Hi Kristina, I am not so sure about answers to your questions. It would be ideal to use SpectraST 5.0 in all your processing if you can easily do this, yes, but in some cases it would not matter much. In general, where peak interpretation is involved, SpectraST 5.0 is better. But I don’t know relative to what you’re doing exactly when peak re-interpretation happens.
Can you just rebuild all your libraries with SpectraST 5.0? That would be safest. Regards, Eric *From:* [email protected] <[email protected]> *On Behalf Of *Kristina Plate *Sent:* Monday, September 23, 2019 4:22 AM *To:* spctools-discuss <[email protected]> *Subject:* Re: [spctools-discuss] Combination of consensus libraries and import in MRM format via spectraST 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:* [email protected] <javascript:> < [email protected] <javascript:>> *On Behalf Of *Kristina Plate *Sent:* Friday, September 20, 2019 6:22 AM *To:* spctools-discuss <[email protected] <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 [email protected] <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. To unsubscribe from this group and stop receiving emails from it, send an email to [email protected]. To view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/d206982e-1892-494c-8762-0c519ae3b10c%40googlegroups.com <https://groups.google.com/d/msgid/spctools-discuss/d206982e-1892-494c-8762-0c519ae3b10c%40googlegroups.com?utm_medium=email&utm_source=footer> . -- 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 [email protected]. To view this discussion on the web visit https://groups.google.com/d/msgid/spctools-discuss/749268edd4136e4cc306381eecf6b5d4%40mail.gmail.com.
