*to complete the picture, here the proteinprophet analysis results*
*
*
*ProteinProphet® analysis results*
Version: Insilicos_LabKey_C++ (TPP v4.5 RAPTURE rev 2, Build 201202031108
(MinGW))
Analysis Date: 2012-07-04T12:21:56
Source Files: c:/Inetpub/wwwroot/ISB/data/120629YEAST2D/interact.pep.xml
Number of input spectra with minimum probability 0.05: 0 1+, 0 2+, and 1324 3+
Reference Database: c:/Inetpub/wwwroot/ISB/data/dbase/Yeast/YEAST.fasta
Residue substitutions: I -> L
Run Options:
Occam's Razor used: Y
Protein Groups: Y
Peptide degeneracies: Y
Number of Sibling Peptides used: Y
Min peptide probability: 0.20
Min peptide weight: 0.50
Original results written to file:
c:/Inetpub/wwwroot/ISB/data/120629YEAST2D/interact.prot.xml
*Analysis Iterations* *EM step**number of iterations*Initial Peptide
Weights2NSP Distributions2Final Peptide Weights1 *Learned Number of Sibling
Peptide Distributions* Neighboring bin smoothing: Y *bin number**nsp
range**positive
freq**negative freq**positive/negative ratio*00.00 <= nsp < 0.100.2080.535
0.3910.10 <= nsp < 0.250.0650.1420.4620.25 <= nsp < 0.500.0580.0421.3830.50
<= nsp < 1.000.1240.0741.6741.00 <= nsp < 2.000.1250.0631.9752.00 <= nsp <
5.000.1660.0592.8265.00 <= nsp < 15.000.2000.0663.05715.00 <= nsp < inf0.054
0.0192.81 (3.05)
*Total Number of Contributing Spectrum IDs (100% share): *660.1
On Wednesday, 4 July 2012 12:32:49 UTC-3, Alex wrote:
>
> Another update to this situation!
>
> I think the problem might actually be in PeptideProphet. I run the same
> dataset using X!Tandem on the GPM.org website, and I do identify doubly
> charged precursor peptide ions. The same dataset only shows triple charged
> precursors, as observed in the pep.shtml file. I select the column: z
> (assumed charge), and ONLY triple charged species appear.
> Here is another twist: When I go to Peptide Prophet Analysis results I
> only see models for 3+, and *No models or data found for charge +2.*
>
> However if I hit on show ALL models, I get the following (which I'm not an
> expert to understand, so hopefully someone can fill me on this information):
>
> FINAL 2+ MODEL after 24 iterations:
> number of spectra: 53628
> using 3+ positive distributions to identify candidates ('-2') above
> background ('0')
> prior: 0.000, total: 0.0
> X! Tandem (k-score) discrim score [fval] negmean: -1.33
> pos: (gaussian mean 1.55, stdev 2.01)
> neg: (evd mean -1.31, stdev 1.14, mu -1.82, beta 0.89)
> no. tolerable trypsin term. [ntt]
> pos: (ntt=0 0.000, ntt=1 0.000, ntt=2 1.000)
> neg: (ntt=0 0.000, ntt=1 0.000, ntt=2 1.000)
> no. missed enz. cleavages [nmc]
> pos: (nmc=0 0.878, 1<=nmc<=2 0.122, nmc>=3 0.000)
> neg: (nmc=0 0.409, 1<=nmc<=2 0.591, nmc>=3 0.000)
> accurate mass diff [massd] (offset: -0.500000)
> pos: (massd=-5.000000 0.000000000, massd=-4.000000 0.000000000,
> massd=-3.000000 0.000000000, massd=-2.000000 0.000154395, massd=-1.000000
> 0.029449633, massd=0.000000 0.390222698, massd=1.000000 0.422314370,
> massd=2.000000 0.128208899, massd=3.000000 0.017069506, massd=4.000000
> 0.012580499, massd=5.000000 0.000000000)
> neg: (massd=-5.000000 0.000000000, massd=-4.000000 0.000000000,
> massd=-3.000000 0.000000000, massd=-2.000000 0.000173670, massd=-1.000000
> 0.179740500, massd=0.000000 0.175030116, massd=1.000000 0.164730014,
> massd=2.000000 0.158361143, massd=3.000000 0.159298718, massd=4.000000
> 0.162665838, massd=5.000000 0.000000000)
>
>
>
> FINAL 3+ MODEL after 30 iterations:
> number of spectra: 30124
> using training data negative distributions
> prior: 0.023, total: 699.6
> X! Tandem (k-score) discrim score [fval] negmean: -1.23
> pos: (gaussian mean 5.15, stdev 2.54)
