Hi,
I´m using OMSSA and the TPP/X!Tandem search (+TPP PeptideProphet/
ProteinProphet) for LC-MS/MS data analysis.
First, I always run OMSSA due to its high performance to have first
information on the sample (for curious colleagues, and to set
priorities), then I make a more thorough analysis using the TPP.
However, OMSSA reports some different peptides as the final TPP
output.
When looking at the *.pep.xml, all peptides found by OMSSA, are also
present in the X!/Tandem-pep.xml file, but not reported at the end of
the pipeline. I already tried to set the p-filter for the
PeptideProphet to very low values (0.0001), but those additional
peptides do not show up.
Example for the trypsin precursor:
TPP reports:
**************
7 gi|136406|sp|P06871.1|TRY1_CANFA gi|136429|sp|P00761.1|TRYP_PIG
0.9565
confidence: 0.28 max coverage: 4.3% num unique peps: 1 tot
indep
spectra: 1 share of spectrum id's: 1.84%
>RecName: Full=Cationic trypsin; Flags: Precursor Length: 246aa
>RecName: Full=Trypsin; Flags: Precursor
weight peptide sequence nsp adj prob init prob
ntt nsp total pep grp
ind
* wt-1.00 2_LSSPATLNSR 0.9565 0.9851 2 0.00
1
OMSSA reports (top redundant hits):
******************************************
Peptide: IITHPnFNGNTLDNDIMLIK Neutral Mass: 2283.3 Da Charge
State:
+3 E-value: 1.203E-006
Peptide: LGEHNIDVLEGNEQFINAAK Neutral Mass: 2211.5 Da Charge
State:
+3 E-value: 4.363E-009
In both searches I included deamidation of Q/N
Searching those peptides in the TPP *.pep.xml gives:
*************************************************************
index spectrum hyperscore nextscore bscore yscore expect
ions
peptide protein
1331 3D-1.01138.01138.3SpectraST 637 558 1 1 0.22
38/76
R.LGEHNIDVLEGNEQFINAAK.IPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG
1339 3D-1.01143.01143.3SpectraST 622 595 1 1 0.71
34/76
R.LGEHNIDVLEGNEQFINAAK.IPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG
1348 3D-1.01149.01149.3SpectraST 632 587 1 1 0.7
36/76
R.LGEHNIDVLEGNEQFINAAK.IPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG
1400 3D-1.01180.01180.3SpectraST 697 649 2 2 0.23
42/76
R.LGEHNIKVLEGNEQFINAAK.IPeptideAtlas gi|209572698|sp|P35030.2|
TRY3_HUMAN
and
index spectrum hyperscore nextscore bscore yscore expect
ions
peptide protein
815 3D-1.00791.00791.2SpectraST 304 299 1 0 15
11/18
K.IITHPNFNGN.TPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG
1472 3D-1.01223.01223.3SpectraST 569 497 1 1 0.65
33/76
K.IITHPNFNGNTLDNDIM147.04LIK.LPeptideAtlas gi|136429|sp|P00761.1|
TRYP_PIG
1490 3D-1.01234.01234.3SpectraST 559 530 1 1 4.9
30/76
K.IITHPNFNGNTLDNDIM147.04LIK.LPeptideAtlas gi|136429|sp|P00761.1|
TRYP_PIG
1545 3D-1.01268.01268.3SpectraST 585 555 1 1 4.2
28/76
K.IITHPNFNGNTLDNDIMLIK.LPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG
1553 3D-1.01273.01273.3SpectraST 581 555 1 1 8.4
24/76
K.IITHPNFNGNTLDNDIMLIK.LPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG
1565 3D-1.01280.01280.3SpectraST 531 522 1 2 7.5
32/76
K.IITHPNFNGNTLDNDIMLIK.LPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG
So, obviously, those peptides did not pass the PeptideProphet filter.
** Any recommendation, how to fix this to obtain a higher sequence
coverage in the TPP? **
Note:
If I tick the PeptideProphet Option " Force the fitting of the mixture
model (bypass automatic mixture model checks) " I increase the
sequence coverage of other proteins, but those trypsin fragments
still do not show up.
Thanks a lot and greetings,
Robert
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