Targeted analysis on MS1 only

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Targeted analysis on MS1 only paramon  2018-06-05 22:43
 

For small molecules, it's not uncommon to perform targeted analysis on MS1 only (high-throughput). However, currently user is required to specify at least one product m/z. At import time:
You must add at least one target transition before importing results.

It is proposed that this restriction is lifted, so that only precursor m/z or formula may be specified (product m/z is not required) for a target.
Probably a warning should be issued in this case, but the program should continue if accepted.

 
 
Nick Shulman responded:  2018-06-05 23:27
In Skyline, monitoring the precursor ion is considered to be a "transition".

In order to get such a transition into your document, one thing you need to do is in:
Settings > Transition Settings > Filter > Small Molecules > Ion Types
add "p" to the list of ion types

The other thing that you need to do is tell Skyline that you want to extract chromatograms from MS1 scans.

To do this, you go to:
Settings > Transition Settings > Full Scan
and under "Isotope Peaks Included" choose something other than "None".

Then, Skyline will add some precursor ions to your Skyline document representing the monoisotopic mass plus maybe some other integers.

If nothing gets added, then you might need to right-click on a Precursor in the Targets tree and choose "Add Children".

Then, you should be able to extract chromatograms using:
File > Import > Results
 
paramon responded:  2018-06-06 05:31
Thank you Nick, the steps you proposed worked nicely!
I could now get my precursor-only targets working on Waters MSe data.
 
Brendan MacLean responded:  2018-06-06 08:12
I will just note that even when you are extracting only precursors from Waters MSe data, you need to tell Skyline that you performed MSe, because the waters data format provides no other way to differentiated MS1 (low energy) from MS2 (high energy) spectra.

So, unless you want to see very spiky chromatograms showing in alternating points the difference between unfragmented precursor between low- and high-energy, you need to also set MS/MS filtering, Acquisition method to DIA and Isolation scheme to All Ions. This tells Skyline that the spectra alternate between low- and high-energy, and if you only have "precursor" targets, it will only extract chromatograms from the low-energy spectra.
 
paramon responded:  2018-06-06 11:39
Thank you for the input, Brendan! I believe I have now figured out how to properly set up the parameters, thanks to your comment yesterday and the message by Nick.

However, closer exploration of the data files revealed that they are not actually MSe but rather interleaving positive/negative polarity scans :-) I did successfully trick Skyline into ignoring negative scans via fake "All Ions" fragmentation setting; but "in ideal world", is it supposed that Skyline can make good use of negative scans also?
 
Brendan MacLean responded:  2018-06-06 12:04
Yes. Skyline should be doing a better job of identifying negative scans, and, yes, it is possible to add negative polarity targets in Skyline and have chromatograms extracted for them as well. If we can get your files (use File > Share - Complete to create a .sky.zip file and a raw data file - also zipped) and post to http://skyline.ms/files.url

Hopefully we can get this sorted out quickly.

--Brendan
 
Brian Pratt responded:  2018-06-08 11:14
Please do provide that data - normally, alternating scan polarity is something Skyline already handles, so there is something strange happening here and I would like to investigate.

Thanks,

Brian Pratt