In earlier versions of Skyline, the Explicit RT value was, as you say, somewhat suggestive. If Skyline didn't find anything that looked like a peak within that window it would go looking elsewhere. Now, though, if it doesn't find anything within that window it just stops looking and records no peak.

It sounds like you wish Skyline would just treat whatever is within that window as signal, regardless of whether it seems peak-like?

Best regards,

Brian Pratt

Hi Brian,
Correct, Skyline's idea of "peak-like" and my idea (in a small molecule world) will likely be very different and will honestly depend on the analyte class. One specific example is in when analyzing PFAS. Some analytes can appear as a branched form or a linear form, with moderate RT differences between the two. Historically, both of these forms are just summed and you report the total. However, some samples will have just linear and some just the branched and some will have both. I imagine this (and other circumstances) would be a hard phenomenon to teach an integration algorithm. If instead, I were able to tell skyline "just integrate everything from 2.3 minutes to 3.4 minutes no matter what" this would save me a TON of time. Then if there is any RT drift or anything else I can go through and manually clean up. Currently, I am going in and correcting every sample (50+) for almost every analyte (30+).

I see some places in the document grid that list what the integration boundaries are but these are return fields and cannot be edited...

Thanks!

Hi Brian,
Just wanted to follow up on this. Is it safe to say that this functionality isn't available at this time, but may be someday? Thanks.

I'll make a formal note of it in our development queue.

There are some barriers to implementation, though: Skyline currently makes a pretty hard assumption about the presence of some kind of peak, for the purpose of background subtraction. In this case, where there's signal from your point of view, absent a peak Skyline would see it as noise and subtract it out, leaving something pretty close to zero.

Luckily I believe there is some unrelated work going on in Skyline that might make this more tractable than it formerly was. Could you furnish some examples of the phenomena you're thinking of? Skyline document (File > Share> Complete), raw data, and a few screen shots that illustrate the problem? This would go a long way toward making this a reality.

Best,
Brian Pratt

Hi Brian,
Thanks for the info - I'll keep an eye out for updates! I've attached some screenshots and a document showing some poor peak shapes that are not handled by Skyline as well as I would hope. Thanks.

Thanks for those images - this is very interesting.

I would have naively looked at those and said Skyline is doing an excellent job of discerning the peak from some major interference. And I'd have (again naively - I'm a software engineer, not a molecular biologist) felt justified because Skyline's integration of the heavy labeled versions seems to be correct.

It almost seems like the branched and linear forms should be treated as different molecules for analysis purposes since they clearly have distinct elution profiles.

What are your thoughts?

Thanks again,

Brian

Hi Brian,
I definitely agree with you - in a perfect world we would make up a calibration curve for the branched form for and a separate calibration curve for the linear form and handle them separately. The problem, however, is the prevalence of these various forms in unknown samples and the availability of standards. I've observed this phenomenon in two different contexts:
-One is in human metabolism where the body can metabolize a chemical by adding a hydrophilic functional group in one of multiple locations on the [drug] molecule (see attached Image 1). The two molecules that result from metabolism of morphine are isobars and although the mass spectrometer cannot tell the difference (except for sometimes with the fragments if you're lucky), these two molecules will have slightly different retention times. Years ago when we were buying the standard for this metabolite (for running a calibration curve) it ended up that it was only possible to synthesize (or isolate) one of these two variants. Therefore we made a calibration curve with only the one form, but the patient samples contained both forms. In this case we integrated the calibration curve like normal (since there was only a single peak there), but then for the samples we integrated both forms and reported the result as "total" morphine-glucuronide. This is not ideal but is generally accepted for structural isomers (with similar retention times) when an exact standard of each form cannot be acquired.
-In the PFAS world, it is a similar issue, the byproducts that are found in water streams contain both several different branched forms as well as a linear form (Image 2). The standard that we are able to purchase is also a mix of branched and linear because they are unable to sufficiently (or economically) separate the linear form from the various branched forms that may be occurring. The current protocol according to government standards is to integrate the whole mess and label it as a "total" quant that incorporates both forms (so long as ion ratios pass). In the end the toxicology to determine whether the branched or linear forms have a greater impact on human health falls to somebody smarter than I - hopefully they are smart enough to figure out how to synthesize pure standards...

Hopefully this sheds some light on the issues - small molecules can be quite tricky like this at times and that's where the ability to just specify a start and end point to the integration window would be really helpful for reducing manual batch processing.