ASAPratio questions and suggestion

[Alex]

Quick question, probably directed to David,

I've used ASAPratio by following through a couple of posts, trying to figure out how to complete the options on Petunia. It still remains a little unclear why or what should be completed in the fields:

Change labeled residues to 

Specified residue mass 1:  

I work with dimethylated labels (formaldehyde reagent targeting free amino groups and its 'heavy' deuterated D2CO form) and  understand I should add K and n as the "change labeled residues" and add the mass of the heavy residues to the "specified residue mass" (n=33.06425 and K=160.15137 for the deuterium modified labels). This has so far worked out to me.

My question is why should they be added to both fields? Is this redundant or is there any case where one could add a modification to one field and not the other? I have added all the modifications (light and heavy labels) to the X!Tandem parameters files, including other modifs as alkylated cystesines and oxidized M and all the peptides have been correctly identified. Judging by the previous posts, this has caused confusion to other ASAPratio users in the past.

And a suggestion: it would be very convenient to have a 'zoom' and 'unzoom' option when viewing the results on the ASAPratio viewer. Sometimes, ASAPratio offers a very narrow window of the integrated peak, and I find viewing the axis in a larger window is good to assess how specific the XIC looks for a particular precursor along the complete chromtographic run.

Thanks TTP people, hope to hearing back from you!!!

Alex 

[David Shteynberg]

Hi Alex,

If your tandem search used monoisotopic masses for the variable mods then you don't really have to specify the masses and it will use the mass in the file.  ASAPRatio allows you to redefine the mass (so you can use average masses for matching fragments and monoisotopic for quantitation), if the pep.xml file alread has the monoisotopic masses listed then you can just specify the labeled amino acids and those will be applied to find the heavy/light pairs.  Let me know if you require further clarification.

Cheers,

-David

Alex 

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[Alex]

Thanks David!

Any chance an XIC zoom in/out option can be introduced in a later version of TTP to ASAPratio?

Cheers

Alex 

-David

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[Brian Hampton]

David,

I am doing very similar work as Alex where light and heavy labels are on K and the N-terminus of the peptides.  In the Tandem parameters file, the modifications are specified according the the API description from Tandem for residue modification mass and allows to search with two different labels on the same residue.

residue, modification mass Lightmod@K, Lightmod@[

residue, potential modification mass Heavymod-Lightmod@K, Heavymod-Lightmod@[

In my case Lightmod=42.024 and Heavymod=48.028

In ASAPRatio,  if I do not specify a modification mass (as you seem to suggest below) for the amino acids it matches up the pairs incorrectly.  Rather than finding labeled pairs of peptides differing by 6u, it finds light labeled and *unlabeled* pairs differing by 42u, which doesn't exist.

So I specify n and K in the "Change modified residues to" since the N-terminus and K are the only labeled residues and then I specify all four labeled residue masses with their respective monoisotopic masses of 42 & 48 for n and 170.0124 and 170.0125 for K.  Then it seems to work.  Maybe it works differently with input from another search engine, I don't know.  

I am guessing the following warning is informational only and doesn't indicate something is wrong:

running: "/usr/local/apps/tpp/bin/ASAPRatioPeptideParser 'BH_2-interact.pep.xml' -lnK -b -r0.5 -mn42.024n48.028K170.0124K176.0125"
WARNING: Found more than one variable mod on 'K'. Please make sure to specify a heavy mass for this residue.

Thanks in advance for any help here.

Brian

[David Shteynberg]

Hi Brian,

Thanks for the input.  Yes, it depends on how the search was performed.  I tested with out specifying the mass for a SILAC sample and it seemed to work.  If you have different mass labels for heavy and light and the light is not specified as a static mod then you have to do what you showed in your commandline.  Ultimately, it is always a good idea to verify for yourself that the masses it uses are the correct ones.  


Cheers,

-David

[Alex]

Brian,

Your approach is interesting, I see you perform only one database search for your sample (looking for both light (static) and heavy labels (variable)). I read some posts that recommend doing two separate searches, one specifying a static modification for the light, and another search specifying a static modification for the heavy label, and then running TTP using both files in the Analyze Peptides-ASAPratio, checking the  Static modification quantification (i.e. each run is either all light or all heavy) option.

Have you tried both options? any comment on the results you obtain?

