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Standards not registering Jiny  2023-10-23 09:49
 

Hi,
I took the advanced small molecule course week before last and I've been trying to get a set of standards to calculate in Skyline following the instructions, I did not run them with internal standard, just trying to test a standard curve and get some idea of my calibration curve, however I keep getting 'Error: All of the external standards are missing one or more peaks. The selected replicate has missing or truncated transitions' no matter what settings I try. I've even tried setting everything as global standards, I've tried putting it on the peptide/molecule mixed, I've tried every quantitation setting combination I can think of, have no idea what I'm missing. You can see in the document grid I've got area counts for everything. Any help would be greatly appreciated.
 
 
Chris Ashwood responded:  2023-10-23 14:58

Hi Jiny,

I'm not a Skyline developer but think I have an idea what the problem might be. The dotted chromatogram lines indicate that the transition is not set as quantitative. I believe that is the reason why the calibration curve can't be generated. In the Transition Settings window and Full-Scan tab, if you have the acquisition method set to DDA, the product ions won't be set to quantitative.

To fix this, you can change the acquisition method to PRM and reimport the data, or set each transition ion as quantitative. To do this:

  1. Right click on the product ion of interest
  2. Click on the Quantitative button from the drop down list.

With either method, the extracted ion chromatogram should now be a solid line instead of a dotted line, and your calibration curve should work

Cheers,
Chris