• Sign In
  1. Requests

Error in QuaSAR- Error in Calculation

support

View Request

view list print
Error in QuaSAR- Error in Calculation patricia fernandez puente  2018-04-09 08:02
 
Today I installed the QuaSAR software to calculate the LOD and LOQ of my experiment in Skyline.
My experiment consists in the quantification of several proteins with the internal standard method.
To make the calibration curve I put, in the same quantity of total digested serum proteins, increasing quantity of heavy peptides.
When I performed the QuaSAR run with my samples this is the error I recived back:
ERROR:
"C:\Program Files\R\R-3.0.1\bin\R.exe" -f "C:\Users\usuario\AppData\Local\Apps\2.0\BRZVJE6R.N0V\6X83QJAC.83W\skyl..tion_e4141a2a22107248_0004.0001_c221031cbe0943a3\Tools\QuaSAR-1_33\QuaSAR-Skyline.R" --slave --no-save --args "C:\Users\usuario\AppData\Local\Apps\2.0\BRZVJE6R.N0V\6X83QJAC.83W\skyl..tion_e4141a2a22107248_0004.0001_c221031cbe0943a3\Tools\QuaSAR-1_33\QuaSAR.R" "C:\Users\usuario\AppData\Local\Apps\2.0\BRZVJE6R.N0V\6X83QJAC.83W\skyl..tion_e4141a2a22107248_0004.0001_c221031cbe0943a3\Tools\QuaSAR-1_33\common.R" "C:\Users\usuario\AppData\Local\Temp\QuaSAR_QuaSAR_Input.csv" NULL "Calibration Curve_09042018" "heavy Area" 1 "light Area" fmol/ul 1 1 -1 1 1 1 0 150 150 1 0.2 0 NULL "Calibration Curve_09042018" 0
[1] $Id: QuaSAR-Skyline.R 149 2014-10-23 13:56:13Z manidr $
[1] $Id: QuaSAR.R 149 2014-10-23 13:56:13Z manidr $
Attaching package: 'boot'
The following object is masked from 'package:lattice':
    melanoma
The following object is masked from 'package:gtools':
    inv.logit, logit
KernSmooth 2.23 loaded
Copyright M. P. Wand 1997-2009
Attaching package: 'gplots'
The following object is masked from 'package:stats':
    lowess
[1] Processing arguments ...
[1] >>> Processing part 1 of 1
[1] Initializing ...
Error in calculate(data.subset, concentrationFile, output.prefix = outputPrefix, :
  Duplicate transitions found -- try including neutral loss
Calls: tryCatch -> tryCatchList -> parse.cmdline -> calculate
Execution halted
Finished!

The folder Calibration Curve_09042018 is empty.

What should I do to fix the problem?
And, is available a workbook with all the possible troubleshoot problems in QuaSAR and the possible ways of solving the problems?

Thank you for your help.
 
 
Nick Shulman responded:  2018-04-09 09:33
There is more info on this error here:
https://skyline.ms/announcements/home/support/thread.view?rowId=31791

Here are some suggestions for what to do:
That error occurs when there are multiple transitions with the same (PrecursorCharge, FragmentIon, ProductCharge) for a given peptide and sample. Possible causes could be:

1. You are monitoring the same fragment with different neutral losses -- in this case (as the error message suggests), include the NeutralLoss column to disambiguate the transitions.

2. You have modified peptides, but are not including the modifications in the ModifiedPeptideSequence column of the Skyline report. In such a case, two different modified peptides could have the same sequence, and hence could result in identical transitions.

3. Your sample specification was incorrect. If you have multiple concentrations, each concentration must be a different "sample". Please see the tutorial on how to specify the sample.
-----------------------


If the above information is not helpful then you could try sending us your Skyline document.
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file is less than 50MB you can attach it to this support request.
Otherwise, you can upload it here:
https://skyline.ms/files.url
 
patricia fernandez puente responded:  2018-04-11 02:17
Hello Nick,
I attached myskyline document for my MRM experiemnt. There are three replicates for the same calibration curve (reverse curve) in a matrix of serum sample, that is kept constant in all the samples. The endogenous light peptide is my internal standand that is the serum matrix for each peptide. I have added different concnetrations of my heavy peptide, that is the one is going to give me the LOD, LOQ and everything.I did not have any duplicate transitions for any light or heavy peptide.
Thank you very much for your help,
 
patricia fernandez puente responded:  2018-04-11 02:23
 
patricia fernandez puente responded:  2018-04-11 02:59
 
Nick Shulman responded:  2018-04-11 13:40
I think QuaSAR is getting confused by the fact that you have three files in each replicate.

That is, you have the files "1.wiff", "1_1.wiff", and "1_2.wiff" and you have put them all inside of the replicate "1".

In Skyline, multiple files per replicate should only be used when you had to spread your data collection out over multiple injections. In your case, it looks like you measured all of your peptides in each of those files, so you should have imported them as separate replicates.