Point mutations?

support
Point mutations? mnt  2021-07-28
 
Hi All

I'm curently working on a project where we want to use Skyline for some validation/relative quantification. I have used X!Tandem in The GPM to search the datafiles and am now attempting to import these into Skyline. However, as part of my search with X!Tandem, i deliberately engaged the "point mutation" option, and now skyline cannot import the data. The generated Error is attached below. So the question is if it is possible or not to to have this option engaged at all? Personally i would hope so since it seems to provide some quite interesting results, however at this point it seems as it is not.

Any suggestions?

ERROR:
---------------------------
Skyline
---------------------------
ERROR: GPM22200000640.xtan.xml(line 12641): Specified modification does not match sequence. Given a modified M at position 0 which is a S in SVQWCAVSQPEATK.
ERROR:
ERROR: GPM22200000641.xtan.xml(line 3397): Specified modification does not match sequence. Given a modified W at position 1 which is a M in VMPVLKSPTIPFFDPQIPK.
ERROR:
ERROR: GPM22200000643.xtan.xml(line 1085): Specified modification does not match sequence. Given a modified C at position 13 which is a Q in VKHEDQQQGEDEHQDK.
ERROR:
ERROR: GPM22200000644.xtan.xml(line 1067): Specified modification does not match sequence. Given a modified C at position 13 which is a Q in VKHEDQQQGEDEHQDK.
ERROR:
ERROR: GPM22200000645.xtan.xml(line 6883): Specified modification does not match sequence. Given a modified W at position 1 which is a M in VMPVLKSPTIPFFDPQIPK.
ERROR:

Command-line:
xxx
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: GPM22200000640.xtan.xml(line 12641): Specified modification does not match sequence. Given a modified M at position 0 which is a S in SVQWCAVSQPEATK.
ERROR:
ERROR: GPM22200000641.xtan.xml(line 3397): Specified modification does not match sequence. Given a modified W at position 1 which is a M in VMPVLKSPTIPFFDPQIPK.
ERROR:
ERROR: GPM22200000643.xtan.xml(line 1085): Specified modification does not match sequence. Given a modified C at position 13 which is a Q in VKHEDQQQGEDEHQDK.
ERROR:
ERROR: GPM22200000644.xtan.xml(line 1067): Specified modification does not match sequence. Given a modified C at position 13 which is a Q in VKHEDQQQGEDEHQDK.
ERROR:
ERROR: GPM22200000645.xtan.xml(line 6883): Specified modification does not match sequence. Given a modified W at position 1 which is a M in VMPVLKSPTIPFFDPQIPK.
ERROR:

Command-line:
xxx
   ved pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) i C:\proj\skyline_21_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:linje 65
   ved pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) i C:\proj\skyline_21_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:linje 201
   ved pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) i C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:linje 157
---------------------------
 
 
Brendan MacLean responded:  2021-07-28
Hi Martin,
We would definitely need an example file to work on this for you. Presumably, there is something in the XML that specifies the mutations, and we are somehow reading the original peptide sequence, when we would need to use the peptide sequence resulting from the point mutation, since that is essentially what X! Tandem is saying it believes you should be looking for.

At the very least, we should be able to get those mutated sequences into a spectral library. It would be more work to encode them in such a way that we can maintain an association with the protein(s) they might originate from. Simple protein sequence matching with the mutated peptide will presumably fail. So, really you would want protein association based on the original sequence and targeting in the mass spec data based on the mutated sequence.

Anyway, it seems you are correct that we do not yet support this. If you give us an example file, we can probably support building a spectral library without errors, but the protein association issue may need to wait a bit longer.

Thanks for reporting the issue.

--Brendan
 
mnt responded:  2021-07-28
Hi Brendan

I cannot giv you the raw data as of now, however, i can do the next best thing.

Below you will find an example data of the 3 different examples. Let me know if there is anything else you need.

