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1
Other / Re: Tandem MS/MS .d files and Proteowizard
Thanks for your help, I had tried using the MS Level 1 filter before, but the files generated are empty.  However, using the msn2xcmsRaw conversion does seem to be working well.  The data in the mzData files looks complete when viewing with plotChrom.  But a new, possibly related, problem is coming up.  The final diffreport doesn’t contain all the data I think it should.  Each xcmsRaw object contains about 2600 mass spectra, but only 10% of these show up in the diffreport file.  This would be OK if the data that seems most important by eye were in the data set, but these are absent.  Instead the file contains many seemingly unimportant fragments.  The .d files contain very clear peaks, with 2-3 abundant m/z values.  Instead of these abundant compounds, which are found in nearly all of our samples, the diffreport is showing some really tiny fragments.  I used the default xcmsSet(files).  I have been changing some of the default values but haven’t had luck yet (I set fwhm = c(5,20)).  Are there any parameters you suspect need to be changed?  We have many different compounds with similar m/z (300.9 is an important fragment at several different retention times).  We’re using a Q-TOF with ppm 2-5.  These are 60 minute runs, 0.2ml/min flow rate.
2
Other / Re: Tandem MS/MS .d files and Proteowizard
Thanks Jan, I will try using the MetShot package.

I'm not sure if I am fully answering your question.  I collected the data in auto ms/ms mode, so yes, my understanding is that the MS2 data is dependent on the results obtained from the MS1 scans.  I think the data we have is a full MS1 trace - where the masshunter software automatically identifies the precursor ions.

Thanks.
3
Other / Re: Tandem MS/MS .d files and Proteowizard
Hi Jan,

Thank you for your reply.  I should have started off with my target, and why I think there may be a problem with reading the data files.  I do want to do profiling on MS2 data.  When I do xcmsRaw(file, includeMSn=TRUE), I receive the warning message: In ‘profStep<-‘(‘*tmp*’,value=1): MS1 scans empty. Skipping profile matrix calculation. 

My goal is to use precursor ion and fragment data to align peaks across many data files, and eventually use metaxcms.  When I use xset<-xcms(mslevel=2), I get the warning message:
 In split.xcmsRaw(object, f=object@msnLevel==mslevel): MSn information will be dropped

Despite this warning, I still get peak grouping when I continue. (I use xset1<-group(xset,bw=10); xset2<-retcor(xset1,family= “symmetric”,plottype= “mdevden”);xset3<-group(xset2,bw=10)).    The final error that I can’t get past is the fillpeaks step (xset4<-fillPeaks(xset3) where I get the following error:
Error in buf[bufidx[imz[1]:imz[2]],iret[1]:iret[2],drop=FALSE:subscript out of bounds
In addition: Warning message:
In FUN(X[[1L]],…): (corrected) retention time vector length mismatch for file.mzXML

Because of the repeated warning messages of missing MS1 data, I thought compatibility between .d and SeeMS may be the root of the problem.  Since viewing the raw Agilent .d files in SeeMS seems to show no MS1 data under the ‘MS Level’ column, I thought I would try this route in order to resolve the warning message after the xcmsSet step.   

Thanks.
4
Other / Tandem MS/MS .d files and Proteowizard
Hi Everyone,

I am trying to convert Agilent .d files to .mzXML files using ProteoWizard (for eventual use in xcms).  When viewing the .d files in SeeMS, the MS Level column only contains '2' data.  When converting the files to .mzXML with MSConvert and using in xcms, I receive errors that there is no MS1 data.  Does anyone have experience reading .d files obtained with tandem MS/MS with the ProteoWizard set of tools?  When viewing the chromatograms in MassHunter, precursor ions are clearly distinguished from fragments, so I am not sure why the Proteowizard tools fail to recognize the MS1 scans. 

Thanks.
5
XCMS / fillPeaks error: subscript out of bounds
Hi everyone,

Has anyone encountered the following error?

Error in buf[bufidx[imz[1]:imz[2]], iret[1]:iret[2],drop=FALSE]: subscript out of bounds
In addition: warning message:
In FUN(X[[1L]],...):
(corrected) retention time vector length mismatch for file

The previous steps seemed to work well (xset<-xcmsSet(mslevel=2);xset1<-group(xset);xset2<-retcor(xset1);xset3<-group(xset2);xset4<-fillPeaks(xset3))

Thanks.