Re: [spctools-discuss] Libra on MS3-based TMT quantification

['David Shteynberg' via spctools-discuss]

Great!  Thank you.

On Wed, Apr 6, 2022, 7:04 AM Shagun Gupta  wrote:

> Hi David
>
> Apologies, for some reason my post that it all worked out never made it
> through. Instead one of my older replies I think got posted again. Sorry
> for the confusion and thank you for your help!
>
> Shagun
>
> On Tuesday, April 5, 2022 at 3:09:59 PM UTC-4 David Shteynberg wrote:
>
>> Hello Shagun,
>>
>> Thank you for sharing your complete dataset.  I still found the following
>> in the conditions file you included:
>> TMT10_VB297695_condition.xml
>>
>>   
>>
>>
>> This should be set to 0.001 for this analysis to work on your type of
>> label.  I am attaching the condition file with this change included so you
>> can rerun Libra using the condition file attached.  Please make sure the
>> tolerance is correct in the file when you try running Libra again and if
>> you still see a problem please provide your TPP results interact* files
>> etc...
>>
>> Cheers,
>> -David
>>
>>
>>
>> On Fri, Apr 1, 2022 at 10:03 PM Shagun Gupta  wrote:
>>
>>> The zip folder for the files can be found here:
>>> https://drive.google.com/drive/folders/1hnR2igUXM_K-Pld2xspikeu_xnLGUdMc?usp=sharing
>>>
>>> Let me know if you have any issues.
>>>
>>> Best
>>> Shagun
>>>
>>> On Friday, March 25, 2022 at 1:34:57 PM UTC-4 David Shteynberg wrote:
>>>
 Please place your files on a shared drive and send me a link.

 On Fri, Mar 25, 2022 at 10:34 AM Shagun Gupta 
 wrote:

> Hi David
>
> Could you suggest a good email to reach you with? I can share the
> pep.xml's and Libra condition file that way?
>
> -Shagun
>
> On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote:
>
>> Hello Shagun,
>>
>> Thank you for your email and interest in the TPP.  I have recently
>> been comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that
>> produced by TPP's Libra.  As far as I can tell, when I run and compare 
>> the
>> quantities (intensities) they are mostly the same between Libra (without
>> isotopic impurity correction and 0 pseudocounts) and the
>> ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the
>> conditions.xml file that has to be defined for each Libra run.  I would 
>> be
>> happy to take a look at your data and analysis to see if it can be placed
>> on the right path for the Libra analysis to work.  Please post your 
>> results
>> somewhere I can download and test.
>>
>> Cheers,
>> -David
>>
>> On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta 
>> wrote:
>>
>>> Hello
>>>
>>> Our lab has been trying to use Libra to quantify MS2 (Ion-trap)
>>> identification and MS3-based quantification of TMT datasets and we were
>>> trying to benchmark with an artificial yeast and mammalian spiked 
>>> dataset
>>> with known fold-change (FC) values. However we observe drastically
>>> different values than expected, something we don't observe with other
>>> search engines for the same dataset.
>>>
>>> Has this issue been encountered before/ is there something obviously
>>> wrong when running with TPP that might cause this? Happy to provide 
>>> further
>>> details on the dataset and parameters used to run with (most of them
>>> default apart from additions like static modification for TMT and
>>> specification of MS3 for quantification among others).
>>>
>>> Thank you,
>>> Shagun
>>>
>>> --
>>> You received this message because you are subscribed to the Google
>>> Groups "spctools-discuss" group.
>>> To unsubscribe from this group and stop receiving emails from it,
>>> send an email to spctools-discu...@googlegroups.com.
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>>> https://groups.google.com/d/msgid/spctools-discuss/ce2c75fb-2da1-4e05-8d08-bbe5a30b0495n%40googlegroups.com
>>> 
>>> .
>>>
>> --
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 To view this discussion on the web visit
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> 
> .
>
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Re: [spctools-discuss] Libra on MS3-based TMT quantification

[Shagun Gupta]

Hi David

Apologies, for some reason my post that it all worked out never made it 
through. Instead one of my older replies I think got posted again. Sorry 
for the confusion and thank you for your help!

