[spctools-discuss] Re: QTOF Premier SILAC data - MassWolf vs PKL

[Dave Trudgian]

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 > > 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 http://tools.proteomecenter.org/wiki/index.php?
>> title=Software:pkl2mzXML
>> 
>> ">Link?
>> 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 > > 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
>>
>> 
>
> >
>   


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[spctools-discuss] Re: QTOF Premier SILAC data - MassWolf vs PKL

[Matthew Chambers]

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  > 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 http://tools.proteomecenter.org/wiki/index.php?
> title=Software:pkl2mzXML
> 
> ">Link?
> 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  > 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
>

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[spctools-discuss] Re: QTOF Premier SILAC data - MassWolf vs PKL

[Brian Pratt]

Would that retain elution time information?  That's important for many types
of analysis.

On Thu, Oct 1, 2009 at 5:56 AM, ChargedPeptide  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 http://tools.proteomecenter.org/wiki/index.php?
> title=Software:pkl2mzXML">Link?
> 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  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
>
> >
>

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[spctools-discuss] Re: QTOF Premier SILAC data - MassWolf vs PKL

[ChargedPeptide]

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 http://tools.proteomecenter.org/wiki/index.php?
title=Software:pkl2mzXML">Link?
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  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

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[spctools-discuss] Re: QTOF Premier SILAC data - MassWolf vs PKL

[Dave Trudgian]

Frederik,

Many thanks for this information, I hadn't come across the Proteios 
convertor. Unfortunately we're working now with SILAC data where we need 
the MS1 for quantitation, but the workflow might be useful for other 
data. I have previously extracted EMRT information from PLGS XML files 
in MS^e experiments, but hadn't thought to look at the XML files for DDA.

Thanks,

DT

fredrik wrote:
> Hi Dave,
>
> An option could be to run PLGS and then convert the PLGS XML files to
> mzML using the Proteios SE converter. Using this workflow the spectrum
> ids and retention times are retained. However, the level 1 MS data is
> lost, so it might not be ideal for your workflow, apart from the
> identifications. But you'll probably gain lock mass correction and
> spectrum summing, compared to the massWolf workflow. The standalone
> converter is found here:
> http://dev.thep.lu.se/fp6-prodac/browser/trunk/mzML/mzMLconverters.zip
> The input PLGS XML files are found in subdirectories of the PLGS
> project directory, and are all named MassSpectrum.xml.
> Here is a perl script that renames the PLGS files to the name of the
> raw data files:
> http://www.proteios.org/trac/wiki/GelBasedReport
>
> Regards
>
> Fredrik
>
> On Sep 14, 1:37 pm, dctrud  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
>> 
>
> >
>   


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[spctools-discuss] Re: QTOF Premier SILAC data - MassWolf vs PKL

[fredrik]

Hi Dave,

An option could be to run PLGS and then convert the PLGS XML files to
mzML using the Proteios SE converter. Using this workflow the spectrum
ids and retention times are retained. However, the level 1 MS data is
lost, so it might not be ideal for your workflow, apart from the
identifications. But you'll probably gain lock mass correction and
spectrum summing, compared to the massWolf workflow. The standalone
converter is found here:
http://dev.thep.lu.se/fp6-prodac/browser/trunk/mzML/mzMLconverters.zip
The input PLGS XML files are found in subdirectories of the PLGS
project directory, and are all named MassSpectrum.xml.
Here is a perl script that renames the PLGS files to the name of the
raw data files:
http://www.proteios.org/trac/wiki/GelBasedReport

Regards

Fredrik

On Sep 14, 1:37 pm, dctrud  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

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[spctools-discuss] Re: QTOF Premier SILAC data - MassWolf vs PKL

[Matthew Chambers]

Can you discern the order the scans are enumerated in the PKLs with only 
the precursor mass and the peaks? I.e. is it:
function=2 scan=1
function=2 scan=2
function=2 scan=3
function=3 scan=1
function=3 scan=2
function=3 scan=3

or

function=2 scan=1
function=3 scan=1
function=2 scan=2
function=3 scan=2
function=2 scan=3
function=3 scan=3

I would expect one of those two orders (with the added complication of 
scan combination: does it combine only within a single function or can 
it also combine scans from multiple functions?). If it's some seemingly 
random order, then it's indeed pretty hopeless to directly compare the 
exports. :(

