I've sent Matt the PLGS xml files now. Unfortunately, as mentioned previously by Frederik, they don't preserve MS1 scans as inidividual scans, so aren't useful for translation to mzXML/mzML with a view to quantitation using ASAPRatio or XPRESS.
We're now getting reasonable IDs and quantitation by doing: 1) Continuous lockmass correction of original RAW using MassLynx 4.1 to create new lockmass corrected RAW 2) Conversion of RAW to mzXML with massWolf 3) Creation of .mgf from RAW using Mascot Distiller with default QTof param file 4) Search with Mascot/Tandem/OMSSA on Distiller .mgf 5) Quantitation with ASAPRatio / XPRESS using the .mzXML - Distiller writes scan numbers into mgf, so search results tie up with the mzXML. This works okay, but is a pain to do, involving much moving of files, clicking buttons, and waiting. The software is spread across different PCs and I can't install all on one due to licenses. Will be a tedious exercise once we start looking at multiple 24 fraction separations soon. Happily this problem should soon disappear as these experiments will move to a new Agilent Q-TOF. There is a very big difference in IDs between a PLGS pkl or Distiller mgf, and searching the mzXML straight off when working with the QTOF, regardless of whether data is acquired as profile or centroid. In contrast our Orbitrap and Bruker HCT Plus tend to give best results when working straight from the mzXML. DT Matthew Chambers wrote: > PKL doesn't retain scan time or scan number. In Dave Trudgian's, case, > it's also merging lots of raw scans without recording which ones and > it's repeating the same fragments many times (at least in the sample he > sent me). It's a nightmare, really: there's no regard for keeping track > of the original spectral evidence. I'd like to get a look at the XML > export from PLGS because I think that's much more likely to have a > tolerable level of metadata. Dave said he'd get those files to me after > he gets back from HUPO, but if anyone else has examples of that export > I'll look at them. > > -Matt > > > Brian Pratt wrote: > >> Would that retain elution time information? That's important for many >> types of analysis. >> >> On Thu, Oct 1, 2009 at 5:56 AM, ChargedPeptide <[email protected] >> <mailto:[email protected]>> wrote: >> >> >> Hmmm... >> Just a thought, while I haven't used it myself , what happens if you >> try to take the route of first exporting a PKL file and then >> converting to mzXML using >> PKL2MZXML <a href="http://tools.proteomecenter.org/wiki/index.php? >> title=Software:pkl2mzXML >> >> <http://tools.proteomecenter.org/wiki/index.php?title=Software:pkl2mzXML>">Link</a>? >> I assume most of the pre processing will already have been carried out >> from the raw data and the conversion is simpler, even though as I >> said, I might be way of the money here. >> >> On Sep 14, 1:37 pm, dctrud <[email protected] >> <mailto:[email protected]>> wrote: >> > Dear all, another query r.e. Waters QTOF data.... >> > >> > We have a QTOF Premier which is now being used for SILAC >> experiments, >> > and we want to quantify using ASAPRatio / Xpress. Have no problems >> > doing this in our pipeline using Orbitrap data, but the QTOF data is >> > causing headaches. Up until now we've mostly been using MassWolf >> just >> > to get MS data into .mzXML for purposes other than ID & quant, PLGS >> > having been the preferred analysis tool for the instrument. >> > >> > If we use .mzXML files and feed them to Mascot/OMSSA/Tandem + TPP we >> > cannot get anywhere near the number of IDs as with .pkl files >> > generated using PLGS. We've tried conversion to mzXML using MassWolf >> > and msconvert, applying lockmass correction in MassLynx prior to >> > conversion, and comparing acquisition of MS/MS scans in centroid >> mode, >> > vs acquisition in profile mode converted to centroid using MassLynx. >> > An example of the differences in IDs: >> > >> > RAW->PLGS->PKL->Mascot->TPP: 874 peptide IDs (1% FDR) >> > RAW->MassWolf->mzXML->mgf->Mascot->TPP: 279 peptide IDs (1 % FDR) >> > >> > No matter what combination of converter / pre-processing in MassLynx >> > we try, the number of IDs is never above 1/3 of that we get from >> a PKL >> > file. We've also tried changes to the acquisition methods, without >> > success. >> > >> > Is anyone able to provide a MassLynx method file, and/or >> procedure for >> > pre-processing / .mzXML conversion they are using successfully >> with a >> > QTOF Premier or similar instrument? >> > >> > Any help would be gratefully appreciated. Many Thanks, >> > >> > DT >> >> > > > > --~--~---------~--~----~------------~-------~--~----~ You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To post to this group, send email to [email protected] To unsubscribe from this group, send email to [email protected] For more options, visit this group at http://groups.google.com/group/spctools-discuss?hl=en -~----------~----~----~----~------~----~------~--~---