Shagun

On Tuesday, April 5, 2022 at 3:09:59 PM UTC-4 David Shteynberg wrote:

> Hello Shagun,
>
> Thank you for sharing your complete dataset.  I still found the following 
> in the conditions file you included:
> TMT10_VB297695_condition.xml
>
>   
>
>
> This should be set to 0.001 for this analysis to work on your type of 
> label.  I am attaching the condition file with this change included so you 
> can rerun Libra using the condition file attached.  Please make sure the 
> tolerance is correct in the file when you try running Libra again and if 
> you still see a problem please provide your TPP results interact* files 
> etc...
>
> Cheers,
> -David
>
>
>
> On Fri, Apr 1, 2022 at 10:03 PM Shagun Gupta  wrote:
>
>> The zip folder for the files can be found here: 
>> https://drive.google.com/drive/folders/1hnR2igUXM_K-Pld2xspikeu_xnLGUdMc?usp=sharing
>>
>> Let me know if you have any issues.
>>
>> Best
>> Shagun
>>
>> On Friday, March 25, 2022 at 1:34:57 PM UTC-4 David Shteynberg wrote:
>>
>>> Please place your files on a shared drive and send me a link.
>>>
>>> On Fri, Mar 25, 2022 at 10:34 AM Shagun Gupta  
>>> wrote:
>>>
 Hi David

 Could you suggest a good email to reach you with? I can share the 
 pep.xml's and Libra condition file that way?

 -Shagun

 On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote:

> Hello Shagun,
>
> Thank you for your email and interest in the TPP.  I have recently 
> been comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that 
> produced by TPP's Libra.  As far as I can tell, when I run and compare 
> the 
> quantities (intensities) they are mostly the same between Libra (without 
> isotopic impurity correction and 0 pseudocounts) and the 
> ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the 
> conditions.xml file that has to be defined for each Libra run.  I would 
> be 
> happy to take a look at your data and analysis to see if it can be placed 
> on the right path for the Libra analysis to work.  Please post your 
> results 
> somewhere I can download and test.
>
> Cheers,
> -David
>
> On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta  
> wrote:
>
>> Hello
>>
>> Our lab has been trying to use Libra to quantify MS2 (Ion-trap) 
>> identification and MS3-based quantification of TMT datasets and we were 
>> trying to benchmark with an artificial yeast and mammalian spiked 
>> dataset 
>> with known fold-change (FC) values. However we observe drastically 
>> different values than expected, something we don't observe with other 
>> search engines for the same dataset. 
>>
>> Has this issue been encountered before/ is there something obviously 
>> wrong when running with TPP that might cause this? Happy to provide 
>> further 
>> details on the dataset and parameters used to run with (most of them 
>> default apart from additions like static modification for TMT and 
>> specification of MS3 for quantification among others).
>>
>> Thank you,
>> Shagun
>>
>> -- 
>> You received this message because you are subscribed to the Google 
>> Groups "spctools-discuss" group.
>> To unsubscribe from this group and stop receiving emails from it, 
>> send an email to spctools-discu...@googlegroups.com.
>> To view this discussion on the web visit 
>> https://groups.google.com/d/msgid/spctools-discuss/ce2c75fb-2da1-4e05-8d08-bbe5a30b0495n%40googlegroups.com
>>  
>> 
>> .
>>
> -- 
 You received this message because you are subscribed to the Google 
 Groups "spctools-discuss" group.
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>>> To view this discussion on the web visit 
 https://groups.google.com/d/msgid/spctools-discuss/7a0b1188-7c5f-4528-9e90-282b860e7627n%40googlegroups.com
  
 
 .

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>>
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>>  
>> 

Re: [spctools-discuss] Libra on MS3-based TMT quantification

['David Shteynberg' via spctools-discuss]

Hello Shagun,

Thank you for sharing your complete dataset.  I still found the following
in the conditions file you included:
TMT10_VB297695_condition.xml

  


This should be set to 0.001 for this analysis to work on your type of
label.  I am attaching the condition file with this change included so you
can rerun Libra using the condition file attached.  Please make sure the
tolerance is correct in the file when you try running Libra again and if
you still see a problem please provide your TPP results interact* files
etc...

Cheers,
-David



On Fri, Apr 1, 2022 at 10:03 PM Shagun Gupta  wrote:

> The zip folder for the files can be found here:
> https://drive.google.com/drive/folders/1hnR2igUXM_K-Pld2xspikeu_xnLGUdMc?usp=sharing
>
> Let me know if you have any issues.
>
> Best
> Shagun
>
> On Friday, March 25, 2022 at 1:34:57 PM UTC-4 David Shteynberg wrote:
>
>> Please place your files on a shared drive and send me a link.
>>
>> On Fri, Mar 25, 2022 at 10:34 AM Shagun Gupta  wrote:
>>
>>> Hi David
>>>
>>> Could you suggest a good email to reach you with? I can share the
>>> pep.xml's and Libra condition file that way?
>>>
>>> -Shagun
>>>
>>> On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote:
>>>
 Hello Shagun,

 Thank you for your email and interest in the TPP.  I have recently been
 comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that produced
 by TPP's Libra.  As far as I can tell, when I run and compare the
 quantities (intensities) they are mostly the same between Libra (without
 isotopic impurity correction and 0 pseudocounts) and the
 ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the
 conditions.xml file that has to be defined for each Libra run.  I would be
 happy to take a look at your data and analysis to see if it can be placed
 on the right path for the Libra analysis to work.  Please post your results
 somewhere I can download and test.

