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

['Alastair Skeffington' via spctools-discuss]

Further updates:

I haven't got any further with getting ASAPRatio to identify what I think 
are the correct mass differences between heavy and light.

The samples I am analysing are from a cellular fractionation experiment 
comparing two conditions where a particular structure is either present or 
absent. Thus if the data from the two conditions samples were 
hypothetically analysed separately I wouldn't necessarily expect a 
massively good alignment between runs. There may be a lot of proteins that 
are only identified in one condition. So my plan to analyse the data and be 
sure of my answers is now:

1. Do separate 15N adn 14N searches and run peptide prophet
2. Combine in iprophet to ensure that 14N peptides are not erroneously 
assigned to 15N spectra and vice versa. 
3. Run protein protphet
4. Run ASAPRatio to get peak areas for the 14N and 15N samples individually
4. Post process the protein prophet output using custom scripts to:
a) identify proteins that are only found with 14M PSMs or only 15N PSMs
b) For proteins with mixed IDs, identify peptides identified in bot ha 14N 
and 15N version. Use the ASAPRatio results to make a ratio for these 
peptides and subsequently calculate a protein ratio.

Does this sound sensible?

Thanks,
Alastair

On Monday, 31 August 2020 at 14:25:46 UTC+2 Alastair Skeffington wrote:

> Hi David,
>
> Thanks for pointing out the wrong mass - there were a couple of others as 
> well. So I've got quite a lot further with the analysis, but I'm not 
> convinced that it's working as it should. Maybe it's easiest if I detail 
> the steps I've gone through and ask some questions as I go. It would be 
> great if you could answer these / annotate with any other thoughts you have:
>
> Step 1: Search using standard 14N labelled masses. The 15N search seems to 
> be fairly useless (see later). No variable modifications in search, because 
> otherwise ASAPRatio complains and fails to run in 'static' mode.
>
> Step 2: InteractParser $intout $in
>
> Step 3: PeptideProphetParser $intout DECOY=XXX_ NONPARAM
>
> Note I get a lot of model failures for this data. I get less model 
> failures when I use fully parametric modelling, but then the decoy search 
> hits are not properly taken into account meaning that in the final prot.xml 
> file I have single -protein protein groups consisting only of a decoy hit. 
> So I continued with the semi-parametric options.
>
> Step 3: ASAPRatioPeptideParser $in  -lACDEFGHIKLMNPQRSTVWY -r0.5 -S 
> -mA72.04581R160.1359N116.0603D116.0356C161.0393E130.0513Q130.076G58.03016H140.085I114.0928L114.0928K130.1124M132.0492F148.0771P98.06146S88.04073T102.0564W188.0967Y164.072V100.0771
>
> So the -m string specifies the modified masses (ie 15N masses) for 
> comparison with the 14N search results. 
>
> This doesn't work the other way round (taking the 15N search results and 
> using the 14N masses in the -m string. This is because of cysteine 
> carbamylation, meaning that ASAPRatio sees the C mass as being a 'heavy' 
> static modification, and the other  residues as being 'light' 
> modifications. This results in an error message that there are a mixture of 
> heavy and light modifications).
>
> Step 4: ProteinProphet $light $out ASAP_PROPHET
>
> I get ratios for reasonable numbers of proteins - but unfortunately I'm 
> not convinced these are reliable. To take one example:
> > L/H ratio given as 2.98
> > Based on peptide: CTTSAAATSTSSGR  
> > Looking at the details in the viewer I see "light +2 m/z 679.3" and 
> "heavy +2 m/z 679.8"
> > There are 17 N atoms in the peptide, 16 with one neutral loss. m/z for 
> the mass difference between light and heavy in the latter case will then be 
> 8. So ASAPRatio should be looking for a mass of about 687.
> > When I look in the 3D data viewer there are candidate peaks in this 
> region.
>
> I also tried xpress and got quantification that were quite different (L/H 
> is 0.66 in this example). The trouble with the xpress output is that there 
> doesn't seem to be a way to click through to the underlying data for 
> inspection. In fact using Petunia I see no way of finding out what peptide 
> or heavy peak identification the ratio is based on. Presumably this 
> information is buried somewhere in the prot.xml / pep.xml files?
>
> So my questions are:
> 1. Should -r be bigger to allow ASAPRatio to find the correct heavy peak?
> 2. Why does ASAPRatio accept a heavy peak that is so obviously the wrong 
> mass given the static modifications? It seems to be completely ignoring 
> them!
> 3. Ideally I would use the 15N search results to validate the identity of 
> the heavy peak where possible. Is there a way to do this other than looking 
> it up manually?
>
> I've uploaded a pdf with the above example, as well as some data files to: 
> https://we.tl/t-uaqpZ4gy7J 
>
> Many thanks for the help!
> Alastair
> On Wednesday, 26 August 2020 at 22:47:57 UTC+2 David Shteynberg wrote:
>
>> Hello Alastair,
>>
>> Unless there is a mistake, I

