ASAPRatio for 15N?

[Jake W]

Hi--

I'm trying to do some 14N/15N quantitation and would like to use
ASAPRatio in addition to XPRESS. I realize ASAPRatio wasn't exactly
designed for 14N/15N quantification, but I'm hoping it might be
possible, since I really like some of its features like charge-state
finding, protein abundance inference and error analysis. I've tried
running ASAPRatio under Xinteract with a command like this:

C:\Inetpub\tpp-bin>run_in c:/Inetpub/wwwroot/ISB/data/
14N15NTesting19Nov08; xint
eract -N240Min_14N15Nrefine_1_quant.interact.pep.xml -p0 -l6 -x20 -OAp
-A-lACDEF
GHIKLMNPQRSTVWY-r0.05-
mC0.99703489G0.99703489A0.99703489S0.99703489P0.99703489T0
.
99703489L0.99703489I0.99703489D0.99703489E0.99703489M0.99703489F0.99703489Y0.99
703489N1.99406978K1.99406978Q1.99406978W1.99406978H2.99110467R3.98813956
c:/Inet
pub/wwwroot/ISB/data/
14N15NTesting19Nov08/240Min_14N15Nrefine_1.tandem.pep.xml

When I do this, though, I get errors that I can't make any sense of
("Trying to compute mass of non-reside"?; the output is at the end of
the message) and no ASAPRatio data is generated. Any advice on this
would be much appreciated.

thank you,
Jake

Here's the Xinteract output:

xinteract (TPP v4.1 JETSTREAM rev 1, Build 200811121809 (MinGW))

running: "C:/Inetpub/tpp-bin/InteractParser
"240Min_14N15Nrefine_1_quant.interac
t.pep.xml" "c:/Inetpub/wwwroot/ISB/data/
14N15NTesting19Nov08/240Min_14N15Nrefine
_1.tandem.pep.xml" -L"6""
file 1: c:/Inetpub/wwwroot/ISB/data/
14N15NTesting19Nov08/240Min_14N15Nrefine_1.
tandem.pep.xml
processed altogether 8124 results


results written to file c:/Inetpub/wwwroot/ISB/data/
14N15NTesting19Nov08/240Min
_14N15Nrefine_1_quant.interact.pep.xml

direct your browser to http://localhost/ISB/data/14N15NTesting19Nov08/240Min_14
N15Nrefine_1_quant.interact.pep.shtml



command completed in 4 sec

running: "C:/Inetpub/tpp-bin/PeptideProphetParser
"240Min_14N15Nrefine_1_quant.i
nteract.pep.xml" MINPROB=0 EXTRAITRS=20 ACCMASS"
using Accurate Mass Bins
(X! Tandem (k-score)) (minprob 0)
adding Accurate Mass mixture distr
init with X! Tandem (k-score) trypsin
MS Instrument info: Manufacturer: Thermo Scientific, Model: LTQ FT,
Ionization:
nanoelectrospray, Analyzer: fourier transform ion cyclotron resonance
mass spect
rometer, Detector: inductive detector

PeptideProphet (TPP v4.1 JETSTREAM rev 1, Build 200811121809 (MinGW))
AKeller@
ISB
read in 0 1+, 4323 2+, 3125 3+, 556 4+, and 120 5+ spectra
Initialising statistical models ...
negmean = 0.0533258
negmean = -0.295923
negmean = -0.716929
negmean = 0.697127
negmean = 1
Iterations: .........10.........20........
WARNING: Mixture model quality test failed for charge (5+). Use
xinteract option
-OF (PeptideProphetParser option FORCEDISTR) to override.
model complete after 29 iterations
command completed in 43 sec

running: "C:/Inetpub/tpp-bin/ProphetModels.pl -i
240Min_14N15Nrefine_1_quant.int
eract.pep.xml"
Analyzing 240Min_14N15Nrefine_1_quant.interact.pep.xml ...
Reading Accurate Mass Model model +1 ...
Reading Accurate Mass Model model +2 ...
Reading Accurate Mass Model model +3 ...
Reading Accurate Mass Model model +4 ...
Reading Accurate Mass Model model +5 ...
Parsing search results "c:/Inetpub/wwwroot/ISB/data/1to1CRun2240Min
(X! Tandem (
k-score))"...
=> Total of 8004 hits.
command completed in 3 sec