> neg: (evd mean -1.23, stdev 1.26, mu -1.80, beta 0.99)
> no. tolerable trypsin term. [ntt]
> pos: (ntt=0 0.000, ntt=1 0.000, ntt=2 1.000)
> neg: (ntt=0 0.000, ntt=1 0.000, ntt=2 1.000)
> no. missed enz. cleavages [nmc]
> pos: (nmc=0 0.878, 1<=nmc<=2 0.122, nmc>=3 0.000)
> neg: (nmc=0 0.409, 1<=nmc<=2 0.591, nmc>=3 0.000)
> accurate mass diff [massd] (offset: -0.500000)
> pos: (massd=-5.000000 0.000000000, massd=-4.000000 0.000000000,
> massd=-3.000000 0.000000000, massd=-2.000000 0.000154395, massd=-1.000000
> 0.029449633, massd=0.000000 0.390222698, massd=1.000000 0.422314370,
> massd=2.000000 0.128208899, massd=3.000000 0.017069506, massd=4.000000
> 0.012580499, massd=5.000000 0.000000000)
> neg: (massd=-5.000000 0.000000000, massd=-4.000000 0.000000000,
> massd=-3.000000 0.000000000, massd=-2.000000 0.000173670, massd=-1.000000
> 0.179740500, massd=0.000000 0.175030116, massd=1.000000 0.164730014,
> massd=2.000000 0.158361143, massd=3.000000 0.159298718, massd=4.000000
> 0.162665838, massd=5.000000 0.000000000)
>
>
>
>
>
> Hope this helps to figure out why I dont get any information for doubly
> charge precursors, which are clearly there on my .wiff data!
>
> Thanks again and hope someone will pick on with this!!!
>
> Alex
>
>
> On Tuesday, 3 July 2012 17:36:50 UTC-3, Alex wrote:
>>
>> I just wanted to add that I checked on previously analyzed samples, and
>> that apparently, with the same settings and experimental conditions, I got
>> the expected distribution of 2+ and 3+ peptides before. This sample was
>> acquired on march of this year, probably with an earlier version of TPP.
>> After that acquisition, I checked my results, and I have only identified 2+
>> OR 3+ peptides in the pep.shtml files (I didn't realize back then on this
>> peculiar observation). Could someone check if this has happened in your set
>> of results?
>>
>> Thanks!
>>
>> Alex
>>
>> On Tuesday, 3 July 2012 17:03:05 UTC-3, Alex wrote:
>>>
>>> Hi TPP community!
>>>
>>> I have an interesting puzzle that I can not figure out!!! I'm running a
>>> 2D-LC-MS/MS experiment of a yeast lysate (peptides labeled by
>>> dimethylation) on a QqLIT (QTrap 3000) instrument acquiring on IDA mode.
>>> I'm analyzing the data on the TPP using X!Tandem search engine with the
>>> params file attached, and interestingly, ALL the peptides identified on the
>>> pep.shtml file are derived from triply charged precursors!!! (ALL of them).
>>> When I check the prot.shtml file all the identified peptides have the
>>> 3_SEQUENCE nomenclature (my understanding is that the 3 identifies the
>>> charge on the precursor, right?). When I look at the raw data (.wiff) file
>>> on Analyst software, I can easily estimate that most (probably a little
>>> more than half) of the MS/MS was actually performed on CLEARLY doubly
>>> charged precursors, according the the ER-MS (enhanced resolution MS
>>> acquired on the LIT)... so it seem very weird that I dont find a single
>>> peptide to spectrum match (PSM) from a doubly charged precursor. Just
>>> wondering if I might have accidentally changed a parameter on the params
>>> file (.xml).
>>>
>>> Has anybody crossed upon this observation before??? Anybody can help
>>> me???
>>>
>>> Thanks!!!
>>>
>>> Alex
>>>
>>
--
You received this message because you are subscribed to the Google Groups
"spctools-discuss" group.
To view this discussion on the web visit
https://groups.google.com/d/msg/spctools-discuss/-/wiv8gSP7xLgJ.
To post to this group, send email to [email protected].
To unsubscribe from this group, send email to
[email protected].
For more options, visit this group at
http://groups.google.com/group/spctools-discuss?hl=en.
