Re: [spctools-discuss] Libra quantification on TMT 16-plex returns zeros only

2022-09-09 Thread 'Luis Mendoza' via spctools-discuss 
Great, glad that it is now working for you!  We will remove references to
that deprecated parameter from our documentation.
Cheers,
--Luis


On Fri, Sep 9, 2022 at 1:54 PM Yasir Ahmed  wrote:

> Thanks Eric and Luis! Turns out I was using the wrong/older syntax in the
> condition file (targetMS level = “3”). The quantification works now. Thanks
> for your help!!!
>
> Cheers,
> Yasir
>
> On Sep 8, 2022, at 6:15 PM, 'Luis Mendoza' via spctools-discuss <
> spctools-discuss@googlegroups.com> wrote:
>
> Hi Yasir,
> Is your data Thermo SPS where the reporter ions are in MS3?  If so, please
> check the relevant box when producing the condition file:
>
> 
>
>
> As Eric noted, it does not seem like your MS2 spectra contain reporter ion
> signals, at least from the one spectrum you posted.  Are there others where
> this is not the case?
>
> Cheers,
> --Luis
>
>
> On Thu, Sep 8, 2022 at 2:28 PM 'Eric Deutsch' via spctools-discuss <
> spctools-discuss@googlegroups.com> wrote:
>
>> I’m thinking that ion trap spectra are usually unsuitable for TMT:
>>
>>
>> 
>>
>>
>> I think you would want to acquire fragmentation spectra with Orbitrap HCD?
>>
>>
>>
>> *From:* spctools-discuss@googlegroups.com <
>> spctools-discuss@googlegroups.com> *On Behalf Of *Yasir Ahmed
>> *Sent:* Thursday, September 8, 2022 2:19 PM
>> *To:* spctools-discuss@googlegroups.com
>> *Subject:* Re: [spctools-discuss] Libra quantification on TMT 16-plex
>> returns zeros only
>>
>>
>> Hi Luis,
>>
>>
>> Just to follow up: I found a handful of cases where one of the channels
>> has super high quantification and observable reporter ions (see screenshots
>> below), albeit it should be way more than this, I believe. I’ll keep
>> digging, but let me know if you have any other ideas.
>>
>>
>> Again, thanks for you suggestions.
>>
>>
>> Cheers,
>>
>> Yasir
>>
>>
>> 
>>
>>
>> 
>>
>>
>> 
>>
>>
>>
>> On Sep 8, 2022, at 5:07 PM, Yasir Ahmed  wrote:
>>
>>
>> Hi Luis,
>>
>>
>> Thanks a lot for the response.
>>
>>
>> Can you try re-running Libra without the centroiding option?  (i.e. set
>> type="0") Or are your mzML data not centroided?
>>
>>
>>
>> I just tried that, but no difference in outcome.
>>
>>
>>
>> Another thing to try: are you able to open the PeptideProphet + Libra
>> results in the PepXMLViewer?  You should be able to see if there are a
>> significant number of quantified PSMs -- you should also be able to examine
>> a few individual spectra and zoom in on the reporter ion signals.
>>
>>
>>
>> I’m able to look at a few individual spectra, and I think they all look
>> mostly good—but frankly I’m not too sure how to evaluate their suitability
>> (see example screenshots below).
>>
>>
>>
>> Lastly, what version of TPP are you running?
>>
>>
>>
>> 6.1.0
>>
>>
>> Thanks again!
>>
>> Yasir
>>
>>
>> 
>>
>>
>> 
>>
>>
>>
>>
>>
>>
>> Cheers,
>>
>> --Luis
>>
>>
>>
>> On Thu, Sep 8, 2022 at 12:02 PM Yasir Ahmed  wrote:
>>
>> Greetings,
>>
>>
>> I'm having difficulty performing TMT quantification using Libra on a
>> 16-plex dataset. Basically, the "quantification.tsv" file that Libra
>> outputs only contains zeros for both peptides and proteins. The Comet
>> search runs just fine. I'm at a loss for where things are going wrong. The
>> Mass Spec facility used ProteomeDiscoverer for the quantification, and it
>> worked fine so the issue is not the raw data.
>>
>>
>> My TPP run repository can be found here
>> <https://github.com/YazBraimah/TPP_TMT_proteomics_pipeline>, and the
>> "condition.xml" file for Libra—along with the Comet params file—is in the
>> "params" folder. The commands used to run the TPP processes can be found in
>> the file called "Snakefile". I'm happy to provide any additional
>> information needed to help troubleshoot this. Any pointers/advise would be
>> greatly appreciated.
>>
>>
>> Cheers,
>>
>> Yasir
>>
>>
>> --
>> 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

Re: [spctools-discuss] Libra quantification on TMT 16-plex returns zeros only

2022-09-09 Thread Yasir Ahmed 
Thanks Eric and Luis! Turns out I was using the wrong/older syntax in the 
condition file (targetMS level = “3”). The quantification works now. Thanks for 
your help!!!