GPM22200000640.xtan.xml : SVQWCAVSQPEATK
<group id="10959" mh="1652.744433" z="2" rt="1446.354" expect="1.0e-15" label="sp|P02788|TRFL_HUMAN Lactotransferrin OS=Homo sapiens GN=LTF PE=1 SV=6" type="model" sumI="4.12" maxI="1082" fI="10.82" act="0" >
<protein expect="-807.2" id="10959.1" uid="62" label="sp|P02788|TRFL_HUMAN Lactotransferrin OS=Homo sapiens GN=LTF PE=1 SV=6" sumI="7.13" >
<note label="description">sp|P02788|TRFL_HUMAN Lactotransferrin OS=Homo sapiens GN=LTF PE=1 SV=6</note>
<file type="peptide" URL="../fasta/HumanMilkProteinDatabase.fasta"/>
<peptide start="1" end="710">
    MKLVFLVLLF LGALGLCLAG RRRSVQWCAV SQPEATKCFQ WQRNMRKVRG
    PPVSCIKRDS PIQCIQAIAE NRADAVTLDG GFIYEAGLAP YKLRPVAAEV
    YGTERQPRTH YYAVAVVKKG GSFQLNELQG LKSCHTGLRR TAGWNVPIGT
    LRPFLNWTGP PEPIEAAVAR FFSASCVPGA DKGQFPNLCR LCAGTGENKC
    AFSSQEPYFS YSGAFKCLRD GAGDVAFIRE STVFEDLSDE AERDEYELLC
    PDNTRKPVDK FKDCHLARVP SHAVVARSVN GKEDAIWNLL RQAQEKFGKD
    KSPKFQLFGS PSGQKDLLFK DSAIGFSRVP PRIDSGLYLG SGYFTAIQNL
    RKSEEEVAAR RARVVWCAVG EQELRKCNQW SGLSEGSVTC SSASTTEDCI
    ALVLKGEADA MSLDGGYVYT AGKCGLVPVL AENYKSQQSS DPDPNCVDRP
    VEGYLAVAVV RRSDTSLTWN SVKGKKSCHT AVDRTAGWNI PMGLLFNQTG
    SCKFDEYFSQ SCAPGSDPRS NLCALCIGDE QGENKCVPNS NERYYGYTGA
    FRCLAENAGD VAFVKDVTVL QNTDGNNNEA WAKDLKLADF ALLCLDGKRK
    PVTEARSCHL AMAPNHAVVS RMDKVERLKQ VLLHQQAKFG RNGSDCPDKF
    CLFQSETKNL LFNDNTECLA RLHGKTTYEK YLGPQYVAGI TNLKKCSTSP
    LLEACEFLRK
<domain id="10959.1.1" start="24" end="37" expect="1.0e-15" mh="1651.745" delta="0.999" hyperscore="23.8" nextscore="23.7" y_score="12.4" y_ions="6" b_score="0.0" b_ions="0" pre="GRRR" post="CFQW" seq="SVQWCAVSQPEATK" missed_cleavages="0">
<aa type="C" at="28" modified="58.00548" />
<aa type="M" at="24" modified="15.99492" />
<aa type="S" at="24" modified="44.00846" pm="M" />
</domain>
</peptide>
</protein>
<group label="supporting data" type="support">
<GAML:trace label="10959.hyper" type="hyperscore expectation function">
<GAML:attribute type="a0">19.9712</GAML:attribute>
<GAML:attribute type="a1">-1.5977</GAML:attribute>
<GAML:Xdata label="10959.hyper" units="score">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="26">
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
</GAML:values>
</GAML:Xdata>
<GAML:Ydata label="10959.hyper" units="counts">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="26">
904 904 904 897 854 833 636 576 198 5 2 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0
</GAML:values>
</GAML:Ydata>
</GAML:trace>
<GAML:trace label="10959.convolute" type="convolution survival function">
<GAML:Xdata label="10959.convolute" units="score">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="15">
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
</GAML:values>
</GAML:Xdata>
<GAML:Ydata label="10959.convolute" units="counts">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="15">
711 711 711 704 661 640 443 379 2 1 1 0 0 1 0
</GAML:values>
</GAML:Ydata>
</GAML:trace>
<GAML:trace label="10959.b" type="b ion histogram">
<GAML:Xdata label="10959.b" units="number of ions">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="4">
0 1 2 3
</GAML:values>
</GAML:Xdata>
<GAML:Ydata label="10959.b" units="counts">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="4">
672 259 5 0
</GAML:values>
</GAML:Ydata>
</GAML:trace>
<GAML:trace label="10959.y" type="y ion histogram">
<GAML:Xdata label="10959.y" units="number of ions">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="8">
0 1 2 3 4 5 6 7
</GAML:values>
</GAML:Xdata>
<GAML:Ydata label="10959.y" units="counts">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="8">
262 640 24 3 0 4 3 0
</GAML:values>
</GAML:Ydata>
</GAML:trace>

