Hi Ankit, before repeating the experiment, I would suggest that you look
through the pepXML file to see if you can find those iRT peptides. Perhaps
they are there and identified, but below your probability threshold? Or
consider if there may be a mis-setting of search parameters that is
preventing them from being identified? (for example, perhaps tolerance
settings that are too small and you’re missing identifications, or ???) In
other words, before rerunning your experiment, it would be good to
understand the root cause why the iRT peptides are not coming through. I
might have nothing to do with the running of the experiment, but rather the
analysis? It would be cheaper to check that first before rereunning.
Regards,
Eric
*From:* spctools-discuss@googlegroups.com <spctools-discuss@googlegroups.com>
*On Behalf Of *Ankit Balhara
*Sent:* Tuesday, January 29, 2019 9:34 AM
*To:* spctools-discuss <spctools-discuss@googlegroups.com>
*Subject:* Re: [spctools-discuss] Problem during spectrast library
generation
Hi Eric,
Thanks for helping. Actually I used 11 iRT peptides (all are present in
iRT.txt file), but unfortunately only two are present in the library. So do
I need to repeat the experiment or there is another way out?
Regards
Ankit
On Tuesday, 29 January 2019 22:10:38 UTC+5:30, Eric Deutsch wrote:
Hi Ankit, I suppose the first question to ask is: how many iRT peptides are
in your iRT.txt file and how many of those have a corresponding entry in
the library?
Regards,
Eric
*From:* spctools...@googlegroups.com <javascript:> <
spctools...@googlegroups.com <javascript:>> *On Behalf Of *Ankit Balhara
*Sent:* Tuesday, January 29, 2019 1:22 AM
*To:* spctools-discuss <spctools...@googlegroups.com <javascript:>>
*Subject:* [spctools-discuss] Problem during spectrast library generation
Hi,
I am trying to build the spectral library using the SpectraST. I have
analyzed the DDA data upto the Mayu FDR determination step, also got the
IPP/PP values corresponding to 1%FDR. After that I am using following
command, but got the error that "Too few landmarks with distinct iRTs to
perform RT normalization". Could anybody please help me out?
c:\TPP\bin>spectrast -cNSpecLib -cICID-QTOF -cf "Protein! ~ 'rev_'"
-cP0.891383 -c_IRTc:\TPP\data\iRT.txt -c_IRR
c:\TPP\data\params\interact.ipro.pep.xml
SpectraST started at Tue Jan 29 14:38:13 2019.
Processing
"c:\TPP\data\params\interact.ipro.pep.xml"...500...1000...1500...DONE!
Importing all spectra with P>=0.891383 ...INFO: Reading file:
c:/TPP/data/HLM_27_DDA.mzXML
10%...20%...30%...40%...50%...60%...70%...80%...90%...DONE!
Library file (BINARY) "SpecLib.splib" created.
Library file (TEXT) "SpecLib.sptxt" created.
M/Z Index file "SpecLib.spidx" created.
Peptide Index file "SpecLib.pepidx" created.
Total number of spectra in library: 1661
Total number of distinct peptide ions in library: 1560
Total number of distinct stripped peptides in library: 1429
CHARGE +1: 0 ; +2: 1187 ; +3: 450 ; +4: 24 ; +5: 0 ; >+5: 0 ;
Unk: 0
TERMINI Tryptic: 1654 ; Semi-tryptic: 7 ; Non-tryptic: 0
PROBABILITY >0.9999: 1347 ; 0.999-0.9999: 147 ; 0.99-0.999: 89 ;
0.9-0.99: 76 ; <0.9: 2
NREPS 20+: 0 ; 10-19: 0 ; 4-9: 0 ; 2-3: 0 ; 1: 1661
MODIFICATIONS C,Carbamidomethyl: 288 ; C,Pyro-carbamidomethyl: 2
M,Oxidation: 12
Q,Gln->pyro-Glu: 4
n,Acetyl: 5
Total Run Time = 24 seconds.
SpectraST finished at Tue Jan 29 14:38:37 2019 with 1 error(s):
PEPXML IMPORT: Too few landmarks with distinct iRTs to perform RT
normalization.
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