Hi Eric, thank you very much for your reply and the detailed and, yes, quite reasonable explanation. Some of the spectral libraries I got for the combination were built with SpectraST 4.0 and the remaining builds as well as the combination and further import steps were done with SpectraST 5.0. But there are two factors bothering me:
1. I thought too, that the "issue" is caused by different SpectraST versions. But when I did the MRM import (spectraST 5.0) on the single spectral libraries which were built with the older version (see above), this phenomenon didn't occur. 2. Even if it's explainable by a smarter interpretation by SpectraST, does that mean that I can use these changed spectra or is it questionable to use spectral libraries of older software versions imported with a newer one? Thank you and kind regards, Kristina Am Freitag, 20. September 2019 20:49:28 UTC+2 schrieb Eric Deutsch: > > Hi Kristina, I’m not certain I fully understand your issue, but maybe I > can answer some questions: > > > > Partial old: > > Name: DIETIIQK/2 *(old entry, original library)* > > 308.1581 226.5 ? > > 309.0826 359.4 ? > > 325.6734 1596.8 b6-35^2/0.000,b6-36^2/0.492 > > 329.1945 141.2 a6^2/0.000 > > 343.1920 668.4 > b6^2/0.000,y3-45/-0.042,y6-45^2/-0.516,y6-46^2/-0.024 > > > > Partial new: > > Name: DIETIIQK/2 *(new entry, combined library)* > > 308.1581 226.5 ? > > 309.0826 359.4 ? > > 326.1680 1596.8 ? > > 328.2231 141.2 m4:6/0.000 > > 344.1816 668.4 m3:5/0.000 > > > > The first two peaks are fine. For the third entry, I am guessing that in > the original library this peak got shifted to exact m/z of the presumed > interpretation b6-35^2. But this is a highly unlikely interpretation. So in > the new entry, that peak retains its original m/z (i.e. not snapped to a > presumed interpretation) and a “?”. SpectraST seems to be smarter about not > assigning a silly interpretation. > > > > For the next peak with intensity 141.2, the old library had an > interpretation of a6^2 and the m/z is snapped to that interpretation. But > a6^2 is a super unlikely interpretation, or maybe let’s call it impossible > and wrong. In the new library, this same peak has been interpreted as an > internal fragmentation ion and the m/z has been snapped to that. The m4:6 > means that the peptide was fragmented in two places such that you get a > little ion that is TII and has a charge so you see it. Internal > fragmentation turns out to be fairly common and so m4:6 is far more > plausible an interpretation than a6^2. Same for m3:5 (ETI). Not necessarily > correct, but far more plausible than an a6^2 and a b6^2. > > > > So I’m guessing that the first spectrum comes from a version where > internal fragmentation was not yet supported, and the second spectrum comes > from a newer version of SpectraST that knows about internal fragmentation > and interprets peaks better. > > > > Does that seem like a reasonable explanation of what’s going on? Henry > might know more. > > > > Regards, > > Eric > > > > > > > > > > > > > > *From:* spctools...@googlegroups.com <javascript:> < > spctools...@googlegroups.com <javascript:>> *On Behalf Of *Kristina Plate > *Sent:* Friday, September 20, 2019 6:22 AM > *To:* spctools-discuss <spctools...@googlegroups.com <javascript:>> > *Subject:* [spctools-discuss] Combination of consensus libraries and > import in MRM format via spectraST > > > > Hello again, > > > > as I described before (topic: *What happens during consensus building?*) > I have a problem with some messed up spectra in my spectral libraries. > > I combined four spectral libraries and built consensus spectra. Afterwards > I reimported the combined consensus spectral library in MRM format and > converted it in an assay library. When I checked the SPTXT file I found > some *slightly changed spectra* and new *"ion types" m*. Please see my > example of an old, normal spectrum and the corresponding new one below: > > > > Name: DIETIIQK/2 *(old entry, original library)* > > LibID: 4160 > > MW: 960.5481 > > PrecursorMZ: 480.2740 > > Status: Normal > > FullName: X.DIETIIQK.X/2 (CID-QTOF) > > Comment: AvePrecursorMz=480.5611 BinaryFileOffset=23385298 > FracUnassigned=0.09,1/5;0.23,11/20;0.06,7/31 MassDiff=-0.0014 Mods=0 NAA=8 > NISTProtein=Id_02 NMC=0 NTT=1 Nreps=1/1 OrigMaxIntensity=11 Parent=480.274 > Pep=Tryptic PrecursorIntensity=0 Prob=1.0000 Protein=1/Id_02 > RawSpectrum=file_02_04.69683.69683 RetentionTime=2745.2,2745.2,2745.2 > Sample=1/sample_02,1,1 Se=1^C1:pb=1.0000/0,fv=2.7040/0 Spec=CREATE > TotalIonCurrent=8.9e+003 iRT=33.4,33.4,33.4 > > NumPeaks: 31 > > 308.1581 226.5 ? > > 309.0826 359.4 ? > > 325.6734 1596.8 b6-35^2/0.000,b6-36^2/0.492 > > 329.1945 141.2 a6^2/0.000 > > 343.1920 668.4 > b6^2/0.000,y3-45/-0.042,y6-45^2/-0.516,y6-46^2/-0.024 > > 353.2183 967.7 y3-35/0.000 > > 354.2183 480.9 y3-35i/0.000 > > 355.2183 481.6 y3-35i/0.000 > > 358.1609 442.5 b3/0.000,y6-17^2/0.456,y6-18^2/0.948 > > 358.1970 96.7 ? > > 365.2030 293.0 ? > > 373.2367 296.2 ? > > 388.2554 1744.6 y3/0.000,b7-36^2/-0.955 > > 424.1714 315.8 b4-35/0.000,b4-36/0.984 > > 442.1820 644.1 b4-17/0.000,b4-18/0.984 > > 457.7633 546.4 p-45^2/0.000,p-46^2/0.492,y4-44/0.414 > > 458.2791 109.5 p-44^2/0.000,b4/-0.929 > > 471.7608 110.9 p-17^2/0.000,p-18^2/0.492 > > 472.2608 111.1 p-17^2i/0.000 > > 477.1321 111.6 ? > > 480.2740 336.0 p^2/0.000 > > 484.3130 224.9 y4-17/0.000,y4-18/0.984 > > 501.3395 523.5 y4/0.000 > > 553.1674 240.4 ? > > 567.3501 487.3 y5-35/0.000 > > 584.3766 247.1 y5-18/0.000 > > 602.3872 2935.1 y5/0.000 > > 696.3927 205.5 y6-35/0.000,y6-36/0.984 > > 714.4032 2211.4 y6-17/0.000,y6-18/0.984 > > 731.4298 10000.0 y6/0.000 > > 732.4298 414.9 y6i/0.000 > > > > Name: DIETIIQK/2 *(new entry, combined library)* > > LibID: 4673 > > MW: 960.5481 > > PrecursorMZ: 480.2740 > > Status: Normal > > FullName: X.DIETIIQK.X/2 (CID-QTOF) > > Comment: AvePrecursorMz=480.5611 BinaryFileOffset=20613807 > FracUnassigned=0.09,1/5;0.25,13/20;0.17,13/31 MassDiff=-0.0014 Mods=0 NAA=8 > NISTProtein=Id_02 NMC=0 NTT=1 Nreps=1/1 OrigMaxIntensity=11 Parent=480.274 > Pep=Tryptic PepContext=1/NTPR_DIHE PrecursorIntensity=0 Prob=1.0000 > Protein=1/Id_02 RawSpectrum=file_02_04.69683.69683 > RetentionTime=2745.2,2745.2,2745.2 Sample=1/sample_02,1,1 > Se=1^C1:pb=1.0000/0,fv=2.7040/0 Spec=CREATE TotalIonCurrent=8.9e+003 > iRT=33.4,33.4,33.4 > > NumPeaks: 31 > > 308.1581 226.5 ? > > 309.0826 359.4 ? > > 326.1680 1596.8 ? > > 328.2231 141.2 m4:6/0.000 > > 344.1816 668.4 m3:5/0.000 > > 353.2183 967.7 y3-35/0.000 > > 354.2183 480.9 y3-35i/0.000 > > 355.2183 481.6 y3-35i/0.000,m5:7/-0.016 > > 358.1609 442.5 b3/0.000 > > 358.1970 96.7 ? > > 365.2030 293.0 ? > > 373.2367 296.2 ? > > 388.2554 1744.6 y3/0.000 > > 424.1714 315.8 b4-35/0.000 > > 441.1980 644.1 b4-18/0.000 > > 457.2713 546.4 p-46^2/0.000,m3:6/0.006 > > 459.2086 109.5 b4/0.000 > > 471.2688 110.9 p-18^2/0.000 > > 472.2512 111.1 ? > > 477.1321 111.6 ? > > 480.2410 336.0 ? > > 484.3130 224.9 y4-17/0.000 > > 501.3395 523.5 y4/0.000 > > 553.1674 240.4 ? > > 568.3124 487.3 ? > > 584.2763 247.1 ? > > 602.3872 2935.1 y5/0.000 > > 695.4087 205.5 y6-36/0.000 > > 713.4192 2211.4 y6-18/0.000 > > 731.4298 10000.0 y6/0.000 > > 732.4675 414.9 ? > > > > I assumed before, that it was an issue with the consensus building, but I > figured out that it took action during the *import via spectraST into MRM > format*. > > > > Now, I just saw that I don't have this problem when I use the single > libraries. Even with consensus libraries built with with an older spectraST > version the MRM import works just fine. > > Maybe there is the *problem in the combination of the spectral libraries?* > I already got them as single consensus libraries and combined them with the > following command > > > > spectrast -cNcombined_lib -cd -cu -cr1 -cy1 -cAC -cJU > Lib_01.splib Lib_01.splib Lib_01.splib Lib_01.splib > > > > Could this be an issue here. Do I have to rebuild all libraries before > combining them? > > > > I would be really happy about any suggestions. Thank you in advance! > > > > Kind regards, > > Kristina > > ____________________ > > Kristina Plate, MSc. Biomathematics > > > > University of Greifswald > > Center for Functional Genomics of Microbes > > Institute for Microbiology > > Department of Microbial Proteomics > > Felix-Hausdorff-Str. 8 > > 17489 Greifswald > > > > phone: +49 3834 420-5925 > > fax: +49 3834 420-5902 > > -- > You received this message because you are subscribed to the Google Groups > "spctools-discuss" group. > To unsubscribe from this group and stop receiving emails from it, send an > email to spctools...@googlegroups.com <javascript:>. > To view this discussion on the web visit > https://groups.google.com/d/msgid/spctools-discuss/1573a2bc-1b4a-4ca7-bc88-75579028df95%40googlegroups.com > > <https://groups.google.com/d/msgid/spctools-discuss/1573a2bc-1b4a-4ca7-bc88-75579028df95%40googlegroups.com?utm_medium=email&utm_source=footer> > . > -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. 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