 Cheers,
 -David

 On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta 
 wrote:

> Hello
>
> Our lab has been trying to use Libra to quantify MS2 (Ion-trap)
> identification and MS3-based quantification of TMT datasets and we were
> trying to benchmark with an artificial yeast and mammalian spiked dataset
> with known fold-change (FC) values. However we observe drastically
> different values than expected, something we don't observe with other
> search engines for the same dataset.
>
> Has this issue been encountered before/ is there something obviously
> wrong when running with TPP that might cause this? Happy to provide 
> further
> details on the dataset and parameters used to run with (most of them
> default apart from additions like static modification for TMT and
> specification of MS3 for quantification among others).
>
> Thank you,
> Shagun
>
> --
> You received this message because you are subscribed to the Google
> Groups "spctools-discuss" group.
> To unsubscribe from this group and stop receiving emails from it, send
> an email to spctools-discu...@googlegroups.com.
> To view this discussion on the web visit
> https://groups.google.com/d/msgid/spctools-discuss/ce2c75fb-2da1-4e05-8d08-bbe5a30b0495n%40googlegroups.com
> 
> .
>
 --
>>> You received this message because you are subscribed to the Google
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>>>
>> To view this discussion on the web visit
>>> https://groups.google.com/d/msgid/spctools-discuss/7a0b1188-7c5f-4528-9e90-282b860e7627n%40googlegroups.com
>>> 
>>> .
>>>
>> --
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> 
> .
>

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Re: [spctools-discuss] Libra on MS3-based TMT quantification

[Shagun Gupta]

Hi David

I've added the files on this drive link:
https://drive.google.com/drive/folders/1hnR2igUXM_K-Pld2xspikeu_xnLGUdMc?usp=sharing

Let me know if you have any issues accessing the files!

-Shagun
On Friday, March 25, 2022 at 1:34:57 PM UTC-4 David Shteynberg wrote:

> Please place your files on a shared drive and send me a link.
>
> On Fri, Mar 25, 2022 at 10:34 AM Shagun Gupta  wrote:
>
>> Hi David
>>
>> Could you suggest a good email to reach you with? I can share the 
>> pep.xml's and Libra condition file that way?
>>
>> -Shagun
>>
>> On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote:
>>
>>> Hello Shagun,
>>>
>>> Thank you for your email and interest in the TPP.  I have recently been 
>>> comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that produced 
>>> by TPP's Libra.  As far as I can tell, when I run and compare the 
>>> quantities (intensities) they are mostly the same between Libra (without 
>>> isotopic impurity correction and 0 pseudocounts) and the 
>>> ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the 
>>> conditions.xml file that has to be defined for each Libra run.  I would be 
>>> happy to take a look at your data and analysis to see if it can be placed 
>>> on the right path for the Libra analysis to work.  Please post your results 
>>> somewhere I can download and test.
>>>
>>> Cheers,
>>> -David
>>>
>>> On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta  wrote:
>>>
 Hello

 Our lab has been trying to use Libra to quantify MS2 (Ion-trap) 
 identification and MS3-based quantification of TMT datasets and we were 
 trying to benchmark with an artificial yeast and mammalian spiked dataset 
 with known fold-change (FC) values. However we observe drastically 
 different values than expected, something we don't observe with other 
 search engines for the same dataset. 

 Has this issue been encountered before/ is there something obviously 
 wrong when running with TPP that might cause this? Happy to provide 
 further 
 details on the dataset and parameters used to run with (most of them 
 default apart from additions like static modification for TMT and 
 specification of MS3 for quantification among others).

 Thank you,
 Shagun

 -- 
 You received this message because you are subscribed to the Google 
 Groups "spctools-discuss" group.
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 To view this discussion on the web visit 
 https://groups.google.com/d/msgid/spctools-discuss/ce2c75fb-2da1-4e05-8d08-bbe5a30b0495n%40googlegroups.com
  
 
 .

>>> -- 
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>>
> To view this discussion on the web visit 
>> https://groups.google.com/d/msgid/spctools-discuss/7a0b1188-7c5f-4528-9e90-282b860e7627n%40googlegroups.com
>>  
>> 
>> .
>>
>

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Re: [spctools-discuss] Libra on MS3-based TMT quantification

[Shagun Gupta]

The zip folder for the files can be found 
here: 
https://drive.google.com/drive/folders/1hnR2igUXM_K-Pld2xspikeu_xnLGUdMc?usp=sharing

Let me know if you have any issues.