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

['Alastair Skeffington' via spctools-discuss]

Hi David,

Thanks for pointing out the wrong mass - there were a couple of others as 
well. So I've got quite a lot further with the analysis, but I'm not 
convinced that it's working as it should. Maybe it's easiest if I detail 
the steps I've gone through and ask some questions as I go. It would be 
great if you could answer these / annotate with any other thoughts you have:

Step 1: Search using standard 14N labelled masses. The 15N search seems to 
be fairly useless (see later). No variable modifications in search, because 
otherwise ASAPRatio complains and fails to run in 'static' mode.

Step 2: InteractParser $intout $in

Step 3: PeptideProphetParser $intout DECOY=XXX_ NONPARAM

Note I get a lot of model failures for this data. I get less model failures 
when I use fully parametric modelling, but then the decoy search hits are 
not properly taken into account meaning that in the final prot.xml file I 
have single -protein protein groups consisting only of a decoy hit. So I 
continued with the semi-parametric options.

Step 3: ASAPRatioPeptideParser $in  -lACDEFGHIKLMNPQRSTVWY -r0.5 -S 
-mA72.04581R160.1359N116.0603D116.0356C161.0393E130.0513Q130.076G58.03016H140.085I114.0928L114.0928K130.1124M132.0492F148.0771P98.06146S88.04073T102.0564W188.0967Y164.072V100.0771

So the -m string specifies the modified masses (ie 15N masses) for 
comparison with the 14N search results. 

This doesn't work the other way round (taking the 15N search results and 
using the 14N masses in the -m string. This is because of cysteine 
carbamylation, meaning that ASAPRatio sees the C mass as being a 'heavy' 
static modification, and the other  residues as being 'light' 
modifications. This results in an error message that there are a mixture of 
heavy and light modifications).

Step 4: ProteinProphet $light $out ASAP_PROPHET

I get ratios for reasonable numbers of proteins - but unfortunately I'm not 
convinced these are reliable. To take one example:
> L/H ratio given as 2.98
> Based on peptide: CTTSAAATSTSSGR  
> Looking at the details in the viewer I see "light +2 m/z 679.3" and 
"heavy +2 m/z 679.8"
> There are 17 N atoms in the peptide, 16 with one neutral loss. m/z for 
the mass difference between light and heavy in the latter case will then be 
8. So ASAPRatio should be looking for a mass of about 687.
> When I look in the 3D data viewer there are candidate peaks in this 
region.

I also tried xpress and got quantification that were quite different (L/H 
is 0.66 in this example). The trouble with the xpress output is that there 
doesn't seem to be a way to click through to the underlying data for 
inspection. In fact using Petunia I see no way of finding out what peptide 
or heavy peak identification the ratio is based on. Presumably this 
information is buried somewhere in the prot.xml / pep.xml files?