running: "C:/Inetpub/tpp-bin/ASAPRatioPeptideParser
"240Min_14N15Nrefine_1_quant
.interact.pep.xml" -lACDEFGHIKLMNPQRSTVWY -r0.05 -
mC0.99703489G0.99703489A0.9970
3489S0.99703489P0.99703489T0.99703489L0.99703489I0.99703489D0.99703489E0.9970348
9M0.99703489F0.99703489Y0.99703489N1.99406978K1.99406978Q1.99406978W1.99406978H2
.99110467R3.98813956"
adjusting 143.004 to 143.059
label A: 0 vs 0
label D: 0 vs 0
label F: 0 vs 0
label G: 0 vs 0
label H: 0 vs 0
label I: 0 vs 0
label K: 0 vs 0
label L: 0 vs 0
label N: 0 vs 0
label P: 0 vs 0
label R: 0 vs 0
label S: 0 vs 0
label T: 0 vs 0
label V: 0 vs 0
label W: 0 vs 0
label Y: 0 vs 0
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
adjusting 143.004 to 143.059
WARNING: Trying to compute mass of non-residue: !
WARNING: Trying to compute mass of non-residue:
WARNING: Trying to compute mass of non-residue: !
WARNING: Trying to compute mass of non-residue:
WARNING: Trying to compute mass of non-residue: !
WARNING: Trying to compute mass of non-residue:
WARNING: Trying to compute mass of non-residue: !
WARNING: Trying to compute mass of non-residue:
adjusting 143.004 to 143.059
command completed in 109 sec

running: "C:/Inetpub/tpp-bin/DatabaseParser
"240Min_14N15Nrefine_1_quant.interac
t.pep.xml""
command completed in 0 sec

running: "C:/Inetpub/tpp-bin/RefreshParser
"240Min_14N15Nrefine_1_quant.interact
.pep.xml" "c:/Inetpub/wwwroot/ISB/data/dbase/MED4revdb.faa""
- Building Commentz-Walter keyword tree... - Searching the tree...
- Linking duplicate entries... - Printing results...

command completed in 5 sec

running: "C:/Inetpub/tpp-bin/PepXMLViewer.cgi -I c:/Inetpub/wwwroot/
ISB/data/14N
15NTesting19Nov08/240Min_14N15Nrefine_1_quant.interact.pep.xml"
command completed in 6 sec

running: "C:/Inetpub/tpp-bin/ProteinProphet
"240Min_14N15Nrefine_1_quant.interac
t.pep.xml" "240Min_14N15Nrefine_1_quant.interact.prot.xml" XML"
ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the
original Pe
rl by A. Keller (TPP v4.1 JETSTREAM rev 1, Build 200811121809 (MinGW))
(xml input) (using degen pep info)
. . . reading in c:/Inetpub/wwwroot/ISB/data/
14N15NTesting19Nov08/240Min_14N15Nr
efine_1_quant.interact.pep.xml. . .
. . . read in 0 1+, 2831 2+, 1585 3+, 190 4+, 0 5+ spectra with min
prob 0.05

protein probabilities written to file c:/Inetpub/wwwroot/ISB/data/
14N15NTesting
19Nov08/240Min_14N15Nrefine_1_quant.interact.prot.xml
direct your browser to http://localhost/ISB/data/14N15NTesting19Nov08/240Min_14
N15Nrefine_1_quant.interact.prot.shtml

command completed in 2 sec

running: "C:/Inetpub/tpp-bin/ProtProphModels.pl -i
240Min_14N15Nrefine_1_quant.i
nteract.prot.xml"
Analyzing 240Min_14N15Nrefine_1_quant.interact.prot.xml ...
command completed in 1 sec

running: "C:/Inetpub/tpp-bin/ASAPRatioProteinRatioParser
"240Min_14N15Nrefine_1_
quant.interact.prot.xml""
command completed in 192 sec

running: "C:/Inetpub/tpp-bin/ASAPRatioPvalueParser
"240Min_14N15Nrefine_1_quant.
interact.prot.xml""
command completed in 12 sec
C:/Inetpub/tpp-bin/InteractParser
"240Min_14N15Nrefine_1_quant.interact.pep.xml"
"c:/Inetpub/wwwroot/ISB/data/
14N15NTesting19Nov08/240Min_14N15Nrefine_1.tandem.
pep.xml" -L"6" 4 sec
C:/Inetpub/tpp-bin/PeptideProphetParser
"240Min_14N15Nrefine_1_quant.interact.pe
p.xml" MINPROB=0 EXTRAITRS=20 ACCMASS 43 sec
C:/Inetpub/tpp-bin/ProphetModels.pl -i
240Min_14N15Nrefine_1_quant.interact.pep.
xml 3 sec
C:/Inetpub/tpp-bin/ASAPRatioPeptideParser
"240Min_14N15Nrefine_1_quant.interact.
pep.xml" -lACDEFGHIKLMNPQRSTVWY -r0.05 -
mC0.99703489G0.99703489A0.99703489S0.997
03489P0.99703489T0.99703489L0.99703489I0.99703489D0.99703489E0.99703489M0.997034
89F0.99703489Y0.99703489N1.99406978K1.99406978Q1.99406978W1.99406978H2.99110467R
3.98813956 109 sec
C:/Inetpub/tpp-bin/RefreshParser
"240Min_14N15Nrefine_1_quant.interact.pep.xml"
"c:/Inetpub/wwwroot/ISB/data/dbase/MED4revdb.faa" 5 sec
C:/Inetpub/tpp-bin/PepXMLViewer.cgi -I c:/Inetpub/wwwroot/ISB/data/
14N15NTesting
19Nov08/240Min_14N15Nrefine_1_quant.interact.pep.xml 6 sec
C:/Inetpub/tpp-bin/ProteinProphet
"240Min_14N15Nrefine_1_quant.interact.pep.xml"
"240Min_14N15Nrefine_1_quant.interact.prot.xml" XML 2 sec
C:/Inetpub/tpp-bin/ProtProphModels.pl -i
240Min_14N15Nrefine_1_quant.interact.pr
ot.xml 1 sec
C:/Inetpub/tpp-bin/ASAPRatioProteinRatioParser
"240Min_14N15Nrefine_1_quant.inte
ract.prot.xml" 192 sec
C:/Inetpub/tpp-bin/ASAPRatioPvalueParser
"240Min_14N15Nrefine_1_quant.interact.p
rot.xml" 12 sec
job completed in 377 sec