Best
Shagun

On Friday, March 25, 2022 at 1:34:57 PM UTC-4 David Shteynberg wrote:

> Please place your files on a shared drive and send me a link.
>
> On Fri, Mar 25, 2022 at 10:34 AM Shagun Gupta  wrote:
>
>> Hi David
>>
>> Could you suggest a good email to reach you with? I can share the 
>> pep.xml's and Libra condition file that way?
>>
>> -Shagun
>>
>> On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote:
>>
>>> Hello Shagun,
>>>
>>> Thank you for your email and interest in the TPP.  I have recently been 
>>> comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that produced 
>>> by TPP's Libra.  As far as I can tell, when I run and compare the 
>>> quantities (intensities) they are mostly the same between Libra (without 
>>> isotopic impurity correction and 0 pseudocounts) and the 
>>> ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the 
>>> conditions.xml file that has to be defined for each Libra run.  I would be 
>>> happy to take a look at your data and analysis to see if it can be placed 
>>> on the right path for the Libra analysis to work.  Please post your results 
>>> somewhere I can download and test.
>>>
>>> Cheers,
>>> -David
>>>
>>> On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta  wrote:
>>>
 Hello

 Our lab has been trying to use Libra to quantify MS2 (Ion-trap) 
 identification and MS3-based quantification of TMT datasets and we were 
 trying to benchmark with an artificial yeast and mammalian spiked dataset 
 with known fold-change (FC) values. However we observe drastically 
 different values than expected, something we don't observe with other 
 search engines for the same dataset. 

 Has this issue been encountered before/ is there something obviously 
 wrong when running with TPP that might cause this? Happy to provide 
 further 
 details on the dataset and parameters used to run with (most of them 
 default apart from additions like static modification for TMT and 
 specification of MS3 for quantification among others).

 Thank you,
 Shagun

 -- 
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 .

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>> https://groups.google.com/d/msgid/spctools-discuss/7a0b1188-7c5f-4528-9e90-282b860e7627n%40googlegroups.com
>>  
>> 
>> .
>>
>

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Re: [spctools-discuss] Libra on MS3-based TMT quantification

['David Shteynberg' via spctools-discuss]

Hello Shagun,

Perhaps I am misunderstand here.  As far as I know, TMT labels are isobaric
and are quantified in the fragment ion spectra.  Any peptide quantified
would have to have the same precursor mass across all TMT labels.
 Presumably there are not that many peptides that are identical between
yeast and human.  The difference in your ratios will only be observable in
the conserved peptides between human and yeast.  Do you have examples of
specific spectra we can consider that work with this approach?  I was able
to run the TPP tools including Libra on this data.

Cheers,
David


On Sat, Mar 26, 2022, 8:11 PM Shagun Gupta  wrote:

> Hi David
>
> So I reran with the changed parameter and the issue still seems to
> persist. I can share the updated results if you'd like as well? Also just
> confirming libra1 - 126, libra2 = 127N and so forth for a TMT10plex for
> example, since the issue is much aggravated in 2 out of three possible
> comparisons. To explain the setup more, yeast proteins are spiked with
> mammalian proteins such that yeast proteins are 10:4:1 (three replicates
> each) with mammalian proteins being (1:1:1) for the same.
>
> Shagun
>
> On Friday, March 25, 2022 at 6:02:55 PM UTC-4 Shagun Gupta wrote:
>
>> Hi David
>>
>> Will do so, thank you! That makes a lot of sense. I have also added the
>> mzXML files but this might be the cause of the discrepancy I see!
>>
>> Thanks
>> Shagun
>>
>> On Friday, March 25, 2022 at 4:42:46 PM UTC-4 David Shteynberg wrote:
>>
>>> Hello Shagun,
>>>
>>> I noticed in your condition file you are using TMT10 with the following
>>> masses:
>>>  
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>>>
>>>
>>> The difference between neighboring channels is <0.01 at the lowest and
>>> yet you are using tolerance of 0.2:
>>>
>>> 
>>>
>>>
>>> I think the appropriate mass tolerance for this type of labeling should
>>> be ~0.001.
>>>
>>> Does that make sense?  Please try running Libra with the mass tolerance
>>> appropriate for this type of label.
>>>
>>> Cheers,
>>> -David
>>>
>>> On Fri, Mar 25, 2022 at 10:34 AM Shagun Gupta 
>>> wrote:
>>>
 Hi David

 Could you suggest a good email to reach you with? I can share the
 pep.xml's and Libra condition file that way?