</group>

GPM22200000641.xtan.xml : VMPVLKSPTIPFFDPQIPK
<group id="31268" mh="3398.899999" z="3" rt="3570.931" expect="1.2e-03" label="sp|P05814|CASB_HUMAN Beta-casein OS=Homo sapiens GN=CSN2 PE=1 SV=4" type="model" sumI="4.65" maxI="1336" fI="13.36" act="0" >
<protein expect="-1038.1" id="31268.1" uid="60" label="sp|P05814|CASB_HUMAN Beta-casein OS=Homo sapiens GN=CSN2 PE=1 SV=4" sumI="7.65" >
<note label="description">sp|P05814|CASB_HUMAN Beta-casein OS=Homo sapiens GN=CSN2 PE=1 SV=4</note>
<file type="peptide" URL="../fasta/HumanMilkProteinDatabase.fasta"/>
<peptide start="1" end="226">
    MKVLILACLV ALALARETIE SLSSSEESIT EYKQKVEKVK HEDQQQGEDE
    HQDKIYPSFQ PQPLIYPFVE PIPYGFLPQN ILPLAQPAVV LPVPQPEIME
    VPKAKDTVYT KGRVMPVLKS PTIPFFDPQI PKLTDLENLH LPLPLLQPLM
    QQVPQPIPQT LALPPQPLWS VPQPKVLPIP QQVVPYPQRA VPVQALLLNQ
    ELLLNPTHQI YPVTQPLAPV HNPISV
<domain id="31268.1.1" start="106" end="132" expect="1.2e-03" mh="3398.961" delta="-0.061" hyperscore="19.0" nextscore="19.0" y_score="13.5" y_ions="4" b_score="6.1" b_ions="1" pre="PKAK" post="LTDL" seq="DTVYTKGRVMPVLKSPTIPFFDPQIPK" missed_cleavages="3">
<aa type="K" at="119" modified="324.28650" />
</domain>
</peptide>
</protein>
<group label="supporting data" type="support">
<GAML:trace label="31268.hyper" type="hyperscore expectation function">
<GAML:attribute type="a0">5.60488</GAML:attribute>
<GAML:attribute type="a1">-0.44839</GAML:attribute>
<GAML:Xdata label="31268.hyper" units="score">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="21">
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
</GAML:values>
</GAML:Xdata>
<GAML:Ydata label="31268.hyper" units="counts">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="21">
601 601 601 601 591 488 384 205 113 26 2 1 1 0 0 0 0 0 0 1 0
</GAML:values>
</GAML:Ydata>
</GAML:trace>
<GAML:trace label="31268.convolute" type="convolution survival function">
<GAML:Xdata label="31268.convolute" units="score">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="15">
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
</GAML:values>
</GAML:Xdata>
<GAML:Ydata label="31268.convolute" units="counts">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="15">
607 607 607 607 597 494 390 210 109 30 3 2 2 1 0
</GAML:values>
</GAML:Ydata>
</GAML:trace>
<GAML:trace label="31268.b" type="b ion histogram">
<GAML:Xdata label="31268.b" units="number of ions">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="5">
0 1 2 3 4
</GAML:values>
</GAML:Xdata>
<GAML:Ydata label="31268.b" units="counts">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="5">
284 331 19 3 0
</GAML:values>
</GAML:Ydata>
</GAML:trace>
<GAML:trace label="31268.y" type="y ion histogram">
<GAML:Xdata label="31268.y" units="number of ions">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="6">
0 1 2 3 4 5
</GAML:values>
</GAML:Xdata>
<GAML:Ydata label="31268.y" units="counts">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="6">
304 300 29 0 4 0
</GAML:values>
</GAML:Ydata>
</GAML:trace>

