Need help with Spectrast and spectrast.usermods

[lgillet]

Hi,
I have been searching yeast N15 datasets. Sequest search went all fine
(after defining the individual aa modifications) and so did the
peptide prophet.
But when I try to compile a spectra library with spectrast, Spectrast
has problems to handle the modifications:

1) First test

> command: spectrast -cNDP-N15-IDA -cP0.9 interact-CommandLine.pep.xml

> output:
SpectraST started at Thu Dec 2 17:05:42 2010.
Importing all spectra with probability >= 0.9...
Processing "interact-CommandLine.pep.xml"...DONE!
Importing spectra...DONE!

Library file (BINARY) "DP-N15-IDA.splib" created.
Library file (TEXT) "DP-N15-IDA.sptxt" created.
M/Z Index file "DP-N15-IDA.spidx" created.
Peptide Index file "DP-N15-IDA.pepidx" created.

Total number of spectra in library: 0
Total number of distinct peptide ions in library: 0
Total number of distinct stripped peptides in library: 0

CHARGE +1: 0 ; +2: 0 ; +3: 0 ; +4: 0 ; +5: 0 ; >+5: 0
TERMINI Tryptic: 0 ; Semi-tryptic: 0 ; Non-tryptic: 0
PROBABILITY >0.9999: 0 ; 0.999-0.9999: 0 ; 0.99-0.999: 0 ;
0.9-0.99: 0 <0.9: 0
NREPS 20+: 0 ; 10-19: 0 ; 4-9: 0 ; 2-3: 0 ; 1: 0
MODIFICATIONS None

Total Run Time = 4 seconds.
SpectraST finished at Thu Dec 2 17:05:46 2010 with 7227 error(s):
PEPXML IMPORT: Peptide ID has unknown modification:
"E[130]D[116]S[88]V[100]Y[164]L[114]A[72]K[130]". Skipped query
"N15-1.0.00966.00966.2".
......................


Second try: specifying a spectrast.usermods file
> command:
spectrast -cNDP-N15-IDA -cP0.9 -Mspectrast.usermods interact-
CommandLine.pep.xml

> output:
SpectraST started at Thu Dec 2 17:08:35 2010.
Importing all spectra with probability >= 0.9...
Processing "interact-CommandLine.pep.xml"...
500...1000...1500...2000...2500...3000...3500...4000...4500...DONE!
Importing spectra...10%...20%...30%...40%...50%...60%...70%...80%...
90%...DONE!

Library file (BINARY) "DP-N15-IDA.splib" created.
Library file (TEXT) "DP-N15-IDA.sptxt" created.
M/Z Index file "DP-N15-IDA.spidx" created.
Peptide Index file "DP-N15-IDA.pepidx" created.

Total number of spectra in library: 4675
Total number of distinct peptide ions in library: 2720
Total number of distinct stripped peptides in library: 2443

CHARGE +1: 0 ; +2: 3370 ; +3: 1305 ; +4: 0 ; +5: 0 ; >+5: 0
TERMINI Tryptic: 4642 ; Semi-tryptic: 33 ; Non-tryptic: 0
PROBABILITY >0.9999: 1325 ; 0.999-0.9999: 1609 ; 0.99-0.999:
1062 ; 0.9-0.99: 679 <0.9: 0
NREPS 20+: 0 ; 10-19: 0 ; 4-9: 0 ; 2-3: 0 ; 1: 4675
MODIFICATIONS A,USM_A_72.034145: 3156
C,Carboxymethyl: 340
D,USM_D_116.023975: 2545
E,USM_E_130.039625: 2959
F,USM_F_148.065445: 2077
G,USM_G_58.018495: 2861
H,USM_H_140.050015: 625
I,USM_I_114.081095: 2906
K,USM_K_130.089030: 2872
L,USM_L_114.081095: 3408
M,USM_M_132.037525: 762
N,USM_N_116.037000: 2431
P,USM_P_98.049795: 2426
Q,USM_Q_130.052650: 1911
R,USM_R_160.089250: 1968
S,USM_S_88.029065: 2735
T,USM_T_102.044715: 2733
V,USM_V_100.065445: 3273
W,USM_W_188.073380: 431

Total Run Time = 17 seconds.
SpectraST finished at Thu Dec 2 17:08:52 2010 with 2541 error(s):
PEPXML IMPORT: Peptide ID has unknown modification:
"E[130]D[116]S[88]V[100]Y[164]L[114]A[72]K[130]". Skipped query
"N15-1.0.00966.00966.2".
..................

My spectrast.usermods was as follows:
G|+0.99703497|
A|+0.99703497|
S|+0.99703497|
P|+0.99703497|
V|+0.99703497|
T|+0.99703497|
C|+58.0184989|
I|+0.99703497|
L|+0.99703497|
D|+0.99703497|
E|+0.99703497|
M|+0.99703497|
F|+0.99703497|
T|+0.99703497|
N|+1.99406994|
Q|+1.99406994|
K|+1.99406994|
W|+1.99406994|
H|+2.99110491|
R|+3.98813988|


I even tried one like this:
G[58]|+0.99703497|
A[72]|+0.99703497|...


But without any success.

Am I doing something wrong?
How to specify the modifications such that spectrast proceeds?

Thanks in advance for your help

Ludovic

[Rachel]

I have encountered the same problem.

My search has Acetylation modifications, but spectrast gives error on such modification as follows,

Can anyone help?

-----------------------------------------------

Total Run Time = 3.056e+004 seconds.

SpectraST finished at Sat Jun 30 19:26:22 2012 with 60305 error(s):

PEPXML IMPORT: Peptide ID has unknown modification: "n[42]SETAISFAK". Skipped query "G_110924_01_Zebrafish_band1_C01.21189.21189.2".

PEPXML IMPORT: Peptide ID has unknown modification: "n[42]SETAISFAK". Skipped query "G_110924_01_Zebrafish_band1_C01.21422.21422.2".

........