[David Shteynberg]

Hi,

The mass given to ASAPRatio should be the total modifiede mass, not
just the mass diff.

-David

[Jake W]

Ah. Thanks for that. Still seem to have trouble, though. So when I
run

run_in c:/Inetpub/wwwroot/ISB/data/ASAPRa
tioTesting20Nov08; xinteract -
N240Min_50ppm_err_ASAP1.interact.pep.xml -p0 -l6
-x20 -OAp -A-lACDEFGHIKLMNPQRSTVWY-r0.05-
mA72.03415R160.10111N118.04293D117.0269
4C105.00919E130.04259Q130.05858G59.02146H140.05891I116.08406L114.08406K130.09496
M133.04049F148.06841P98.05276S88.03203T102.04768W187.07931Y165.06333V101.06841
c
:/Inetpub/wwwroot/ISB/data/
ASAPRatioTesting20Nov08/240Min_50ppm_err.tandem.pep.x
ml

I get: (I'll just put the ASAPRatio Parser output here, everything
else seems OK):

running: "C:/Inetpub/tpp-bin/ASAPRatioPeptideParser
"240Min_50ppm_err_ASAP1.inte
ract.pep.xml" -lACDEFGHIKLMNPQRSTVWY -r0.05 -
mA72.03415R160.10111N118.04293D117.
02694C105.00919E130.04259Q130.05858G59.02146H140.05891I116.08406L114.08406K130.0
9496M133.04049F148.06841P98.05276S88.03203T102.04768W187.07931Y165.06333V101.068
41"

adjusting 143.004 to 143.059
label A: 0 vs 0
label D: 0 vs 0
label F: 0 vs 0
label G: 0 vs 0
label H: 0 vs 0
label I: 0 vs 0
label K: 0 vs 0
label L: 0 vs 0
label N: 0 vs 0
label P: 0 vs 0
label R: 0 vs 0
label S: 0 vs 0
label T: 0 vs 0
label V: 0 vs 0
label W: 0 vs 0
label Y: 0 vs 0
adjusting 143.004 to 143.059

(same repeated ~15 times)
adjusting 143.004 to 143.059
WARNING: Trying to compute mass of non-residue: r
WARNING: Trying to compute mass of non-residue: o
WARNING: Trying to compute mass of non-residue: t
WARNING: Trying to compute mass of non-residue: e
WARNING: Trying to compute mass of non-residue: i
adjusting 143.004 to 143.059
(same repeated ~20 times)
adjusting 143.004 to 143.059
command completed in 200 sec

running: "C:/Inetpub/tpp-bin/ASAPRatioProteinRatioParser
"240Min_50ppm_err_ASAP1
.interact.prot.xml""
command completed in 297 sec

running: "C:/Inetpub/tpp-bin/ASAPRatioPvalueParser
"240Min_50ppm_err_ASAP1.inter
act.prot.xml""
no pvalues available for this dataset

command completed in 1 sec

Any further ideas?

many thanks,
Jake

[Carlo de Oliveira Martins]

I was taking a look at Jake's table of amino acid masses and found some differences for 15N labelled:

Maybe I made some mistake, but I think it is worth to take a look:

A 72.03415

R 160.10111

N 116.04293

D 116.02694

C 161.0307

E 130.04259

Q 130.05858

G 58.02146

H 140.05891

I 114.08406

L 114.08406

K 130.09496
M 132.04049

F 148.06841

P 98.05276

S 88.03203

T 102.04768

W 188.07931

Y 164.06333

V 100.06841

All the best,

Carlo