Cheers,
Yasir

> On Sep 8, 2022, at 6:15 PM, 'Luis Mendoza' via spctools-discuss 
>  wrote:
> 
> Hi Yasir,
> Is your data Thermo SPS where the reporter ions are in MS3?  If so, please 
> check the relevant box when producing the condition file:
> 
> 
> 
> 
> As Eric noted, it does not seem like your MS2 spectra contain reporter ion 
> signals, at least from the one spectrum you posted.  Are there others where 
> this is not the case?
> 
> Cheers,
> --Luis
> 
> 
> On Thu, Sep 8, 2022 at 2:28 PM 'Eric Deutsch' via spctools-discuss 
>  <mailto:spctools-discuss@googlegroups.com>> wrote:
> I’m thinking that ion trap spectra are usually unsuitable for TMT:
> 
>  
> 
> 
>  
> I think you would want to acquire fragmentation spectra with Orbitrap HCD?
> 
>  
>  
> From: spctools-discuss@googlegroups.com 
> <mailto:spctools-discuss@googlegroups.com>  <mailto:spctools-discuss@googlegroups.com>> On Behalf Of Yasir Ahmed
> Sent: Thursday, September 8, 2022 2:19 PM
> To: spctools-discuss@googlegroups.com 
> <mailto:spctools-discuss@googlegroups.com>
> Subject: Re: [spctools-discuss] Libra quantification on TMT 16-plex returns 
> zeros only
> 
>  
> Hi Luis,
> 
>  
> Just to follow up: I found a handful of cases where one of the channels has 
> super high quantification and observable reporter ions (see screenshots 
> below), albeit it should be way more than this, I believe. I’ll keep digging, 
> but let me know if you have any other ideas.
> 
>  
> Again, thanks for you suggestions.
> 
>  
> Cheers,
> 
> Yasir
> 
>  
> 
> 
>  
> 
> 
>  
> 
> 
> 
> 
> 
> On Sep 8, 2022, at 5:07 PM, Yasir Ahmed  <mailto:yazah...@gmail.com>> wrote:
> 
>  
> Hi Luis,
> 
>  
> Thanks a lot for the response. 
> 
>  
> Can you try re-running Libra without the centroiding option?  (i.e. set 
> type="0") Or are your mzML data not centroided? 
> 
>  
> I just tried that, but no difference in outcome.
> 
> 
> 
> 
> Another thing to try: are you able to open the PeptideProphet + Libra results 
> in the PepXMLViewer?  You should be able to see if there are a significant 
> number of quantified PSMs -- you should also be able to examine a few 
> individual spectra and zoom in on the reporter ion signals.
> 
>  
> I’m able to look at a few individual spectra, and I think they all look 
> mostly good—but frankly I’m not too sure how to evaluate their suitability 
> (see example screenshots below). 
> 
> 
> 
> 
> Lastly, what version of TPP are you running?
> 
>  
> 6.1.0
> 
>  
> Thanks again!
> 
> Yasir
> 
>  
> 
> 
>  
> 
> 
>  
> 
> 
> 
>  
> Cheers,
> 
> --Luis
> 
>  
>  
> On Thu, Sep 8, 2022 at 12:02 PM Yasir Ahmed  <mailto:yazah...@gmail.com>> wrote:
> 
> Greetings,
> 
>  
> I'm having difficulty performing TMT quantification using Libra on a 16-plex 
> dataset. Basically, the "quantification.tsv" file that Libra outputs only 
> contains zeros for both peptides and proteins. The Comet search runs just 
> fine. I'm at a loss for where things are going wrong. The Mass Spec facility 
> used ProteomeDiscoverer for the quantification, and it worked fine so the 
> issue is not the raw data.
> 
>  
> My TPP run repository can be found here 
> <https://github.com/YazBraimah/TPP_TMT_proteomics_pipeline>, and the 
> "condition.xml" file for Libra—along with the Comet params file—is in the 
> "params" folder. The commands used to run the TPP processes can be found in 
> the file called "Snakefile". I'm happy to provide any additional information 
> needed to help troubleshoot this. Any pointers/advise would be greatly 
> appreciated.
> 
>  
> Cheers,
> 
> Yasir
> 
>  
> -- 
> 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-discuss+unsubscr...@googlegroups.com 
> <mailto:spctools-discuss+unsubscr...@googlegroups.com>.
> To view this discussion on the web visit 
> https://groups.google.com/d/msgid/spctools-discuss/7cb42706-53ae-4c94-acd2-ea0293fea8b2n%40googlegroups.com
>  
> <https://groups.google.com/d/msgid/spctools-discuss/7cb42706-53ae-4c94-acd2-ea0293fea8b2n%40googlegroups.com?utm_medium=email_source=footer>.
> 
>  
&g

Re: [spctools-discuss] Libra quantification on TMT 16-plex returns zeros only

2022-09-08 Thread 'Luis Mendoza' via spctools-discuss 
Hello Yasir,

Can you try re-running Libra without the centroiding option?  (i.e. set
type="0") Or are your mzML data not centroided?
Another thing to try: are you able to open the PeptideProphet + Libra
results in the PepXMLViewer?  You should be able to see if there are a
significant number of quantified PSMs -- you should also be able to examine
a few individual spectra and zoom in on the reporter ion signals.
Lastly, what version of TPP are you running?

Cheers,
--Luis


On Thu, Sep 8, 2022 at 12:02 PM Yasir Ahmed  wrote:

> Greetings,
>
> I'm having difficulty performing TMT quantification using Libra on a
> 16-plex dataset. Basically, the "quantification.tsv" file that Libra
> outputs only contains zeros for both peptides and proteins. The Comet
> search runs just fine. I'm at a loss for where things are going wrong. The
> Mass Spec facility used ProteomeDiscoverer for the quantification, and it
> worked fine so the issue is not the raw data.
>
> My TPP run repository can be found here
> , and the
> "condition.xml" file for Libra—along with the Comet params file—is in the
> "params" folder. The commands used to run the TPP processes can be found in
> the file called "Snakefile". I'm happy to provide any additional
> information needed to help troubleshoot this. Any pointers/advise would be
> greatly appreciated.
>
> Cheers,
> Yasir
>
> --
> 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-discuss+unsubscr...@googlegroups.com.
> To view this discussion on the web visit
> https://groups.google.com/d/msgid/spctools-discuss/7cb42706-53ae-4c94-acd2-ea0293fea8b2n%40googlegroups.com
> 
> .
>

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