</group>

GPM22200000643.xtan.xml : VKHEDQQQGEDEHQDK
<group id="921" mh="2307.069921" z="3" rt="119.835" expect="1.3e-06" label="sp|P05814|CASB_HUMAN Beta-casein OS=Homo sapiens GN=CSN2 PE=1 SV=4" type="model" sumI="4.91" maxI="6170" fI="61.7" act="0" >
<protein expect="-1405.4" id="921.1" uid="60" label="sp|P05814|CASB_HUMAN Beta-casein OS=Homo sapiens GN=CSN2 PE=1 SV=4" sumI="7.96" >
<note label="description">sp|P05814|CASB_HUMAN Beta-casein OS=Homo sapiens GN=CSN2 PE=1 SV=4</note>
<file type="peptide" URL="../fasta/HumanMilkProteinDatabase.fasta"/>
<peptide start="1" end="226">
    MKVLILACLV ALALARETIE SLSSSEESIT EYKQKVEKVK HEDQQQGEDE
    HQDKIYPSFQ PQPLIYPFVE PIPYGFLPQN ILPLAQPAVV LPVPQPEIME
    VPKAKDTVYT KGRVMPVLKS PTIPFFDPQI PKLTDLENLH LPLPLLQPLM
    QQVPQPIPQT LALPPQPLWS VPQPKVLPIP QQVVPYPQRA VPVQALLLNQ
    ELLLNPTHQI YPVTQPLAPV HNPISV
<domain id="921.1.1" start="39" end="54" expect="1.3e-06" mh="2307.101" delta="-0.031" hyperscore="30.0" nextscore="14.6" y_score="0.0" y_ions="0" b_score="9.7" b_ions="11" pre="KVEK" post="IYPS" seq="VKHEDQQQGEDEHQDK" missed_cleavages="1">
<aa type="C" at="52" modified="58.00548" />
<aa type="K" at="54" modified="324.28650" />
<aa type="Q" at="52" modified="-25.04939" pm="C" />
</domain>
</peptide>
</protein>
<group label="supporting data" type="support">
<GAML:trace label="921.hyper" type="hyperscore expectation function">
<GAML:attribute type="a0">6.26765</GAML:attribute>
<GAML:attribute type="a1">-0.404365</GAML:attribute>
<GAML:Xdata label="921.hyper" units="score">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="63">
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
60 61 62
</GAML:values>
</GAML:Xdata>
<GAML:Ydata label="921.hyper" units="counts">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="63">
16698 16698 16230 14588 11119 8643 4563 2722 1144 612 289 109 5 2 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 1 0
</GAML:values>
</GAML:Ydata>
</GAML:trace>
<GAML:trace label="921.convolute" type="convolution survival function">
<GAML:Xdata label="921.convolute" units="score">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="15">
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
</GAML:values>
</GAML:Xdata>
<GAML:Ydata label="921.convolute" units="counts">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="15">
16715 16715 16247 14603 11085 8490 4369 2520 895 444 249 96 3 1 0
</GAML:values>
</GAML:Ydata>
</GAML:trace>
<GAML:trace label="921.b" type="b ion histogram">
<GAML:Xdata label="921.b" units="number of ions">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="8">
0 1 2 3 4 5 6 7
</GAML:values>
</GAML:Xdata>
<GAML:Ydata label="921.b" units="counts">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="8">
6467 9002 1108 125 12 1 0 3
</GAML:values>
</GAML:Ydata>
</GAML:trace>
<GAML:trace label="921.y" type="y ion histogram">
<GAML:Xdata label="921.y" units="number of ions">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="8">
0 1 2 3 4 5 6 7
</GAML:values>
</GAML:Xdata>
<GAML:Ydata label="921.y" units="counts">
<GAML:values byteorder="INTEL" format="ASCII" numvalues="8">
8459 7115 1070 69 1 0 2 2
</GAML:values>
</GAML:Ydata>
</GAML:trace>

