Hi Gerard,
If you would like to see only the XIC for a single transition, on the View menu, choose Transitions and click Single. After that, when you select individual transitions in the Peptide View tree, you will see only the XIC for that transition.

I highly recommend spending time with the tutorials and videos. The cover a lot of useful functionality.

Hope I have correctly identified your concern.

--Brendan

Hi Brendan, and thanks again for the (super)fast answer,

As far as I know, but I could be totally wrong, the XIC refers to the extracted chromatogram based on the TIC, not on the BPC.

Therefore, I would expect the XIC to be done, in Xcalibur, with the "Mass Range" option. However, Skyline gives the extracted ion based on the Base peak, the one that you obtain in Xcalibur selecting "Base peak" and then, introducing the correspondent mass range.

I attach an image to make it clearer. However, I could be wrong and the XIC could actually refer to the BPC XIC indeed.

Gerard

Hi Gerard,
I am not super familiar with Xcalibur, but asking other folks in the lab who are, and even playing with Xcalibur a little, we did not see any difference between BPC and Mass Range with our SRM data.

I have debugged pretty carefully through Skyline reading Thermo raw files from triple quadrupole instruments (Quantum Ultra and Vantage). From this, I know that the data is composed of MS/MS spectra with individual intensity values at each product m/z value you have specified in your transition list. If you specified only 2 product ions for your precursor, then the SRM MS/MS spectrum should contain only 2 m/z, intensity pairs per scan.

If you are getting a difference between your base peak and an extracted mass range, my colleagues tell me, that could only happen if there are more than one m/z, intensity pairs within the mass range in question, in your case (530.00-533.00). Which, I believe, means that there is something about your data that we don't fully understand yet.

Skyline will only use 1 intensity value in the data for each transition. It will always choose the intensity value with the m/z value closest to the one specified in the document. It seems you are getting another peak within your mass range, which I can't explain, given the transitions you say you have measured, unless, you also measured another precursor with the same precursor m/z, which happens to have a product m/z within your XIC range.

The answer lies in your data. Skyline is matching intensities to transitions correctly. Using an XIC of a mass range is not the way you want to do this, precisely because of the potential interference from other measurements in your method.

I would be interested to hear what you find to be the cause of the difference between BPC and XIC in your data.

--Brendan

So, here it is the list of transitions for this file

577.287148    769.32975
577.287148    185.128454
->-> 405.193042    531.288536
->-> 405.193042    460.251423
391.219394    573.335487
391.219394    209.103302
366.703577    501.314357
366.703577    331.208829
313.163222    512.235104
313.163222    425.203075
483.256174    607.356222
483.256174    508.287808
560.823692    423.260196
560.823692    276.134267
337.163222    423.223811
337.163222    223.107718

It should not be possible to mix different masses, at least it seems that these transitions are quite separated from the others.

I try to attach here the RAW file, maybe you can check it in Xcalibur the differences between TIC and BPC, and between a XIC from the TIC (mass range) and a XIC from the BPC (BPC).

What I can say is that the TIC and the BPC of the different precursors, in all cases (except those that the transition is not detected) the TIC gives a significantly clearer peak with better S/N ratio.

I would say that a BPC of a peak where two masses show a high intensity, should give more noise, since it only selects the most intense and the other, then, is understood as background.

Gerard

Hi Gerard,
Thanks for the data. Would you mind posting the Skyline document you are using with this data?

Thanks.

--Brendan

Hi Gerard,
Also, you should be able to see the TIC for the precursor in Skyline if on the View menu, you choose Transitions and click Total.

I still don't understand the difference between product ion XIC and BPC, but if all you are looking for is precursor TIC plotting, that will give it to you.

--Brendan

Here it goes the file.

This is the thing. I would say that in skyline, when I select "Total", what I see is the BPC and not the TIC. Just now I have in front of me both Xcalibur and Skyline and I am sure that Skyline (Transitions -> Total) is showing the BPC.

Well, at least I believe I am sure :)

Hi Gerard,
Indeed you are right that the ProteoWizard library Skyline uses to access Thermo (and all vendor) raw data files was using the base peak instead of summing a mass range. And this mostly proves we just haven't seen any SRM profile data before.

Yes, your RAW file was acquired in profile mode, and that is the "something about your data that we [didn't] fully understand." It explains a lot, though, like why your file is 27M for such a small number of transitions, and, of course, why BPC and XIC yield different values.

If you were to acquire in centroid mode, you would get a single peak in each scan for each transition in your precursor, at exactly the specified Q3 m/z. And, then, BPC and XIC would be equivalent, as we have seen trying your filters on data collected in our lab, all of which is centroided. Your files would also be smaller.

I am told that a fix for profile mode SRM has been implemented in ProteoWizard. That should be released to the public release of Skyline in version 1.2 sometime this fall. Until then, you are right that Skyline does not handle profile mode SRM correctly. You will have to acquire Thermo SRM data in centroid mode, or wait for the fix to be released.

Thanks for pointing out this issue.

--Brendan