Re: [spctools-discuss] how to specify heavy label for oxidised methionine in ASAPRatio

2020-08-17 Thread 'Alastair Skeffington' via spctools-discuss 
Hi David,

Ah sorry - my mistake. Here's a new link with a tarball including the mxXML 
files.

https://we.tl/t-UnX74n4qod

Many thanks!
Alastair

On Monday, 17 August 2020 20:51:54 UTC+2, David Shteynberg wrote:
>
> Hello Alastair,
>
> I downloaded the file you shared with me, however, there were no mzML 
> files to match the pepXML files so I couldn't actually try the analysis.  
>  I will make another attempt if you can provide the mass spec data. 
>
> Thanks,
> -David
>
> On Mon, Aug 17, 2020 at 11:21 AM 'Alastair Skeffington' via 
> spctools-discuss > wrote:
>
>> Hi David,
>>
>> The link to the data has now expired, Let me know if you are still 
>> willing to have a look and I can send it again.
>>
>> Otherwise maybe you could briefly describe the process you would go 
>> through?
>>
>> Many thanks,
>> Alastair
>>
>> On Thursday, 6 August 2020 22:17:00 UTC+2, Alastair Skeffington wrote:
>>>
>>> Hi David,
>>>
>>> Many thanks for your reply.
>>>
>>> So it any peptides with modifications will simply be ignored for 
>>> quantification and I can ignore the warning message?
>>>
>>> Yes - each search was either light or heavy as defined in the static 
>>> modifications. 
>>>
>>> And the -r parameter is then the window to search in the RT dimension? 
>>> Because the command line option says 'range around precursor m/z to search 
>>> for peak' I assumed this was for the m/z dimension. I thought that the 
>>> shift of the peak would also mostly be in the m/z dimension - but I'm no 
>>> mass spectrometrist!
>>>
>>> I would be amazing if you had a moment to have a look at the data - 
>>> thanks very much for offering. I've put two example pairs of files here: 
>>> https://we.tl/t-cVDD1MVDOr 
>>>
>>> For each sample there is a light 'L' version of the search results and a 
>>> heavy 'H' version. I've also included the search database (based on some 
>>> PacBio data some I'm afraid it's quite big with a lot of isoforms).
>>>
>>> Many thanks,
>>> Alastair
>>>
>>> On Wednesday, 5 August 2020 20:37:16 UTC+2, David Shteynberg wrote:

 Hi Alastair,

 If your search results are either all heavy or all light (not variable 
 mod searched) then you should also use option -S.  

 1). You cannot specify anything but single amino acids in this string.  
 Your quantitation will be based on peptides without PTMs in this dataset.

 2). -r8 is a MUCH too wide window to recover the MS1 signal in 
 RTspace.  The lower this number the more selective the tool is at 
 isolating 
 your target signal.  With -r8 you will not be quantifying the correct 
 signal, unless you have a very bare sample.

 If you are able to share this data I can try running it to help you 
 optimize your settings.

 Thanks,
 -David

 On Wed, Aug 5, 2020 at 11:02 AM 'Alastair Skeffington' via 
 spctools-discuss  wrote:

> Hello,
>
> I'm trying to run a 14N/15N labelling experiment through ASAPRatio. 
> I've been running the first steps like this - here for the results of a 
> database search with heavy masses:
>
> InteractParser sample_interact.pep.xml sample.pep.xml
>
> PeptideProphetParser sample_interact.pep.xml
>
> RefreshParser sample_interact.pep.xml ./EhuxAllproteins_MCC_decoy.fasta
>
> ASAPRatioPeptideParser sample_interact.pep.xml  -lACDEFGHIKLMNPQRSTVWY 
> -r8 
> -mA72.0779R160.1857N116.1026D116.0874C104.1429E130.1140Q130.1292G58.0513H140.1393I114.1576L114.1576K130.1723M132.1961F148.1739P99.1152S89.0773T102.1038W188.0793Y164.0633V101.1311
>
> At this point I get a warning:
>
> WARNING: Found more than one variable mod on 'M'. Please make sure to 
> specify a heavy mass for this residue
>
> So I have two questions:
>
> 1) How do I specify the heavy mass for oxidised methionine? Mox ? And 
> is phosphorylated serine coded Sp ?
>
> 2) I've used -r8 instead of the default 0.5. My reasoning is that a 
> medium sized heavy peptide could easily differ from the 14N counterpart 
> by 
> 16 Da. Assuming charge +2, then using a m/z range of 8. Does this sound 
> remotely sensible?
>
> Many thanks!
> Alastair
>
> -- 
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> https://groups.google.com/d/msgid/spctools-discuss/4853fda5-cc2f-48e2-a9e2-2ae93225b288o%40googlegroups.com
>  
> 
> .
>
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Re: [spctools-discuss] how to specify heavy label for oxidised methionine in ASAPRatio

