Different peak height, area in Skyline and in Xcalibur

Different peak height, area in Skyline and in Xcalibur hilaire  2020-01-02

I am analyzing Q-Exactive HF PRM Data with Skyline.
In Skyline after importing of raw data, for compound G, the Skyline XIC is totally different from what I can see in Xcalibur.
For 78.9585, the height in Skyline is 880, while in Xcalibur it is 5.02e4.
For 492.9919, the height in Skyline is 75346, while in Xcalibur it is 1.98e4.
What is the cause of the error and how can I improve it??

I have attached the skyline file, transition setting and the XIC, MSMS snapshot from Xcalibur for your reference.

Brian Pratt responded:  2020-01-02

Have you had a look at previous questions along this line?


may cast some light.

If not, please respond here and we'll dig in further.

Best Regards

Brian Pratt

Brendan MacLean responded:  2020-01-02

That thread is probably not applicable here, because you are expecting to see 493 showing up 2.5x the height of 79, and it is essentially missing instead. So, there clearly is not the expected linear relationship.

I would generally start by clicking on the chromatogram to look at the underlying spectra that have produced the chromatograms you see in Skyline. Though, I can't do that without the raw data files, you still could. It might become immediately obvious what is going on.

My first (wild) guess would be that your 240,000 at 200 resolving power produces too narrow an extraction width (a.k.a Full Scan Filter Width in the reports or results view), which is 0.000413 for 79 and 0.000645 for 493.

Kind of makes me realize that when resolving power is that high, the extraction width really needs to include both resolving power and mass accuracy. You may be able to resolve to 0.5 PPM, but your mass accuracy still is likely not up to supporting a 1 PPM extraction window.

I would be surprised if relaxing your resolving power somewhat doesn't improve what you are seeing.


hilaire responded:  2020-01-03

Hi Brendan and Brian,

Thank you for your rapid reply and helpful information!

After reducing the resolution in transition setting from 240,000 to 20,000, the peak height of 79 has increased, and then I have observed the said linear relationship between the peak height calculated in Skyline vs Xcalibur, where the peak height in Skyline for 493 and 79 are both roughly 3.8 times that in Xcalibur.

Best regards,