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
>>
>
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