Error when importing raw file

support
Error when importing raw file davidz  2019-05-29
 

I got this error when importing the raw file from Q-Exactive:
At 1:41 PM:
Failed importing results file 'E:\Users\WeiZ\Downloads\qe\72828_90397.RAW'.
The time interval 1 to 0 is not valid.

What does it mean?
Thanks,

David

 
 
Brian Pratt responded:  2019-05-29
Hi David,

There is something unusual going on, certainly. Can you provide that file, along with the Skyline document? For the Skyline document, please use Skyline's File>Share>Complete menu item to create a .sky.zip file. You can upload the two files to http://skyline.ms/files.url .

Thanks,
Brian Pratt
 
davidz responded:  2019-05-30
Hi Brian,

I changed the transition settings and peptide settings and made the import work, but I don't why, here is the skyline document, and these are q-exactive PRM runs, what are the best settings for transition and peptide for q-exact files?
Thanks,

David
 
Nick Shulman responded:  2019-05-30
Hi, David,

That error that you ran into "The time interval 1 to 0 is not valid" can sometimes happen in Skyline 4.2 if Skyline ends up with a chromatogram that has only one point in it. This can happen when extracting MS2 chromatograms from DDA data if there really might be only one MS2 spectrum whose isolation window is close enough to the precursor for Skyline to use it for a chromatogram point.

We have fixed this in Skyline-Daily so that single point chromatograms will no longer cause this particular error message.

By the way, your chromatograms in that file "AX9989_humanUniref100_72828_90397.mzXML" look like they came from DDA data, not PRM.
When I am looking at the chromatogram for DVKPENFLVGRPGTK+++, there are only 8 chromatogram points in the time range from 15 to 65 minutes.

If this is DDA data, then you should only be trying to extract chromatograms from the MS1 scans. In Skyline-Daily, we added a new MS/MS filtering acquisition method called "DDA" on "Settings > Transition Settings > Full Scan". When it is set to "DDA", Skyline extracts MS2 chromatograms but does not use them for quantification.

Your replicate "AX9989_humanUniref100_72927_90533" does look like a real scheduled PRM file. For that same peptide, there are 42 chromatogram points between 25 and 32 minutes.
-- Nick
 
davidz responded:  2019-05-30
Hi Nick,

Thanks for your reply, and you're right, I just realized that 72828 file is a DDA + PRM run, where some mzs are targeted in a time window, some are targeted throughout the run.
BTW, can you have a look at the transition and peptide settings, and see if I've set them up correctly for processing Q-Exactive PRM runs, given I have the correct PRM runs next time?
Thanks,

David
 
Nick Shulman responded:  2019-05-30
The settings look pretty good. Here are a couple of things that might be mistakes, but also might be perfectly fine:

I noticed that the peptide "LIKDDEYNPCQGSKFPIK" does not have any chromatograms. That might be happening because it's m/z is 764.396194, which is really close to "SYPYLFKGLEDLHLDER" which is 764.395071.
When your acquisition method is "Targeted" (i.e. PRM) then Skyline assigns each MS2 scan to whichever peptide has an m/z which is the absolute closest to what the mass spectrometer says was isolated. If you want Skyline to allow spectra to be assigned to all of the peptides that fall within the isolation window, then you should say that the acqusition method is "DIA".

We recommend using "Centroided" for both the precursor and product mass analyzer. This is because Thermo's software centroiding algorithm is better than what Skyline does which is just summing the profile intensities across a m/z channel. You have it set to "Orbitrap" for your MS2 scans. "Centroided" is probably better, but it might not make any observable difference at all.

For the MS1 filtering, you have the mass accuracy set to 5ppm. If your mass spectrometer is not actually that accurate, then what you will see some spikly chromatograms which alternate between the correct value and zero, depending on whether the centroid happens to fall within Skyline's m/z extraction window.
-- Nick
 
davidz responded:  2019-05-30
Thanks, Nick! I will make changes to the setting accordingly. For LIKDDEYNPCQGSKFPIK and SYPYLFKGLEDLHLDER peptides, LIKDDEYNPCQGSKFPIK has a carbamidomethyl C @10, so the mz will be different, and the they elute at different times. However, for
TVAVKILK m/z 534.402 @21.0m VAIKIISK m/z 534.402 @21.9 minute
what's the best way to separate them? setting acquisition method to "DIA"? will it affect others though?
Thanks,

David
 
Nick Shulman responded:  2019-05-30
David,

If your PRM method uses exactly the same masses as Skyline is expecting, and it has enough digits of precision, then Skyline will have no trouble figuring out which spectrum belongs to which peptide.
But, if one of those two peptides is hogging all the spectra then something might be off about either the precision or accuracy of your method.
I would have to see your .raw file in order to be sure whether something like that is going on with the data you collected.
-- Nick
 
davidz responded:  2019-05-31
Hi Nick,

Just uploaded the raw file, please have a look. Also, I've tried the DIA setting for these PRM files, peaks finding are, uh, different, I need to examine the result closely. One general question though, can I run samples in DIA mode, then import peptides I'm looking for, and quantitate in Skyline this way?
Thanks,

David
 
Nick Shulman responded:  2019-05-31
Thanks for sending that .raw file.

I see that your isolation windows are 3 units wide.

For PRM, we normally recommend a narrower isolation window which only targets the monoisotopic peak of the intact peptide.
One reason for this is that Skyline only extracts the MS2 chromatogram from the monoisotopic peak of the fragment ion. The monoisotopic precursor is usually the bigger contributor of monoisotopic fragments. The M+1 peak sometimes fragments into a monoisotopic fragment, and sometimes produces a fragment that is one Dalton heavier. For this reason, we do not think that the signal gains from widening the precursor window is not worth the loss of selectivity.
The other reason not to have wide isolation window is that Skyline thinks that you are trying to target whatever m/z is in the middle of the window, and that might not be your intention. For instance, maybe you have a charge 1 peptide whose m/z is 500. You might want your mass spectrometer to isolate the entire isotope envelope, so you make the window go from 499.5 to 502.5. In that way, you will get MS2 signal from the monoisotopic precursor, as well as M+1, and M+2. However, when Skyline looks at that window, Skyline thinks its best match would be a peptide whose m/z was 501, the exact middle of that range. It might be that we should add a feature in the future to enable you want Skyline to consider the entire isotope envelope when deciding which peptide most closely matches a targeted MS/MS scan, but for now, wide isolation windows in targeted MS/MS will not do what you want because Skyline thinks you only care about the monoisotopic peak.

I hope that makes sense.

What was your intention, for instance, for the isolation window centered on 764.39478? The window spans from [762.89478-765.89478]. Skyline matches that window to SYPYLFKGLEDLHLDER with an m/z of 764.3951, which is why SYPYLFKGLEDLHLDER+++ has a chromatogram. But, it sounds like you were hoping those scans would be used for extracting chromatograms for some other analyte.
What software did you use to produce your isolation list? I believe that if you export isolations lists from Skyline the windows will always be exactly on one of your monoisotopic precursors, but I don't see an exact match in your document.

It is not going to work very well to tell Skyline that this is DIA data. The problem with that is that then Skyline will think that any peptide whose monoisotopic precursor falls within the isolation window should get a chromatogram point extracted from that MS2 scan. Peptides will match to more than one isolation window, but those different isolation windows will all include different amounts of the precursor isotope envelope. Because of that, the intensity in the chromatogram will jump up and down between scans depending on how the isolation window intersects with the isotope distribution.

-- Nick