the same file and run it through Proteome Discoverer with and without first deconvoluting the MS/MS spectra so all the fragments are +1 (this was in PD 2.1, due to the fact I'm using an older PC today thanks to some weird malware issues that I think are Zoom related)

It's safe to assume in a tryptic digest that you've always got your single basic residue at the terminus. There's one. In a +2 you've got one "mobile proton" so probably +1 fragments make sense, but in a +3?

I'm too bored with this post to dig up some MS/MS spectra. (I went down a rabbit hole reading about Trans Am specs to make sure I was right. My Craigslist hybrid is faster 0-60 than a 1985 Trans Am when the two were brand new. (The Trans Am looks way cooler, though).

CV -75 file number 1. No deconvolution/SeQuest+ Percolator

Same file. Changed nothing except added deconvolution of the MS/MS spectra

FAIMS Pro results may not be exactly what everyone wants. If you're looking for the highest coverage, maybe you want to take it off and run without it. If what you want is the highest number of protein IDs because you are willing to sacrifice some coverage of the higher abundance ones to see some of the lower abundance ones....

----particularly if you're willing to tweak your workflow toward this newish kind of data!

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