However, even in the worst case it might be informative or at least 
interesting to see what a spectral-duplicate-finder application could do 
for comparing the two spectrum lists. Presumably, the combined scans in 
the PKLs would score highest with the several uncombined scans in the 
mzML that went into the combination. Deisotoping might throw a wrench in 
the duplicate comparison, but the question would be does it throw a 
smaller wrench into the comparison between the combined PKL scan and its 
uncombined mzML counterparts than it does for other comparisons. We have 
an as-yet unpublished tool that has several spectral duplicate scoring 
algorithms, so I'll still take a look at the data if you want. We have 
Waters raw data but none of it is processed with PLGS to compare 
msconvert's processing to, so that's my motivation to help. :)

-Matt


Dave Trudgian wrote:
> Matt,
>
> Thanks for your comments. Unfortunately the pkl format doesn't retain 
> any scan numbering or retention time information. I've tried comparing 
> the output, but it's pretty much impossible to match things up. Trying 
> to match precursor masses to compare associated MS/MS spectra doesn't 
> work, due to the PLGS mass correction resulting in different precursor 
> masses in the pkl vs mzXML, even if the RAW file is lockmass corrected 
> with MassLynx prior to mzXML conversion. Thanks for the offer of having 
> a look though.
>
> Nice to hear that PLGS should feature mzML export soon. We're eagerly 
> awaiting PLGS 2.4 for many reasons. A new Aglient QTOF arrives here 
> soon, and the Waters machine will then most likely be used mainly for 
> label-free MSe work with PLGS, but would be nice to have the option of 
> putting Waters DDA data through our pipeline. We now have 5 instruments 
> from 5 different vendors... all good fun!
>
> Many Thanks,
>
> DT
>
> Matt Chambers wrote:
>   
>> It usually comes down to spectrum processing - specifically scan 
>> combination, deisotoping, and centroiding. MassWolf and msconvert are 
>> unlikely to be able to do as well as the vendor's own processing without 
>> significant effort devoted toward optimizing on that platform. I'll take 
>> a look at the data if you want: I'd compare the results of the PKL 
>> export (assuming they are named in a way that retains function/scan 
>> information, or at least retention time) to msconvert's mzML export 
>> (which I know preserves function/scan information) to try to determine 
>> what is the difference in processing of the data. I know they're working 
>> on an mzML export for PLGS; if they preserve scan combination metadata 
>> in their output then that would be a great help as well.
>>
>> -Matt
>>
>>
>> dctrud 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

--~--~-~--

[spctools-discuss] Re: QTOF Premier SILAC data - MassWolf vs PKL

[Dave Trudgian]

Matt,

I'll check with the owner of the data about sending it to you. Could you 
let me know how's best to send it? Feel free to contact me off the list 
at this address.

The spectral comparison software sounds interesting!

