Re: [spctools-discuss] ProteinProphet output number of unique peptides vs total number of peptides

[Hampton, Brian]

Hi David, thanks for the quick reply.

I think I see now.  When there is some ambiguity which prevents determining a 
precise peptide sequence, for example incomplete fragment ion series, or 
isobaric AA assignments etc. then there must be a probability calculation 
performed, the result of which attempts to assign this peptide to the more 
likely protein candidate.  The result of which is recorded in the "total num 
peptides" value.  In the case of a tie, is the value added to all possible 
protein hits or not used?

Thanks a bunch!

Brian

From: 'David Shteynberg' via spctools-discuss 

Sent: Wednesday, June 28, 2023 12:37 PM
To: spctools-discuss@googlegroups.com 
Subject: Re: [spctools-discuss] ProteinProphet output number of unique peptides 
vs total number of peptides

Hello Brian,

Thanks for the discussion.  The way this is possible is because there are 
degenerate peptides that map to multiple proteins, in which case only those 
that have sufficient weight will be counted as "is contributing evidence" 
peptides.  Only those PSM  instances that are contributing evidence are counted 
in the "tot num peps" value.   Please let me know if further clarification 
would be helpful or if you have other questions.

Cheers,
-David

On Wed, Jun 28, 2023 at 9:27 AM Hampton, Brian 
mailto:bhamp...@som.umaryland.edu>> wrote:
Hello David,

I have difficulty understanding this.  I had always operated under the 
assumption that the "num unique peps" would be less than or equal to the "tot 
num peps" - because - I thought "tot num peps" = "num unique peps" + number of 
(shared) peptides also mapping to given protein.  Is this not the case and if 
so how would "tot num peps" ever be less than "num unique peps"?

Best regards,
Brian




Brian Hampton
Proteomics Core Lab

Center for Vascular and Inflammatory Diseases

University of Maryland School of Medicine

BioPark One Rm 307

800 West Baltimore Street

Baltimore, MD. 21201

(410)706-8207


From: 'David Shteynberg' via spctools-discuss 
mailto:spctools-discuss@googlegroups.com>>
Sent: Wednesday, June 28, 2023 11:33 AM
To: spctools-discuss@googlegroups.com<mailto:spctools-discuss@googlegroups.com> 
mailto:spctools-discuss@googlegroups.com>>
Subject: Re: [spctools-discuss] ProteinProphet output number of unique peptides 
vs total number of peptides

Hi Murielle,

Sorry this is a little confusing.  Column "num unique peps" represents total 
number of unique peptides mapping to the protein.  The "tot num peps" counts 
the PSMs that are "contributing evidence" (last column) to the protein, 
sometimes this number can be lower because not all peptides mapping are 
contributing evidence and spectral counts can be low per peptide.

The answer to your second question is yes, the "tot num peps" is the spectral 
count for "is contributing evidence" peptides.

Cheers,
-David


On Wed, Jun 28, 2023 at 7:27 AM 
muriell...@gmail.com<mailto:muriell...@gmail.com> 
mailto:murielle.al...@gmail.com>> wrote:
Hi David,

Thank you for such a quick reply!

I used tpp version 6.2.0 run via a singularity image and I produced the table 
by including the EXCELXX option in proteinprophet.

Cheers,

Murielle

On Wednesday, June 28, 2023 at 12:44:03 AM UTC+2 David Shteynberg wrote:
Hi Murielle,

Thanks for using the TPP and submitting your question here.   When I try the 
latest version of the TPP and run ProteinProphet through there, the columns 
exported in my tab separated file from ProteinProphet are different from yours. 
 Can you please provide a bit more information about how you generated this 
file and which version of the software you used?

Cheers,
-David

On Tue, Jun 27, 2023 at 7:10 AM muriell...@gmail.com  
wrote:
Hi all,

I have two questions concerning the output of ProteinProphet :

1) Why do I have some entries for which the total number of peptides is lower 
than the number of unique peptides?  (see columns 5 and 6 below, an extract of 
my output table)

[protein prophet output.jpeg]
2) Does the column "total number of peptides" correspond to spectral counts?

Thank you very much ,

Murielle

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Re: [spctools-discuss] ProteinProphet output number of unique peptides vs total number of peptides

[Hampton, Brian]

Hello David,

I have difficulty understanding this.  I had always operated under the 
assumption that the "num unique peps" would be less than or equal to the "tot 
num peps" - because - I thought "tot num peps" = "num unique peps" + number of 
(shared) peptides also mapping to given protein.  Is this not the case and if 
so how would "tot num peps" ever be less than "num unique peps"?

Best regards,
Brian




Brian Hampton
Proteomics Core Lab

Center for Vascular and Inflammatory Diseases

University of Maryland School of Medicine

BioPark One Rm 307

800 West Baltimore Street

Baltimore, MD. 21201

(410)706-8207


From: 'David Shteynberg' via spctools-discuss 

Sent: Wednesday, June 28, 2023 11:33 AM
To: spctools-discuss@googlegroups.com 
Subject: Re: [spctools-discuss] ProteinProphet output number of unique peptides 
vs total number of peptides

Hi Murielle,

Sorry this is a little confusing.  Column "num unique peps" represents total 
number of unique peptides mapping to the protein.  The "tot num peps" counts 
the PSMs that are "contributing evidence" (last column) to the protein, 
sometimes this number can be lower because not all peptides mapping are 
contributing evidence and spectral counts can be low per peptide.

