Monday, January 28, 2013

Optimizing your nanoLC conditions part 2: Calculating your peak width at threshold (PWAT)

This is part 2 of this who-knows-how-many parts monologue on optimizing your nanoLC conditions.  Part 1 was yesterday, or you can click here to go straight to it.

We're going to talk about the next thing I consider when I'm setting up nanoLC flow conditions:  peak width and cycle time.  By peak width, I mean how wide your average peak is.  Classically, we consider the 1/2 peak width of an  HPLC peaks like the one below (stolen from

It is important to note that the 1/2 peak width isn't that useful in mass spectrometry.  What you are interested in is what I will call the peak width at threshold (PWAT).  Somewhere in your method, regardless of your instrument, you have set a fragmentation threshold.  You don't just want to be trying to fragment everything you saw in your MS1 spectra, so you set this threshold to fragment things that are unlikely to just be noise.  Hence your threshold.  In discovery experiments, these are often set low (1e3 or lower, though 5e3 is probably the setting I see the most on hybrid instruments) just in case that biomarker is down there in the noise.  So, if your peak looks like the one above, and the peak is 1e6 in intensity, the you fragment it clear down at the base.  If you use dynamic exclusion to then ignore that peak for the duration of the elution, then you will not fragment it again.  If your threshold is higher, say 1e5, then you will fragment that eluting ion first (and perhaps only) at 10% of the way up the peak.  Now, these settings sound like they are hurting you, but only in the case of this peak.  If you set the threshold too high, like 1e5, and you biomarker peptide only elutes with a peak height of 5e4, then you've missed it.  Again, this discussion is well beyond the scope of what I am writing here.  I also refuse to turn this into another monologue on appropriate dynamic exclusion settings, you can read one of those here.

Back on topic:  What you need to know is your PWAT.  The best way to find this is to measure a few peaks in your RAW data and get a good estimate of your PWAT at your LC conditions.  Once you know this, you have two choices -- you can either change your LC conditions to match the MS settings, or you can change the MS settings to match you LC conditions.

Sorry this is so short, I ran out of time this morning.  Next up:  cycle time vs peak width (link here!)

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