Thanks,

DT


Matthew Chambers wrote:
> Can you discern the order the scans are enumerated in the PKLs with only 
> the precursor mass and the peaks? I.e. is it:
> function=2 scan=1
> function=2 scan=2
> function=2 scan=3
> function=3 scan=1
> function=3 scan=2
> function=3 scan=3
>
> or
>
> function=2 scan=1
> function=3 scan=1
> function=2 scan=2
> function=3 scan=2
> function=2 scan=3
> function=3 scan=3
>
> I would expect one of those two orders (with the added complication of 
> scan combination: does it combine only within a single function or can 
> it also combine scans from multiple functions?). If it's some seemingly 
> random order, then it's indeed pretty hopeless to directly compare the 
> exports. :(
>
> However, even in the worst case it might be informative or at least 
> interesting to see what a spectral-duplicate-finder application could do 
> for comparing the two spectrum lists. Presumably, the combined scans in 
> the PKLs would score highest with the several uncombined scans in the 
> mzML that went into the combination. Deisotoping might throw a wrench in 
> the duplicate comparison, but the question would be does it throw a 
> smaller wrench into the comparison between the combined PKL scan and its 
> uncombined mzML counterparts than it does for other comparisons. We have 
> an as-yet unpublished tool that has several spectral duplicate scoring 
> algorithms, so I'll still take a look at the data if you want. We have 
> Waters raw data but none of it is processed with PLGS to compare 
> msconvert's processing to, so that's my motivation to help. :)
>
> -Matt
>
>
> Dave Trudgian wrote:
>   
>> Matt,
>>
>> Thanks for your comments. Unfortunately the pkl format doesn't retain 
>> any scan numbering or retention time information. I've tried comparing 
>> the output, but it's pretty much impossible to match things up. Trying 
>> to match precursor masses to compare associated MS/MS spectra doesn't 
>> work, due to the PLGS mass correction resulting in different precursor 
>> masses in the pkl vs mzXML, even if the RAW file is lockmass corrected 
>> with MassLynx prior to mzXML conversion. Thanks for the offer of having 
>> a look though.
>>
>> Nice to hear that PLGS should feature mzML export soon. We're eagerly 
>> awaiting PLGS 2.4 for many reasons. A new Aglient QTOF arrives here 
>> soon, and the Waters machine will then most likely be used mainly for 
>> label-free MSe work with PLGS, but would be nice to have the option of 
>> putting Waters DDA data through our pipeline. We now have 5 instruments 
>> from 5 different vendors... all good fun!
>>
>> Many Thanks,
>>
>> DT
>>
>> Matt Chambers wrote:
>>   
>> 
>>> It usually comes down to spectrum processing - specifically scan 
>>> combination, deisotoping, and centroiding. MassWolf and msconvert are 
>>> unlikely to be able to do as well as the vendor's own processing without 
>>> significant effort devoted toward optimizing on that platform. I'll take 
>>> a look at the data if you want: I'd compare the results of the PKL 
>>> export (assuming they are named in a way that retains function/scan 
>>> information, or at least retention time) to msconvert's mzML export 
>>> (which I know preserves function/scan information) to try to determine 
>>> what is the difference in processing of the data. I know they're working 
>>> on an mzML export for PLGS; if they preserve scan combination metadata 
>>> in their output then that would be a great help as well.
>>>
>>> -Matt
>>>
>>>
>>> dctrud 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 nu

[spctools-discuss] Re: QTOF Premier SILAC data - MassWolf vs PKL

[Dave Trudgian]

Matt,

Thanks for your comments. Unfortunately the pkl format doesn't retain 
any scan numbering or retention time information. I've tried comparing 
the output, but it's pretty much impossible to match things up. Trying 
to match precursor masses to compare associated MS/MS spectra doesn't 
work, due to the PLGS mass correction resulting in different precursor 
masses in the pkl vs mzXML, even if the RAW file is lockmass corrected 
with MassLynx prior to mzXML conversion. Thanks for the offer of having 
a look though.

Nice to hear that PLGS should feature mzML export soon. We're eagerly 
awaiting PLGS 2.4 for many reasons. A new Aglient QTOF arrives here 
soon, and the Waters machine will then most likely be used mainly for 
label-free MSe work with PLGS, but would be nice to have the option of 
putting Waters DDA data through our pipeline. We now have 5 instruments 
from 5 different vendors... all good fun!

Many Thanks,

DT

Matt Chambers wrote:
> It usually comes down to spectrum processing - specifically scan 
> combination, deisotoping, and centroiding. MassWolf and msconvert are 
> unlikely to be able to do as well as the vendor's own processing without 
> significant effort devoted toward optimizing on that platform. I'll take 
> a look at the data if you want: I'd compare the results of the PKL 
> export (assuming they are named in a way that retains function/scan 
> information, or at least retention time) to msconvert's mzML export 
> (which I know preserves function/scan information) to try to determine 
> what is the difference in processing of the data. I know they're working 
> on an mzML export for PLGS; if they preserve scan combination metadata 
> in their output then that would be a great help as well.
>
> -Matt
>
>
> dctrud 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
>> 
>
> >
>   


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[spctools-discuss] Re: QTOF Premier SILAC data - MassWolf vs PKL

[Matt Chambers]

It usually comes down to spectrum processing - specifically scan 
combination, deisotoping, and centroiding. MassWolf and msconvert are 
unlikely to be able to do as well as the vendor's own processing without 
significant effort devoted toward optimizing on that platform. I'll take 
a look at the data if you want: I'd compare the results of the PKL 
export (assuming they are named in a way that retains function/scan 
information, or at least retention time) to msconvert's mzML export 
(which I know preserves function/scan information) to try to determine 
what is the difference in processing of the data. I know they're working 
on an mzML export for PLGS; if they preserve scan combination metadata 
in their output then that would be a great help as well.

-Matt


dctrud 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

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