2020-08-17 Thread 'David Shteynberg' via spctools-discuss 
Hello Alastair,

I downloaded the file you shared with me, however, there were no mzML files
to match the pepXML files so I couldn't actually try the analysis.   I will
make another attempt if you can provide the mass spec data.

Thanks,
-David

On Mon, Aug 17, 2020 at 11:21 AM 'Alastair Skeffington' via
spctools-discuss  wrote:

> Hi David,
>
> The link to the data has now expired, Let me know if you are still willing
> to have a look and I can send it again.
>
> Otherwise maybe you could briefly describe the process you would go
> through?
>
> Many thanks,
> Alastair
>
> On Thursday, 6 August 2020 22:17:00 UTC+2, Alastair Skeffington wrote:
>>
>> Hi David,
>>
>> Many thanks for your reply.
>>
>> So it any peptides with modifications will simply be ignored for
>> quantification and I can ignore the warning message?
>>
>> Yes - each search was either light or heavy as defined in the static
>> modifications.
>>
>> And the -r parameter is then the window to search in the RT dimension?
>> Because the command line option says 'range around precursor m/z to search
>> for peak' I assumed this was for the m/z dimension. I thought that the
>> shift of the peak would also mostly be in the m/z dimension - but I'm no
>> mass spectrometrist!
>>
>> I would be amazing if you had a moment to have a look at the data -
>> thanks very much for offering. I've put two example pairs of files here:
>> https://we.tl/t-cVDD1MVDOr
>>
>> For each sample there is a light 'L' version of the search results and a
>> heavy 'H' version. I've also included the search database (based on some
>> PacBio data some I'm afraid it's quite big with a lot of isoforms).
>>
>> Many thanks,
>> Alastair
>>
>> On Wednesday, 5 August 2020 20:37:16 UTC+2, David Shteynberg wrote:
>>>
>>> Hi Alastair,
>>>
>>> If your search results are either all heavy or all light (not variable
>>> mod searched) then you should also use option -S.
>>>
>>> 1). You cannot specify anything but single amino acids in this string.
>>> Your quantitation will be based on peptides without PTMs in this dataset.
>>>
>>> 2). -r8 is a MUCH too wide window to recover the MS1 signal in RTspace.
>>> The lower this number the more selective the tool is at isolating your
>>> target signal.  With -r8 you will not be quantifying the correct signal,
>>> unless you have a very bare sample.
>>>
>>> If you are able to share this data I can try running it to help you
>>> optimize your settings.
>>>
>>> Thanks,
>>> -David
>>>
>>> On Wed, Aug 5, 2020 at 11:02 AM 'Alastair Skeffington' via
>>> spctools-discuss  wrote:
>>>
 Hello,

 I'm trying to run a 14N/15N labelling experiment through ASAPRatio.
 I've been running the first steps like this - here for the results of a
 database search with heavy masses:

 InteractParser sample_interact.pep.xml sample.pep.xml

 PeptideProphetParser sample_interact.pep.xml

 RefreshParser sample_interact.pep.xml ./EhuxAllproteins_MCC_decoy.fasta

 ASAPRatioPeptideParser sample_interact.pep.xml  -lACDEFGHIKLMNPQRSTVWY
 -r8
 -mA72.0779R160.1857N116.1026D116.0874C104.1429E130.1140Q130.1292G58.0513H140.1393I114.1576L114.1576K130.1723M132.1961F148.1739P99.1152S89.0773T102.1038W188.0793Y164.0633V101.1311

 At this point I get a warning:

 WARNING: Found more than one variable mod on 'M'. Please make sure to
 specify a heavy mass for this residue

 So I have two questions:

 1) How do I specify the heavy mass for oxidised methionine? Mox ? And
 is phosphorylated serine coded Sp ?

 2) I've used -r8 instead of the default 0.5. My reasoning is that a
 medium sized heavy peptide could easily differ from the 14N counterpart by
 16 Da. Assuming charge +2, then using a m/z range of 8. Does this sound
 remotely sensible?