The answer to your second question is yes, the "tot num peps" is the spectral 
count for "is contributing evidence" peptides.

Cheers,
-David


On Wed, Jun 28, 2023 at 7:27 AM 
muriell...@gmail.com 
mailto:murielle.al...@gmail.com>> wrote:
Hi David,

Thank you for such a quick reply!

I used tpp version 6.2.0 run via a singularity image and I produced the table 
by including the EXCELXX option in proteinprophet.

Cheers,

Murielle

On Wednesday, June 28, 2023 at 12:44:03 AM UTC+2 David Shteynberg wrote:
Hi Murielle,

Thanks for using the TPP and submitting your question here.   When I try the 
latest version of the TPP and run ProteinProphet through there, the columns 
exported in my tab separated file from ProteinProphet are different from yours. 
 Can you please provide a bit more information about how you generated this 
file and which version of the software you used?

Cheers,
-David

On Tue, Jun 27, 2023 at 7:10 AM muriell...@gmail.com  
wrote:
Hi all,

I have two questions concerning the output of ProteinProphet :

1) Why do I have some entries for which the total number of peptides is lower 
than the number of unique peptides?  (see columns 5 and 6 below, an extract of 
my output table)

[protein prophet output.jpeg]
2) Does the column "total number of peptides" correspond to spectral counts?

Thank you very much ,

Murielle

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Re: [spctools-discuss] RefreshParser reports number of unmapped entries - why are there unmapped entries?

[Hampton, Brian]

Hi David,


Thank you for checking this.  I'll redo the database and search and report back 
what happens.


Thanks,


Brian


From: spctools-discuss@googlegroups.com  on 
behalf of David Shteynberg 
Sent: Tuesday, January 29, 2019 6:55:03 PM
To: spctools-discuss
Subject: Re: [spctools-discuss] RefreshParser reports number of unmapped 
entries - why are there unmapped entries?

Hi Brian,

It appears that all of the  UNMAPPED results are to DECOY proteins.  Is it 
possible that you regenerated the DECOYS at some point but kept the database 
name the same?  DECOYS get reshuffled so you are not guaranteed to have the 
same DECOYS peptides that you had the first time you created the DECOYS.  I 
would suggest you apply RefreshParser using the same database you used in the 
search or redo the search.

Thanks,
-David

On Tue, Jan 29, 2019 at 10:45 AM Hampton, Brian 
mailto:bhamp...@som.umaryland.edu>> wrote:
Hi David,

Thanks for having a look.  I sent you a link to the interact…pep.xml and the 
database files to download at your convenience.

Brian

On Jan 29, 2019, at 1:09 PM, David Shteynberg 
mailto:david.shteynb...@systemsbiology.org>>
 wrote:

Hi Brian,

Can you post the files for me to trace the execution of RefreshParser and 
troubleshoot the issue?

Thanks,
-David

On Tue, Jan 29, 2019 at 7:24 AM Hampton, Brian 
mailto:bhamp...@som.umaryland.edu>> wrote:
Hello,

I cannot determine why RefreshParser would not be able to remap all entries. 
Since the PSMs are derived from the fasta db used in the search, I would expect 
all PSMs to map to their respective database entries.  For example:


running: "/usr/local/tpp/bin/RefreshParser 
'/tmp/X33oJO/data/SMC/16-18-27-6094/source/interact-a-2.pep.xml' 
'/usr/local/tpp/data/dbase/human-reviewed-190128_DECOY-crap.fasta'"
  - Building Commentz-Walter keyword tree...
  - Searching the tree...
  - Linking duplicate entries...
  - Printing results...
Warning: could not map 3081 entries

command completed in 5 sec

Thanks in advance for any help.

Brian

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Re: [spctools-discuss] RefreshParser reports number of unmapped entries - why are there unmapped entries?

[Hampton, Brian]

Hi David,

Thanks for having a look.  I sent you a link to the interact…pep.xml and the 
database files to download at your convenience.

Brian

On Jan 29, 2019, at 1:09 PM, David Shteynberg 
mailto:david.shteynb...@systemsbiology.org>>
 wrote:

Hi Brian,

Can you post the files for me to trace the execution of RefreshParser and 
troubleshoot the issue?

Thanks,
-David

On Tue, Jan 29, 2019 at 7:24 AM Hampton, Brian 
mailto:bhamp...@som.umaryland.edu>> wrote:
Hello,

I cannot determine why RefreshParser would not be able to remap all entries. 
Since the PSMs are derived from the fasta db used in the search, I would expect 
all PSMs to map to their respective database entries.  For example:


running: "/usr/local/tpp/bin/RefreshParser 
'/tmp/X33oJO/data/SMC/16-18-27-6094/source/interact-a-2.pep.xml' 
'/usr/local/tpp/data/dbase/human-reviewed-190128_DECOY-crap.fasta'"
  - Building Commentz-Walter keyword tree...
  - Searching the tree...
  - Linking duplicate entries...
  - Printing results...
Warning: could not map 3081 entries

command completed in 5 sec

Thanks in advance for any help.

