Isobar tool and file format

[niki...@gmail.com]

Hi there,

I wanted to try isobar to quantify TMT 10-plex data from MaxQuant. I am struggling with the format of the files I need to provide. Does anyone know which files do I need to use for the isobar quantification? Does anyone know which files have the spectrum identifier in MaxQuant? Is it the same with scan number or scan index?

I can find some of the information in the MSMS.txt and peptides.txt files, but not all of them, in order to create the ID.csv and IBSPECTRA.csv manually. I suppose it is OK to create these files manually?

Thank you for your help.

Best wishes

Nikiana

[Veit Schwämmle]

Hi Nikiana,

You can create the files manually and they need to follow a certain standard (description hidden somewhere in the end of the isobar documentation). I have no idea how to reformat the MaxQuant output but you can try to identify the right information for the "spectrum" columns in the orignial mgf files as isobar will search them (TITLE lines).

Below you find an example to change the format of the PeptideShaker output (report in tsv format). And avoid quotes in the column headers as this will create an error.

Best regards

Veit

# filenames
ident_file <- "Default PSM Report.txt"
mgf_files <- list.files("./",pattern=".mgf$",full.names = T)
psms <- read.csv(ident_file,sep="\t")

# filter for confidence 100
psms <- psms[psms$Confidence.... == 100,]

names(psms)
names(psms)[c(2:3,7:11)] <- c("accession","sequence", "peptide","modif","fixed", "file","spectrum")
write.table(psms[,c("accession","peptide","modif","spectrum","file")],"t.corr.csv",sep="\t", row.names = F,quote=F)

ib <- readIBSpectra("iTRAQ4plexSpectra","t.corr.csv", mgf_files, decode.titles=T)

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[Michael Wagner]

Hi Veit,

I know it's been a while since this issue has come up, but I am having similar issues as Nikiana in importing some iodoTMT6plex data processed by Proteome Discoverer 1.4 to the Isobar package. I decided to try and manually create the id.csv file that includes the quantification data. I have looked in the Isobar documentation and cannot seem to find the description of the format standard (i.e., what column labels and in what order) of the ID.csv file. I try to create the IBSpectra object using the following: 

> ib33834 <-readIBSpectra("TMT6plexSpectra","full33834.csv")
  reading id file full33834.csv [type: ibspectra] ... done
Error in `[.data.frame`(id.data, , .SPECTRUM.COLS["SPECTRUM"]) : 
  undefined columns selected

Does this error occur because of the column formatting of the ID.csv file, or is there a different issue? If so, how do you get around this? I can also attach a snippet of the .csv I am trying to import. Thank you for your time and your help!

Best,

Mike

PS-- I have also tried importing the data as an mzIdentML file (created by converting the .msf file from PD to .mzid using the ProCon converter tool), but had this error code: 

> ib33834 <- readIBSpectra("TMT6plexSpectra","/Users/michaelwagner/Desktop/33834.mzid")
  reading id file /Users/michaelwagner/Desktop/33834.mzid [type: mzid] ...Error in data.frame(ref = xpathSApply(doc, paste0(root, "/x:DataCollection/x:Inputs/x:SearchDatabase"),  : 
  arguments imply differing number of rows: 1, 0

Thanks again!

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[Veit Schwämmle]

Hi Michael,

Isobar is not maintained anymore for a longer time and I suggest you to use e.g. the R package MSnbase as it contains much more functionality and you also get fast feedback by the developers.

I checked in my old files and the columns of the id file should be

"accession","peptide","modif","spectrum","file"

You seem to miss the "file" and I also would remove the other columns to be sure.

But be aware that any quote in the spectrum ids could make trouble.

Best regards

Veit

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