 -Shagun

 On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote:

> Hello Shagun,
>
> Thank you for your email and interest in the TPP.  I have recently
> been comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that
> produced by TPP's Libra.  As far as I can tell, when I run and compare the
> quantities (intensities) they are mostly the same between Libra (without
> isotopic impurity correction and 0 pseudocounts) and the
> ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the
> conditions.xml file that has to be defined for each Libra run.  I would be
> happy to take a look at your data and analysis to see if it can be placed
> on the right path for the Libra analysis to work.  Please post your 
> results
> somewhere I can download and test.
>
> Cheers,
> -David
>
> On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta 
> wrote:
>
>> Hello
>>
>> Our lab has been trying to use Libra to quantify MS2 (Ion-trap)
>> identification and MS3-based quantification of TMT datasets and we were
>> trying to benchmark with an artificial yeast and mammalian spiked dataset
>> with known fold-change (FC) values. However we observe drastically
>> different values than expected, something we don't observe with other
>> search engines for the same dataset.
>>
>> Has this issue been encountered before/ is there something obviously
>> wrong when running with TPP that might cause this? Happy to provide 
>> further
>> details on the dataset and parameters used to run with (most of them
>> default apart from additions like static modification for TMT and
>> specification of MS3 for quantification among others).
>>
>> Thank you,
>> Shagun
>>
>> --
>> You received this message because you are subscribed to the Google
>> Groups "spctools-discuss" group.
>> To unsubscribe from this group and stop receiving emails from it,
>> send an email to spctools-discu...@googlegroups.com.
>> To view this discussion on the web visit
>> https://groups.google.com/d/msgid/spctools-discuss/ce2c75fb-2da1-4e05-8d08-bbe5a30b0495n%40googlegroups.com
>> 
>> .
>>
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>>> To view this di

Re: [spctools-discuss] Libra on MS3-based TMT quantification

[Shagun Gupta]

Hi David

So I reran with the changed parameter and the issue still seems to persist. 
I can share the updated results if you'd like as well? Also just confirming 
libra1 - 126, libra2 = 127N and so forth for a TMT10plex for example, since 
the issue is much aggravated in 2 out of three possible comparisons. To 
explain the setup more, yeast proteins are spiked with mammalian proteins 
such that yeast proteins are 10:4:1 (three replicates each) with mammalian 
proteins being (1:1:1) for the same.

Shagun

On Friday, March 25, 2022 at 6:02:55 PM UTC-4 Shagun Gupta wrote:

> Hi David
>
> Will do so, thank you! That makes a lot of sense. I have also added the 
> mzXML files but this might be the cause of the discrepancy I see!
>
> Thanks
> Shagun
>
> On Friday, March 25, 2022 at 4:42:46 PM UTC-4 David Shteynberg wrote:
>
>> Hello Shagun,
>>
>> I noticed in your condition file you are using TMT10 with the following 
>> masses:
>>  
>> 
>> 
>> 
>> 
>> 
>> 
>> 
>> 
>> 
>>
>>
>> The difference between neighboring channels is <0.01 at the lowest and 
>> yet you are using tolerance of 0.2:
>>
>> 
>>
>>
>> I think the appropriate mass tolerance for this type of labeling should 
>> be ~0.001.
>>
>> Does that make sense?  Please try running Libra with the mass tolerance 
>> appropriate for this type of label. 
>>
>> Cheers,
>> -David
>>
>> On Fri, Mar 25, 2022 at 10:34 AM Shagun Gupta  wrote:
>>
>>> Hi David
>>>
>>> Could you suggest a good email to reach you with? I can share the 
>>> pep.xml's and Libra condition file that way?
>>>
>>> -Shagun
>>>
>>> On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote:
>>>
 Hello Shagun,

 Thank you for your email and interest in the TPP.  I have recently been 
 comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that 
 produced 
 by TPP's Libra.  As far as I can tell, when I run and compare the 
 quantities (intensities) they are mostly the same between Libra (without 
 isotopic impurity correction and 0 pseudocounts) and the 
 ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the 
 conditions.xml file that has to be defined for each Libra run.  I would be 
 happy to take a look at your data and analysis to see if it can be placed 
 on the right path for the Libra analysis to work.  Please post your 
 results 
 somewhere I can download and test.

 Cheers,
 -David

 On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta  
 wrote:

> Hello
>
> Our lab has been trying to use Libra to quantify MS2 (Ion-trap) 
> identification and MS3-based quantification of TMT datasets and we were 
> trying to benchmark with an artificial yeast and mammalian spiked dataset 
> with known fold-change (FC) values. However we observe drastically 
> different values than expected, something we don't observe with other 
> search engines for the same dataset. 
>
> Has this issue been encountered before/ is there something obviously 
> wrong when running with TPP that might cause this? Happy to provide 
> further 
> details on the dataset and parameters used to run with (most of them 
> default apart from additions like static modification for TMT and 
> specification of MS3 for quantification among others).
>
> Thank you,
> Shagun
>
> -- 
> You received this message because you are subscribed to the Google 
> Groups "spctools-discuss" group.
> To unsubscribe from this group and stop receiving emails from it, send 
> an email to spctools-discu...@googlegroups.com.
> To view this discussion on the web visit 
> https://groups.google.com/d/msgid/spctools-discuss/ce2c75fb-2da1-4e05-8d08-bbe5a30b0495n%40googlegroups.com
>  
> 
> .
>
 -- 
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>>>
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>>> https://groups.google.com/d/msgid/spctools-discuss/7a0b1188-7c5f-4528-9e90-282b860e7627n%40googlegroups.com
>>>  
>>> 
>>> .
>>>
>>