Brian

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[spctools-discuss] Quantic vs St. Peter

[Hampton, Brian]

Hello again,

Is there a publication or other information that explains Quantic?  Why would 
one choose Quantic or St. Peter for LFQ analysis?  
I have looked for this on the Wiki and in the release notes, as well as a 
keyword search but can’t find the information.  Maybe I missed something.

Thanks,

Brian

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[spctools-discuss] RefreshParser reports number of unmapped entries - why are there unmapped entries?

[Hampton, Brian]

Hello,

I cannot determine why RefreshParser would not be able to remap all entries. 
Since the PSMs are derived from the fasta db used in the search, I would expect 
all PSMs to map to their respective database entries.  For example:


running: "/usr/local/tpp/bin/RefreshParser 
'/tmp/X33oJO/data/SMC/16-18-27-6094/source/interact-a-2.pep.xml' 
'/usr/local/tpp/data/dbase/human-reviewed-190128_DECOY-crap.fasta'"
  - Building Commentz-Walter keyword tree...
  - Searching the tree...
  - Linking duplicate entries...
  - Printing results...
Warning: could not map 3081 entries

command completed in 5 sec

Thanks in advance for any help.

Brian

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Re: [spctools-discuss] TPP documentation issue

[Hampton, Brian]

Hello Paul,

The quickest way to greater understanding on the use of the tools is to execute 
the binaries in question at the command line with no options specified.  The 
output will show information about their usage.  For example on my old Linux 
install of TPP 4.8, at the command line, I change directory to the …/tpp/bin 
directory and type in e.g. ./xinteract.  There is quite a bit of usage 
information output for xinteract since it is a wrapper for many of the binaries 
to execute them in succession to form the pipeline.  If you page down (in my 
version) after running the xinteract command there is this output which 
references PTMProphet:

PTMProphet options [run PTMProphet on the iProphet result with options that 
follow the '-M' (e.g. -M-STY,79.9663-MZTOL=0.4)]:
 -{,,...}  [specify mod 
masses (e.g. -STY,79.9663,K,114.0429,M,15.9949)]
 -MZTOL= [Use specified +/- 
mz tolerance on site specific ions (default=0.1 dalton)]
 -NOUPDATE   [Don't update 
modification_info tags in pepXML]



Also running the binaries at the command line can sometimes aid in 
troubleshooting since they tend to output more information about what is going 
on than is displayed via the GUI web interface.

Aside from this, you can also look up the published papers on many of the 
tools, attend the periodic TPP training courses, and ask questions to this 
forum.  I am personally not aware of any tutorials for PTMProphet or most of 
the other tools for that matter.

It would be a wonderful thing were the TPP developer team to provide tutorials 
(online not just the courses which you must attend in person) that cover the 
breadth and depth of the TPP toolset much like what the developers of Skyline 
and Maxquant have done. This would enable many more people to learn and use the 
TPP tools at their own pace, in their own environment.   I think that would 
markedly increase TPP usage around the world.

Cheers,

Brian


On Jan 5, 2018, at 10:29 AM, Paul Abraham 
mailto:pabraham6...@gmail.com>> wrote:

Hello,

I am having a difficult time locating documentation for several of the TPP 
tools. For example, the PTMProphet tool referenced on the wiki page does not 
have any supporting documentation. Am I missing some obscure landing page 
somewhere? The only information I find for how to efficiently utilize this tool 
is by scouring this forum and this is by no means a good use of time, 
considering most posts highlight the issues.

Can anyone point to documentation for this tool particularly, but other TPP 
tools as well?

Regards,
Paul

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Re: [spctools-discuss] Re: StPeter documentation

[Hampton, Brian]

Thanks Jason!  Once your manuscript is published it would be great to announce 
that on this list.

Cheers!

Brian

On Jul 12, 2017, at 8:13 AM, Jason Winget 
mailto:jwin...@gmail.com>> wrote:

Hi Brian,
StPeter is a direct implementation of the Normalized Spectral Index, described 
by Griffin et. al: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805705/
It uses MS2 intensities for protein-level quantification.

We are working on publishing it independently but so far journal editors have 
not been very receptive...

As for usage, if you run StPeter from the command line with no arguments you 
will see a usage statement. By default it uses only non-degenerate peptides and 
a 1% FDR cutoff. Typically the default values are fine, so you should just be 
able to run it against your ProtXML results. It will write the quantification 
values back into the ProtXML and will also output some simple CSV results.

At the moment PTMs are hard-coded and contain only a limited set, so the most 
common issue that I run into is that the program will crash when searching 
"unusual" mods.

-Jason

On Friday, June 16, 2017 at 5:33:59 PM UTC-4, Brian Hampton wrote:
Hello,

I would be appreciative if someone could point me to documentation that 
describes StPeter which is used for label-free quantification in TPP. I've 
tried the usual searching with Google, PubMed and looking through the SPC Tools 
sites without success.

Thanks,

Brian


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