</group>
 
Brendan MacLean responded:  2021-07-28
Thanks. I think this is the critical line for our spectral library to understand:

<aa type="S" at="24" modified="44.00846" pm="M" />

Once it is understood correctly, you should get a valid spectral library without errors. I have posted the following issue to Kaipo who may be able to implement a simple fix relatively quickly.

https://skyline.ms/issues/home/issues/details.view?issueId=827
 
mnt responded:  2021-07-28
Thanks a lot.

The way i see it there is two things going on;
1) A point mutation from S to M.
2) The M is oxidized, thus an additional 16Da is added to the M.

The combination of this, seems to be the issue.

Also, as a followup question.

When/If I am to then "allign/assign" the peptides to a protein using the same fasta file as used in the orriginal search, would this particular peptide be able to match or not?

I'm posting here since I cannot respond to the issue reported to Kaipo.
 
Brendan MacLean responded:  2021-07-29
A change to support the point mutation has been added to a pull request on GitHub.

You are right that the error message you have reported is caused by the combination of those two things, but cases where the point mutation is not causing an error are still wrong in the current Skyline implementation. They are still incorrectly assigning a confident identification to the original peptide sequence for the spectrum which actually identified the point mutated peptide. Imagine there had not been any oxidation on the M. You would not have gotten an error, but Skyline would have matched the spectrum to the original S-containing peptide, which is not what X! Tandem intended.

Until this fix is released in a Skyline-daily release, I would not recommend building libraries with searches that allowed point mutations.

Thanks for reporting this and supplying your example.

--Brendan
 
mnt responded:  2021-07-29
Hi Brendan

Thanks a lot for the explanation, makes sense. :)

Just out of curiosity, do you have any idea about when this "Skyline-daily release" with this fix is released? Sorry for being pushy, i just have some very exiting data that i want to explore further...

Cheers
Martin
 
Kaipo responded:  2021-07-29
Hi Martin,
I don't have an answer about the new release, but here is a version with the fix (actually it would be good if you could test this for me):
https://teamcity.labkey.org/viewLog.html?buildId=1493043&buildTypeId=bt209&tab=artifacts

See this page about running the test build: https://skyline.ms/wiki/home/software/Skyline/page.view?name=Running%20Skyline%20TeamCity%20Artifact%20Builds

Thanks,
Kaipo
 
mnt responded:  2021-08-01
Hi Kaipo

I would love to do this, however I don't know how to. I cannot find any pull-request at github.. Maybe it's not there yet, or i got no clue how to do it. I guess the latter are the more likely option.

I hope you can provide more details on how to.

Cheers
Martin
 
Kaipo responded:  2021-08-02
Hi Martin,

Here is the direct link to the download: https://teamcity.labkey.org/repository/download/bt209/1493043:id/SkylineTester.zip
Once you extract the contents of that zip file, run the extracted file "SkylineTester Files\Skyline-daily.exe". That version of Skyline should support building a library containing the point mutations.

Thanks,
Kaipo
 
mnt responded:  2021-08-03
Hi Kaipo

Thanks a lot for the help. However, the link you provided doesn't work. I get an error 404.