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Re: [spctools-discuss] Libra on MS3-based TMT quantification

[Shagun Gupta]

Hi David

Will do so, thank you! That makes a lot of sense. I have also added the 
mzXML files but this might be the cause of the discrepancy I see!

Thanks
Shagun

On Friday, March 25, 2022 at 4:42:46 PM UTC-4 David Shteynberg wrote:

> Hello Shagun,
>
> I noticed in your condition file you are using TMT10 with the following 
> masses:
>  
> 
> 
> 
> 
> 
> 
> 
> 
> 
>
>
> The difference between neighboring channels is <0.01 at the lowest and yet 
> you are using tolerance of 0.2:
>
> 
>
>
> I think the appropriate mass tolerance for this type of labeling should be 
> ~0.001.
>
> Does that make sense?  Please try running Libra with the mass tolerance 
> appropriate for this type of label. 
>
> Cheers,
> -David
>
> On Fri, Mar 25, 2022 at 10:34 AM Shagun Gupta  wrote:
>
>> Hi David
>>
>> Could you suggest a good email to reach you with? I can share the 
>> pep.xml's and Libra condition file that way?
>>
>> -Shagun
>>
>> On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote:
>>
>>> Hello Shagun,
>>>
>>> Thank you for your email and interest in the TPP.  I have recently been 
>>> comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that produced 
>>> by TPP's Libra.  As far as I can tell, when I run and compare the 
>>> quantities (intensities) they are mostly the same between Libra (without 
>>> isotopic impurity correction and 0 pseudocounts) and the 
>>> ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the 
>>> conditions.xml file that has to be defined for each Libra run.  I would be 
>>> happy to take a look at your data and analysis to see if it can be placed 
>>> on the right path for the Libra analysis to work.  Please post your results 
>>> somewhere I can download and test.
>>>
>>> Cheers,
>>> -David
>>>
>>> On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta  wrote:
>>>
 Hello

 Our lab has been trying to use Libra to quantify MS2 (Ion-trap) 
 identification and MS3-based quantification of TMT datasets and we were 
 trying to benchmark with an artificial yeast and mammalian spiked dataset 
 with known fold-change (FC) values. However we observe drastically 
 different values than expected, something we don't observe with other 
 search engines for the same dataset. 

 Has this issue been encountered before/ is there something obviously 
 wrong when running with TPP that might cause this? Happy to provide 
 further 
 details on the dataset and parameters used to run with (most of them 
 default apart from additions like static modification for TMT and 
 specification of MS3 for quantification among others).

 Thank you,
 Shagun

 -- 
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 Groups "spctools-discuss" group.
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 .

>>> -- 
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>> https://groups.google.com/d/msgid/spctools-discuss/7a0b1188-7c5f-4528-9e90-282b860e7627n%40googlegroups.com
>>  
>> 
>> .
>>
>

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Re: [spctools-discuss] Libra on MS3-based TMT quantification

['David Shteynberg' via spctools-discuss]

Hello Shagun,

I noticed in your condition file you are using TMT10 with the following
masses:
 











The difference between neighboring channels is <0.01 at the lowest and yet
you are using tolerance of 0.2:




I think the appropriate mass tolerance for this type of labeling should be
~0.001.

Does that make sense?  Please try running Libra with the mass tolerance
appropriate for this type of label.

Cheers,
-David

On Fri, Mar 25, 2022 at 10:34 AM Shagun Gupta  wrote:

> Hi David
>
> Could you suggest a good email to reach you with? I can share the
> pep.xml's and Libra condition file that way?
>
> -Shagun
>
> On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote:
>
>> Hello Shagun,
>>
>> Thank you for your email and interest in the TPP.  I have recently been
>> comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that produced
>> by TPP's Libra.  As far as I can tell, when I run and compare the
>> quantities (intensities) they are mostly the same between Libra (without
>> isotopic impurity correction and 0 pseudocounts) and the
>> ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the
>> conditions.xml file that has to be defined for each Libra run.  I would be
>> happy to take a look at your data and analysis to see if it can be placed
>> on the right path for the Libra analysis to work.  Please post your results
>> somewhere I can download and test.
>>
>> Cheers,
>> -David
>>
>> On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta  wrote:
>>
>>> Hello
>>>
>>> Our lab has been trying to use Libra to quantify MS2 (Ion-trap)
>>> identification and MS3-based quantification of TMT datasets and we were
>>> trying to benchmark with an artificial yeast and mammalian spiked dataset
>>> with known fold-change (FC) values. However we observe drastically
>>> different values than expected, something we don't observe with other
>>> search engines for the same dataset.
>>>
>>> Has this issue been encountered before/ is there something obviously
>>> wrong when running with TPP that might cause this? Happy to provide further
>>> details on the dataset and parameters used to run with (most of them
>>> default apart from additions like static modification for TMT and
>>> specification of MS3 for quantification among others).
>>>
>>> Thank you,
>>> Shagun
>>>
>>> --
>>> You received this message because you are subscribed to the Google
>>> Groups "spctools-discuss" group.
>>> To unsubscribe from this group and stop receiving emails from it, send
>>> an email to spctools-discu...@googlegroups.com.
>>> To view this discussion on the web visit
>>> https://groups.google.com/d/msgid/spctools-discuss/ce2c75fb-2da1-4e05-8d08-bbe5a30b0495n%40googlegroups.com
>>> 
>>> .
>>>
>> --
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> 
> .
>

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Re: [spctools-discuss] Libra on MS3-based TMT quantification

['David Shteynberg' via spctools-discuss]

Hello Shagun,

Also please include your mzML files, which contain the actual data that
Libra quantifies.

Thanks!
-David

On Fri, Mar 25, 2022 at 10:34 AM David Shteynberg <
david.shteynb...@isbscience.org> wrote:

> Please place your files on a shared drive and send me a link.
>
> On Fri, Mar 25, 2022 at 10:34 AM Shagun Gupta  wrote:
>
>> Hi David
>>
>> Could you suggest a good email to reach you with? I can share the
>> pep.xml's and Libra condition file that way?
>>
>> -Shagun
>>
>> On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote:
>>
>>> Hello Shagun,
>>>
>>> Thank you for your email and interest in the TPP.  I have recently been
>>> comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that produced
>>> by TPP's Libra.  As far as I can tell, when I run and compare the
>>> quantities (intensities) they are mostly the same between Libra (without
>>> isotopic impurity correction and 0 pseudocounts) and the
>>> ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the
>>> conditions.xml file that has to be defined for each Libra run.  I would be
>>> happy to take a look at your data and analysis to see if it can be placed
>>> on the right path for the Libra analysis to work.  Please post your results
>>> somewhere I can download and test.
>>>
>>> Cheers,
>>> -David
>>>
>>> On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta  wrote:
>>>
 Hello

 Our lab has been trying to use Libra to quantify MS2 (Ion-trap)
 identification and MS3-based quantification of TMT datasets and we were
 trying to benchmark with an artificial yeast and mammalian spiked dataset
 with known fold-change (FC) values. However we observe drastically
 different values than expected, something we don't observe with other
 search engines for the same dataset.

 Has this issue been encountered before/ is there something obviously
 wrong when running with TPP that might cause this? Happy to provide further
 details on the dataset and parameters used to run with (most of them
 default apart from additions like static modification for TMT and
 specification of MS3 for quantification among others).

 Thank you,
 Shagun

 --
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 To view this discussion on the web visit
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 .

>>> --
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>> 
>> .
>>
>

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Re: [spctools-discuss] Libra on MS3-based TMT quantification

['David Shteynberg' via spctools-discuss]

Please place your files on a shared drive and send me a link.

On Fri, Mar 25, 2022 at 10:34 AM Shagun Gupta  wrote:

> Hi David
>
> Could you suggest a good email to reach you with? I can share the
> pep.xml's and Libra condition file that way?
>
> -Shagun
>
> On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote:
>
>> Hello Shagun,
>>
>> Thank you for your email and interest in the TPP.  I have recently been
>> comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that produced
>> by TPP's Libra.  As far as I can tell, when I run and compare the
>> quantities (intensities) they are mostly the same between Libra (without
>> isotopic impurity correction and 0 pseudocounts) and the
>> ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the
>> conditions.xml file that has to be defined for each Libra run.  I would be
>> happy to take a look at your data and analysis to see if it can be placed
>> on the right path for the Libra analysis to work.  Please post your results
>> somewhere I can download and test.
>>
>> Cheers,
>> -David
>>
>> On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta  wrote:
>>
>>> Hello
>>>
>>> Our lab has been trying to use Libra to quantify MS2 (Ion-trap)
>>> identification and MS3-based quantification of TMT datasets and we were
>>> trying to benchmark with an artificial yeast and mammalian spiked dataset
>>> with known fold-change (FC) values. However we observe drastically
>>> different values than expected, something we don't observe with other
>>> search engines for the same dataset.
>>>
>>> Has this issue been encountered before/ is there something obviously
>>> wrong when running with TPP that might cause this? Happy to provide further
>>> details on the dataset and parameters used to run with (most of them
>>> default apart from additions like static modification for TMT and
>>> specification of MS3 for quantification among others).
>>>
>>> Thank you,
>>> Shagun
>>>
>>> --
>>> You received this message because you are subscribed to the Google
>>> Groups "spctools-discuss" group.
>>> To unsubscribe from this group and stop receiving emails from it, send
>>> an email to spctools-discu...@googlegroups.com.
>>> To view this discussion on the web visit
>>> https://groups.google.com/d/msgid/spctools-discuss/ce2c75fb-2da1-4e05-8d08-bbe5a30b0495n%40googlegroups.com
>>> 
>>> .
>>>
>> --
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> 
> .
>