So my questions are:
1. Should -r be bigger to allow ASAPRatio to find the correct heavy peak?
2. Why does ASAPRatio accept a heavy peak that is so obviously the wrong 
mass given the static modifications? It seems to be completely ignoring 
them!
3. Ideally I would use the 15N search results to validate the identity of 
the heavy peak where possible. Is there a way to do this other than looking 
it up manually?

I've uploaded a pdf with the above example, as well as some data files 
to: https://we.tl/t-uaqpZ4gy7J 

Many thanks for the help!
Alastair
On Wednesday, 26 August 2020 at 22:47:57 UTC+2 David Shteynberg wrote:

> Hello Alastair,
>
> Unless there is a mistake, I think the N15 mass string should be:
>
>
>  
> -mA72.03415R160.10111N116.04293D116.02694C161.0307E130.04259Q130.05858G58.02146H140.05891I114.08406L114.08406K130.09496M132.04049F148.06841P
> 98.05276S88.03203T102.04768W188.07931Y164.06333V100.06841
>
>
> See the following post:
>
> https://groups.google.com/g/spctools-discuss/c/O-Ude_j6Nss/m/6Tl2Q-_hAAAJ
>
> However, I am not detecting correct results in your heavy 'H' search 
> results.  I would start your troubleshooting there, beginning with the 
> heavy mass of proline.
>
> Sorry this has taken me some time to get to and keep me posted to your 
> progress.
>
> Cheers,
> -David
>
>
>
>
>
>
> On Wed, Aug 26, 2020 at 2:49 AM 'Alastair Skeffington' via 
> spctools-discuss  wrote:
>
>> Hi David,
>>
>> Did you manage to grab the data before the link expired?
>>
>> Thanks,
>> Alastair
>>
>> On Monday, 17 August 2020 at 21:23:55 UTC+2 Alastair Skeffington wrote:
>>
>>> 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

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

['David Shteynberg' via spctools-discuss]

Hello Alastair,

Unless there is a mistake, I think the N15 mass string should be:

 
-mA72.03415R160.10111N116.04293D116.02694C161.0307E130.04259Q130.05858G58.02146H140.05891I114.08406L114.08406K130.09496M132.04049F148.06841P
98.05276S88.03203T102.04768W188.07931Y164.06333V100.06841


See the following post:

https://groups.google.com/g/spctools-discuss/c/O-Ude_j6Nss/m/6Tl2Q-_hAAAJ

However, I am not detecting correct results in your heavy 'H' search
results.  I would start your troubleshooting there, beginning with the
heavy mass of proline.

Sorry this has taken me some time to get to and keep me posted to your
progress.

Cheers,
-David






On Wed, Aug 26, 2020 at 2:49 AM 'Alastair Skeffington' via spctools-discuss
 wrote:

> Hi David,
>
> Did you manage to grab the data before the link expired?
>
> Thanks,
> Alastair
>
> On Monday, 17 August 2020 at 21:23:55 UTC+2 Alastair Skeffington wrote:
>
>> 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 oxidi

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

['Alastair Skeffington' via spctools-discuss]

Hi David,

Did you manage to grab the data before the link expired?

Thanks,
Alastair

On Monday, 17 August 2020 at 21:23:55 UTC+2 Alastair Skeffington wrote:

> 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
>>
>> -- 
>> 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
>>  
>

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

['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
>
> -- 
> 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
>  
> 
> .
>
 -- 
>> You received this message because you are subscribed to the Google Groups 
>> "spctools-discuss" group.
>> To unsubscribe from this group an

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

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

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

['Alastair Skeffington' via spctools-discuss]

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

['David Shteynberg' via spctools-discuss]

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-discuss+unsubscr...@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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[spctools-discuss] how to specify heavy label for oxidised methionine in ASAPRatio

['Alastair Skeffington' via spctools-discuss]

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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