 Many thanks!
 Alastair

 --
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 Groups "spctools-discuss" group.
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 .

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> 
> .
>

-- 
You

Re: [spctools-discuss] how to specify heavy label for oxidised methionine in ASAPRatio

2020-08-17 Thread 'Alastair Skeffington' via spctools-discuss 
Hi David,

The link to the data has now expired, Let me know if you are still willing 
to have a look and I can send it again.

Otherwise maybe you could briefly describe the process you would go through?

Many thanks,
Alastair

On Thursday, 6 August 2020 22:17:00 UTC+2, Alastair Skeffington wrote:
>
> Hi David,
>
> Many thanks for your reply.
>
> So it any peptides with modifications will simply be ignored for 
> quantification and I can ignore the warning message?
>
> Yes - each search was either light or heavy as defined in the static 
> modifications. 
>
> And the -r parameter is then the window to search in the RT dimension? 
> Because the command line option says 'range around precursor m/z to search 
> for peak' I assumed this was for the m/z dimension. I thought that the 
> shift of the peak would also mostly be in the m/z dimension - but I'm no 
> mass spectrometrist!
>
> I would be amazing if you had a moment to have a look at the data - thanks 
> very much for offering. I've put two example pairs of files here: 
> https://we.tl/t-cVDD1MVDOr 
>
> For each sample there is a light 'L' version of the search results and a 
> heavy 'H' version. I've also included the search database (based on some 
> PacBio data some I'm afraid it's quite big with a lot of isoforms).
>
> Many thanks,
> Alastair
>
> On Wednesday, 5 August 2020 20:37:16 UTC+2, David Shteynberg wrote:
>>
>> Hi Alastair,
>>
>> If your search results are either all heavy or all light (not variable 
>> mod searched) then you should also use option -S.  
>>
>> 1). You cannot specify anything but single amino acids in this string.  
>> Your quantitation will be based on peptides without PTMs in this dataset.
>>
>> 2). -r8 is a MUCH too wide window to recover the MS1 signal in RTspace.  
>> The lower this number the more selective the tool is at isolating your 
>> target signal.  With -r8 you will not be quantifying the correct signal, 
>> unless you have a very bare sample.
>>
>> If you are able to share this data I can try running it to help you 
>> optimize your settings.
>>
>> Thanks,
>> -David
>>
>> On Wed, Aug 5, 2020 at 11:02 AM 'Alastair Skeffington' via 
>> spctools-discuss  wrote:
>>
>>> Hello,
>>>
>>> I'm trying to run a 14N/15N labelling experiment through ASAPRatio. I've 
>>> been running the first steps like this - here for the results of a database 
>>> search with heavy masses:
>>>
>>> InteractParser sample_interact.pep.xml sample.pep.xml
>>>
>>> PeptideProphetParser sample_interact.pep.xml
>>>
>>> RefreshParser sample_interact.pep.xml ./EhuxAllproteins_MCC_decoy.fasta
>>>
>>> ASAPRatioPeptideParser sample_interact.pep.xml  -lACDEFGHIKLMNPQRSTVWY 
>>> -r8 
>>> -mA72.0779R160.1857N116.1026D116.0874C104.1429E130.1140Q130.1292G58.0513H140.1393I114.1576L114.1576K130.1723M132.1961F148.1739P99.1152S89.0773T102.1038W188.0793Y164.0633V101.1311
>>>
>>> At this point I get a warning:
>>>
>>> WARNING: Found more than one variable mod on 'M'. Please make sure to 
>>> specify a heavy mass for this residue
>>>
>>> So I have two questions:
>>>
>>> 1) How do I specify the heavy mass for oxidised methionine? Mox ? And is 
>>> phosphorylated serine coded Sp ?
>>>
>>> 2) I've used -r8 instead of the default 0.5. My reasoning is that a 
>>> medium sized heavy peptide could easily differ from the 14N counterpart by 
>>> 16 Da. Assuming charge +2, then using a m/z range of 8. Does this sound 
>>> remotely sensible?
>>>
>>> Many thanks!
>>> Alastair
>>>
>>> -- 
>>> 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...@googlegroups.com.
>>> To view this discussion on the web visit 
>>> https://groups.google.com/d/msgid/spctools-discuss/4853fda5-cc2f-48e2-a9e2-2ae93225b288o%40googlegroups.com
>>>  
>>> 
>>> .
>>>
>>

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