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Re: [spctools-discuss] Libra on MS3-based TMT quantification

[Shagun Gupta]

Hi David

Could you suggest a good email to reach you with? I can share the pep.xml's 
and Libra condition file that way?

-Shagun

On Friday, March 25, 2022 at 11:00:38 AM UTC-4 David Shteynberg wrote:

> Hello Shagun,
>
> Thank you for your email and interest in the TPP.  I have recently been 
> comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that produced 
> by TPP's Libra.  As far as I can tell, when I run and compare the 
> quantities (intensities) they are mostly the same between Libra (without 
> isotopic impurity correction and 0 pseudocounts) and the 
> ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the 
> conditions.xml file that has to be defined for each Libra run.  I would be 
> happy to take a look at your data and analysis to see if it can be placed 
> on the right path for the Libra analysis to work.  Please post your results 
> somewhere I can download and test.
>
> Cheers,
> -David
>
> On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta  wrote:
>
>> Hello
>>
>> Our lab has been trying to use Libra to quantify MS2 (Ion-trap) 
>> identification and MS3-based quantification of TMT datasets and we were 
>> trying to benchmark with an artificial yeast and mammalian spiked dataset 
>> with known fold-change (FC) values. However we observe drastically 
>> different values than expected, something we don't observe with other 
>> search engines for the same dataset. 
>>
>> Has this issue been encountered before/ is there something obviously 
>> wrong when running with TPP that might cause this? Happy to provide further 
>> details on the dataset and parameters used to run with (most of them 
>> default apart from additions like static modification for TMT and 
>> specification of MS3 for quantification among others).
>>
>> Thank you,
>> Shagun
>>
>> -- 
>> You received this message because you are subscribed to the Google Groups 
>> "spctools-discuss" group.
>> To unsubscribe from this group and stop receiving emails from it, send an 
>> email to spctools-discu...@googlegroups.com.
>> To view this discussion on the web visit 
>> https://groups.google.com/d/msgid/spctools-discuss/ce2c75fb-2da1-4e05-8d08-bbe5a30b0495n%40googlegroups.com
>>  
>> 
>> .
>>
>

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Re: [spctools-discuss] Libra on MS3-based TMT quantification

['David Shteynberg' via spctools-discuss]

Hello Shagun,

Thank you for your email and interest in the TPP.  I have recently been
comparing TMTPro analysis by ProteomeDiscoverer and Byonic to that produced
by TPP's Libra.  As far as I can tell, when I run and compare the
quantities (intensities) they are mostly the same between Libra (without
isotopic impurity correction and 0 pseudocounts) and the
ProteomeDiscoverer/Byonic.  Execution of Libra is defined in the
conditions.xml file that has to be defined for each Libra run.  I would be
happy to take a look at your data and analysis to see if it can be placed
on the right path for the Libra analysis to work.  Please post your results
somewhere I can download and test.

Cheers,
-David

On Fri, Mar 25, 2022 at 6:36 AM Shagun Gupta  wrote:

> Hello
>
> Our lab has been trying to use Libra to quantify MS2 (Ion-trap)
> identification and MS3-based quantification of TMT datasets and we were
> trying to benchmark with an artificial yeast and mammalian spiked dataset
> with known fold-change (FC) values. However we observe drastically
> different values than expected, something we don't observe with other
> search engines for the same dataset.
>
> Has this issue been encountered before/ is there something obviously wrong
> when running with TPP that might cause this? Happy to provide further
> details on the dataset and parameters used to run with (most of them
> default apart from additions like static modification for TMT and
> specification of MS3 for quantification among others).
>
> Thank you,
> Shagun
>
> --
> You received this message because you are subscribed to the Google Groups
> "spctools-discuss" group.
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> https://groups.google.com/d/msgid/spctools-discuss/ce2c75fb-2da1-4e05-8d08-bbe5a30b0495n%40googlegroups.com
> 
> .
>

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