Anyhow, when browsing through the overview for #9644, it seems as all tests are passed? If so, when is it released? :D

Cheers
Martin
 
Kaipo responded:  2021-08-04
Hi Martin,

Sorry, not sure why the link didn't work. I uploaded it here instead: https://proteome.gs.washington.edu/~kaipot/share/pointmutations.zip

Thanks,
Kaipo
 
mnt responded:  2021-08-05
Hi Kaipo

I just managed to test it, I've set it up with the needed PTMs but it is unable to build the library. The type of errors are the same as above, now I'm using some other files so it is not the exact same error. I have attached the errors here.

---------------------------
Skyline-daily
---------------------------
ERROR: GPM22200000680.xtan.xml(line 5477): Specified modification does not match sequence. Given a modified M at position 2 which is a T in SPTIPFFDPQIPK.
ERROR:
ERROR: GPM22200000681.xtan.xml(line 22698): Specified modification does not match sequence. Given a modified C at position 16 which is a P in AVPVQALLLNQELLLNPTHQIYPVTQPLAPVHNPISV.
ERROR:
ERROR: GPM22200000682.xtan.xml(line 700): Specified modification does not match sequence. Given a modified A at position 0 which is a E in ETIESLSSSEESITEYK.
ERROR:
ERROR: GPM22200000683.xtan.xml(line 2131): Specified modification does not match sequence. Given a modified C at position 9 which is a F in ADAVTLDGGFIYEAGLAPYK.
ERROR:
ERROR: GPM22200000684.xtan.xml(line 3642): Specified modification does not match sequence. Given a modified W at position 3 which is a M in GRVMPVLKSPTIPFFDPQIPK.
ERROR:
ERROR: GPM22200000685.xtan.xml(line 1268): Specified modification does not match sequence. Given a modified M at position 0 which is a T in THYYAVAVVKKGGSFQLNELQGLK.
ERROR:
ERROR: GPM22200000686.xtan.xml(line 54562): Specified modification does not match sequence. Given a modified C at position 6 which is a N in YPERLQNPSESSEPIPLESR.
ERROR:
ERROR: GPM22200000687.xtan.xml(line 8809): Specified modification does not match sequence. Given a modified W at position 12 which is a T in GQFPNLCRLCAGTGENKCAFSSQEPYFSYSGAFK.
ERROR:
ERROR: GPM22200000688.xtan.xml(line 6053): Specified modification does not match sequence. Given a modified T at position 1 which is a M in VMPVLKSPTIPFFDPQIPK.
ERROR:
ERROR: GPM22200000689.xtan.xml(line 1593): Specified modification does not match sequence. Given a modified K at position 19 which is a T in RDSPIQCIQAIAENRADAVTLDGGFIYEAGLAPYK.
ERROR:
ERROR: GPM22200000690.xtan.xml(line 10700): Specified modification does not match sequence. Given a modified Y at position 3 which is a M in GRVMPVLKSPTIPFFDPQIPK.
ERROR:
ERROR: GPM22200000691.xtan.xml(line 8536): Specified modification does not match sequence. Given a modified C at position 11 which is a G in THYYAVAVVKKGGSFQLNELQGLK.
ERROR:

Command-line:
XXX
---------------------------
OK More Info
---------------------------
System.IO.IOException: ERROR: GPM22200000680.xtan.xml(line 5477): Specified modification does not match sequence. Given a modified M at position 2 which is a T in SPTIPFFDPQIPK.
ERROR:
ERROR: GPM22200000681.xtan.xml(line 22698): Specified modification does not match sequence. Given a modified C at position 16 which is a P in AVPVQALLLNQELLLNPTHQIYPVTQPLAPVHNPISV.
ERROR:
ERROR: GPM22200000682.xtan.xml(line 700): Specified modification does not match sequence. Given a modified A at position 0 which is a E in ETIESLSSSEESITEYK.
ERROR:
ERROR: GPM22200000683.xtan.xml(line 2131): Specified modification does not match sequence. Given a modified C at position 9 which is a F in ADAVTLDGGFIYEAGLAPYK.
ERROR:
ERROR: GPM22200000684.xtan.xml(line 3642): Specified modification does not match sequence. Given a modified W at position 3 which is a M in GRVMPVLKSPTIPFFDPQIPK.
ERROR:
ERROR: GPM22200000685.xtan.xml(line 1268): Specified modification does not match sequence. Given a modified M at position 0 which is a T in THYYAVAVVKKGGSFQLNELQGLK.
ERROR:
ERROR: GPM22200000686.xtan.xml(line 54562): Specified modification does not match sequence. Given a modified C at position 6 which is a N in YPERLQNPSESSEPIPLESR.
ERROR:
ERROR: GPM22200000687.xtan.xml(line 8809): Specified modification does not match sequence. Given a modified W at position 12 which is a T in GQFPNLCRLCAGTGENKCAFSSQEPYFSYSGAFK.
ERROR:
ERROR: GPM22200000688.xtan.xml(line 6053): Specified modification does not match sequence. Given a modified T at position 1 which is a M in VMPVLKSPTIPFFDPQIPK.
ERROR:
ERROR: GPM22200000689.xtan.xml(line 1593): Specified modification does not match sequence. Given a modified K at position 19 which is a T in RDSPIQCIQAIAENRADAVTLDGGFIYEAGLAPYK.
ERROR:
ERROR: GPM22200000690.xtan.xml(line 10700): Specified modification does not match sequence. Given a modified Y at position 3 which is a M in GRVMPVLKSPTIPFFDPQIPK.
ERROR:
ERROR: GPM22200000691.xtan.xml(line 8536): Specified modification does not match sequence. Given a modified C at position 11 which is a G in THYYAVAVVKKGGSFQLNELQGLK.
ERROR:

Command-line:
XXX
   ved pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) i Z:\pwiz\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:linje 65
   ved pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) i Z:\pwiz\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:linje 201
   ved pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) i Z:\pwiz\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:linje 157
---------------------------
When looking at the error, there is one thing that seems diferent, here it refferes to a Z drive. I don't have a Z drive, i guess this is a remnent of something on your end? But could this be what is causeing the issue?

Cheers
Martin
 
Kaipo responded:  2021-08-05
Hi Martin,
It's difficult for me to diagnose or fix the problem without an example file. Are you able to share it with me privately or generate a small one that can be shared?

Thanks,
Kaipo
 
mnt responded:  2021-08-20
Hi Kaipo

First of all, i'm very sorry about the delay. I got way too much on my plate and i now have to spend my vacation on catching up... Well, i guess we all have something to do. :)

Unfortunately i cannot share the file with you. The best i can do is to provide some examples. Hare you have 3 examples. Let me know if you need more. Do note though, that the lines with "***" in the end is the line number which is mentioned above with the "complaint", this is counted in notepad, so i'm not sure it skyline line counts is zero or one based. Please let me knwo if there is anything else i can do.

GPM22200000680.xtan.xml
<peptide start="1" end="226">
    MKVLILACLV ALALARETIE SLSSSEESIT EYKQKVEKVK HEDQQQGEDE
    HQDKIYPSFQ PQPLIYPFVE PIPYGFLPQN ILPLAQPAVV LPVPQPEIME
    VPKAKDTVYT KGRVMPVLKS PTIPFFDPQI PKLTDLENLH LPLPLLQPLM
    QQVPQPIPQT LALPPQPLWS VPQPKVLPIP QQVVPYPQRA VPVQALLLNQ
    ELLLNPTHQI YPVTQPLAPV HNPISV
<domain id="27905.1.1" start="120" end="132" expect="1.4e-04" mh="1514.7712" delta="-0.0063" hyperscore="36.4" nextscore="36.2" y_score="14.2" y_ions="9" b_score="0.0" b_ions="0" pre="PVLK" post="LTDL" seq="SPTIPFFDPQIPK" missed_cleavages="0">
<aa type="M" at="122" modified="15.99492" />    ***
<aa type="S" at="120" modified="-18.01056" />
<aa type="T" at="122" modified="29.99281" pm="M" />
</domain>
</peptide>

