Multiple injections in a single LC-MRM file, aka RapidFire

Multiple injections in a single LC-MRM file, aka RapidFire echan  2020-02-21

Hello Skyline team,

Skyline has worked out great for our small molecule analysis by target MRM/PRM.

However, we're having trouble figuring out how to import data from our screening datasets in a RapidFire-like workflow as described in Figure 5 of this SCIEX technote.

We're monitoring the same 4 transitions (for molecules) in 30 second injections across one 96-well plate (4 * 96 = 384 transitions), with all the results saved to one .wiff file. We can process the data using SCIEX MultiQuant as shown in the attached screenshot.

However, the closest I have come to with Skyline is to import the .wiff file as 96 .sky files, each with different explicit retention time values (incremented by 30 seconds). I can manually duplicate the same molecules 96 times and edit the RT (and RT windows) in the XML document, but all of my edits get overwritten with all the RT (and RT windows) reverting to the same values after re-importing my results (i.e. re-importing my .wiff file).

It would be fantastic if we can use Skyline to manage our MRM data, regardless of whether we're monitoring a single molecule/single injection or single molecules/multiple injections in a single file. Could you give us some pointers? Thanks. -Eric

Nick Shulman responded:  2020-02-21

Can you send us one of your .wiff files (and the .wiff.scan that goes with it)?

Skyline can handle .wiff files that have multiple "Samples" in them, and each Sample sort of looks like a different file within Skyline.

It sounds like your .wiff files are different, and they look like a single injection that is really long. Is there any way that you could make them look like Samples in the .wiff file. (I'm not an expert on wiff file formats. Someone else might be able to phrase my question in a way that makes sense).

Skyline keeps track of which peaks you have manually adjusted so that the peak boundaries are preserved when you do "Edit > Manage Results > Reimport". If you are going to be adjusting peaks by modifying the XML file, you should add the attribute:
to the <transition_peak> and <precursor_peak> elements so that Skyline knows not to change them if Skyline does a reimport.

Usually we don't recommend manually editing the XML file, and would try to suggest that you use "File > Import > Peak Boundaries" instead.
-- Nick
echan responded:  2020-02-21
Thanks for your reply, Nick.
I've uploaded an example file ("") to the file sharing site.
The retention times are at 7.22 min, 7.72 min, 8.22 min, 8.72 min ... 51.72 min, 52.22 min.