Thanks

Alex

On Tuesday, 12 June 2012 17:08:32 UTC-3, Brian Hampton wrote:

David,

I am doing very similar work as Alex where light and heavy labels are on K and the N-terminus of the peptides.  In the Tandem parameters file, the modifications are specified according the the API description from Tandem for residue modification mass and allows to search with two different labels on the same residue.

residue, modification mass Lightmod@K, Lightmod@[

residue, potential modification mass Heavymod-Lightmod@K, Heavymod-Lightmod@[

In my case Lightmod=42.024 and Heavymod=48.028

In ASAPRatio,  if I do not specify a modification mass (as you seem to suggest below) for the amino acids it matches up the pairs incorrectly.  Rather than finding labeled pairs of peptides differing by 6u, it finds light labeled and *unlabeled* pairs differing by 42u, which doesn't exist.

So I specify n and K in the "Change modified residues to" since the N-terminus and K are the only labeled residues and then I specify all four labeled residue masses with their respective monoisotopic masses of 42 & 48 for n and 170.0124 and 170.0125 for K.  Then it seems to work.  Maybe it works differently with input from another search engine, I don't know.  

I am guessing the following warning is informational only and doesn't indicate something is wrong:

running: "/usr/local/apps/tpp/bin/ASAPRatioPeptideParser 'BH_2-interact.pep.xml' -lnK -b -r0.5 -mn42.024n48.028K170.0124K176.0125"
WARNING: Found more than one variable mod on 'K'. Please make sure to specify a heavy mass for this residue.

Thanks in advance for any help here.

Brian

On Mon, Jun 11, 2012 at 4:02 PM, David Shteynberg <David.Shteynberg@systemsbiology.org> wrote:

Hi Alex,

If your tandem search used monoisotopic masses for the variable mods then you don't really have to specify the masses and it will use the mass in the file.  ASAPRatio allows you to redefine the mass (so you can use average masses for matching fragments and monoisotopic for quantitation), if the pep.xml file alread has the monoisotopic masses listed then you can just specify the labeled amino acids and those will be applied to find the heavy/light pairs.  Let me know if you require further clarification.

Cheers,

-David

On Mon, Jun 11, 2012 at 12:47 PM, Alex <ale.c...@gmail.com> wrote:

Quick question, probably directed to David,

I've used ASAPratio by following through a couple of posts, trying to figure out how to complete the options on Petunia. It still remains a little unclear why or what should be completed in the fields:

Change labeled residues to 

Specified residue mass 1:  

I work with dimethylated labels (formaldehyde reagent targeting free amino groups and its 'heavy' deuterated D2CO form) and  understand I should add K and n as the "change labeled residues" and add the mass of the heavy residues to the "specified residue mass" (n=33.06425 and K=160.15137 for the deuterium modified labels). This has so far worked out to me.

My question is why should they be added to both fields? Is this redundant or is there any case where one could add a modification to one field and not the other? I have added all the modifications (light and heavy labels) to the X!Tandem parameters files, including other modifs as alkylated cystesines and oxidized M and all the peptides have been correctly identified. Judging by the previous posts, this has caused confusion to other ASAPratio users in the past.

And a suggestion: it would be very convenient to have a 'zoom' and 'unzoom' option when viewing the results on the ASAPratio viewer. Sometimes, ASAPratio offers a very narrow window of the integrated peak, and I find viewing the axis in a larger window is good to assess how specific the XIC looks for a particular precursor along the complete chromtographic run.

Thanks TTP people, hope to hearing back from you!!!

Alex 

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[Brian Hampton]

Hi Alex,

Thanks for pointing out what should have been obvious.. Do two searches one with light the other with heavy as a static modification.  Everything works fine now.

Well there is a dramatic difference between the results from the two search types.  I think using the method specified in the Tandem API for searching with two mods on the same residue in the same search may not be working well at all.  I think because that method puts a potential mod (the heavy label) on the static mod (light) and that increases the number of peptide spectral matches some of which are not correct.  In my case it increased the number of peptides that match to a protein present in the sample and identified proteins that may not be in the sample.

The results from doing two searches one with light static and the other with heavy static mods gave fewer peptides/protein hit and it gave a different list of protein IDs.  There was some overlap between the two search results but the differences were many.

I am using a quadrupole ion trap so mass accuracy isn't its strong point.  Maybe using data collected with a HMA instrument, the static and variable mod single search would work better?

Brian

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