GPM22200000681.xtan.xml
<peptide start="1" end="226">
    MKVLILACLV ALALARETIE SLSSSEESIT EYKQKVEKVK HEDQQQGEDE
    HQDKIYPSFQ PQPLIYPFVE PIPYGFLPQN ILPLAQPAVV LPVPQPEIME
    VPKAKDTVYT KGRVMPVLKS PTIPFFDPQI PKLTDLENLH LPLPLLQPLM
    QQVPQPIPQT LALPPQPLWS VPQPKVLPIP QQVVPYPQRA VPVQALLLNQ
    ELLLNPTHQI YPVTQPLAPV HNPISV
<domain id="43289.1.1" start="190" end="226" expect="2.5e-05" mh="4181.187" delta="2.012" hyperscore="43.2" nextscore="43.0" y_score="11.3" y_ions="5" b_score="11.5" b_ions="10" pre="YPQR" post="]" seq="AVPVQALLLNQELLLNPTHQIYPVTQPLAPVHNPISV" missed_cleavages="0">
<aa type="C" at="206" modified="58.00548" />    ***
<aa type="Y" at="211" modified="79.96633" />
<aa type="P" at="206" modified="5.95642" pm="C" />
</domain>
</peptide>

GPM22200000685.xtan.xml
<peptide start="1" end="710">
    MKLVFLVLLF LGALGLCLAG RRRSVQWCAV SQPEATKCFQ WQRNMRKVRG
    PPVSCIKRDS PIQCIQAIAE NRADAVTLDG GFIYEAGLAP YKLRPVAAEV
    YGTERQPRTH YYAVAVVKKG GSFQLNELQG LKSCHTGLRR TAGWNVPIGT
    LRPFLNWTGP PEPIEAAVAR FFSASCVPGA DKGQFPNLCR LCAGTGENKC
    AFSSQEPYFS YSGAFKCLRD GAGDVAFIRE STVFEDLSDE AERDEYELLC
    PDNTRKPVDK FKDCHLARVP SHAVVARSVN GKEDAIWNLL RQAQEKFGKD
    KSPKFQLFGS PSGQKDLLFK DSAIGFSRVP PRIDSGLYLG SGYFTAIQNL
    RKSEEEVAAR RARVVWCAVG EQELRKCNQW SGLSEGSVTC SSASTTEDCI
    ALVLKGEADA MSLDGGYVYT AGKCGLVPVL AENYKSQQSS DPDPNCVDRP
    VEGYLAVAVV RRSDTSLTWN SVKGKKSCHT AVDRTAGWNI PMGLLFNQTG
    SCKFDEYFSQ SCAPGSDPRS NLCALCIGDE QGENKCVPNS NERYYGYTGA
    FRCLAENAGD VAFVKDVTVL QNTDGNNNEA WAKDLKLADF ALLCLDGKRK
    PVTEARSCHL AMAPNHAVVS RMDKVERLKQ VLLHQQAKFG RNGSDCPDKF
    CLFQSETKNL LFNDNTECLA RLHGKTTYEK YLGPQYVAGI TNLKKCSTSP
    LLEACEFLRK
<domain id="24656.1.1" start="109" end="132" expect="6.6e-05" mh="2856.355" delta="2.027" hyperscore="27.7" nextscore="27.5" y_score="11.6" y_ions="7" b_score="6.5" b_ions="2" pre="RQPR" post="SCHT" seq="THYYAVAVVKKGGSFQLNELQGLK" missed_cleavages="2">
<aa type="M" at="109" modified="15.99492" />    ***
<aa type="Y" at="111" modified="79.96633" />
<aa type="Y" at="112" modified="79.96633" />
<aa type="T" at="109" modified="29.99281" pm="M" />
</domain>
</peptide>

Cheers
Martin
 
Kaipo responded:  2021-08-23
Hi Martin,
I've hacked together a small file (attached) with these 3 examples and it works for me with the test build I linked earlier. Can you try building a library with the attached file and see if it works for you?

Thanks,
Kaipo