Targetted Isotope Quantification using mzMatch and xcms pipeline

[Josh Goldford]

Hello!

I am attempting to use R packages xcms and mzMatch on a Mac to identify 13C labeled isotopes of HPLC separated peptides from an LTQ-Orbitrap Velos.

I am very new to R, but I have successfully worked through the example provided on the following webpage: http://mzmatch.sourceforge.net/isotopes-targetted.php.

My problem differs from the provided tutorial in that I do not have biological replicates.  That is, I simply have one .RAW file of MS1 spectra collected in profile mode.  I was given a list of peptides to search for in the spectra, which came from an unlabeled MS/MS orbitrap experiment.

Currently, I am converting the .RAW file into an mzXML in centroid mode using msconvert.  I am using the following parameters for peak picking: method="centWave",ppm=5,peakwidth=c(9,50),snthresh=1.  

From my understanding of all the functions presented in the tutorial, I have no further processing steps left given I only have one .RAW file (I was hoping someone could verify this!)

After this, the .peakml file is generated using PeakML.xcms.write.SingleMeasurement.  I then attempt to run PeakML.Isotope.TargettedIsotopes and get the following error:

Error in .jcall(java_project, returnSig = "D", method = "formulaToMass",  : 

  java.lang.RuntimeException: Unknown element: A

I have attached the .csv file containing the peptides I am searching for.  I have checked to see if the .csv file was the issue, but when I use the example .csv file "metlist_example.csv," I receive the same error.  This makes me think that there is a problem with the generation of the peakml file.

Again, I am fairly new to R and Java, so I don't possess proper debugging skills yet.  Any help would be greatly appreciated!

Sincerely,

Josh

[Gavin Blackburn]

Hi Josh,

 

I’ve not used the targeted isotopes function, the error doesn’t look like a single file problem, I’m also unsure how well it works for peptides.

 

If you have a list of peptides that you want to look for and you only have one file then I’d suggest you either use Xcalibur or, as you are on a Mac, MZMine. Both of these programmes will allow you to pull out RICs for specific mass ranges (I don’t have much experience with MZMine and recommend MZMatch/IDEOM for processing larger data sets), so all you need to do is calculate your expected mass ions and look for a peak. Xcalibur is the best programme to do this and is what I would suggest you use. From a Mac you could use Teamviewer or Logmein and remote desktop to a windows machine to do it.

 

Cheers,

 

Gavin.

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[Andris Jankevics]

Hi Josh,

Something is wrong in your peptides.csv file. After quick check I can't see what excatly, but error message what you copied say that it tries to find a mass for chemical element "A" which of course is not existing. I hope Unni will be able to help you.

Cheers,

Andris

Andris Jankevics

(1) Research associate

     Faculty of Life Sciences, Manchester Institute of Biotechnology, 

     University of Manchester,

     Manchester, United Kingdom

(2) Groningen Bioinformatics Center,
   University of Groningen, The Netherlands

[Vincent Bonifay]

Hi

I am also new with IDEOM using the targeted isotopic function. I just started to use it yesterday. The IDEOM tool, "isotopes search" worked fine. I wanted to use PeakML for a more precise search of my mzXML file. But i got the same type of error:

Error in .jcall(java_project, returnSig = "D", method = "formulaToMass",  : 

  java.lang.ArrayIndexOutOfBoundsException: 0 >= 0

I have been working on trying to resolve it since yesterday. But i have not found the issue yet.

Regards

[Vincent Bonifay]

Furthermore, i thought that it might have been a problem of version. So I also updated R and rjava to the latest version, but unfortunately it did not fix the issue

One more precision i do have triplicate for 2 groups. The peakml file i am using is the highintensity gapfilled. 

[Darren Creek]

Hi Vincent,

It looks like a similar problem to Josh's - an error in reading formulas from your input file.

Please find the isotopelist.txt file and see if there is a formula missing - or an unusual character in the formula column.  

If there is no obvious issue please post that text file on the forum and we will check it for you. If it is strictly confidential then please send to Unni and myself personally. 

all the best,

Darren

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Dr Darren Creek

NHMRC Training Fellow
Department of Biochemistry and Molecular Biology, Bio21 Institute

University of Melbourne

dcr...@unimelb.edu.au

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(+61) 3 83442351

[Vincent Bonifay]

Hi Darren,

Thank you for the tips. After filtering and removing the masses which did not have an assigned formula, I did not get the error message. The process is really slow at this point, and R keep going to a non-responding mode for 1 sec, then works for 10 sec. But it seems after 8-9 hours i should be able to have a good list of the isotopes for the 80-90 formula.

Thank you again

Regards