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Nesvilab / FragPipe

A cross-platform proteomics data analysis suite
http://fragpipe.nesvilab.org
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biotinylated search #371

Closed giorgiolivi closed 3 years ago

giorgiolivi commented 3 years ago

Dear all,

I have just a curiosity, Recently I’ve performed histone pulldown, And I would like to analyse my sample using LC-MS/MS. The sequence are: Histone H3K9 Peptide - biotinylated ARTKQTARKSTGGKAPRKQLA - GGYK(Biotin) - NH2 Histone H3K9me3 Peptide - biotinylated ARTKQTAR-Kme3- STGGKAPRKQLA - GGYK (Biotin) - NH2 A synthetic peptide derived from Histone H3.1, amino acids 1-21, with an added lysine residue containing a biotin moiety and a C-terminal amide group (CONH2). C-term biotinylated Lys residue is separated with two Gly (provide short spacer) and one Tyr (provides accurate UV quantification) from the rest of the peptide sequence.

Which would be the best way to search the data using fragpipe, Variable modification, or fixed modification or is there any specific functionality at PTMs level bar? Thanks, Regards

anesvi commented 3 years ago

I am not sure I fully understand. If you just care about the interactors of your bait peptides, then do a normal search. Are you asking what is the best way to identify the bait peptides themselves? I do not know. In addition to the modifications on K, they have a lot of trypsin cleavage sites. Are you digesting samples from the pulldowns with trypsin? We do not have any special functionality. How would you search with other tools? Alexey

From: giorgiolivi @.> Sent: Tuesday, May 18, 2021 4:23 AM To: Nesvilab/FragPipe @.> Cc: Subscribed @.***> Subject: [Nesvilab/FragPipe] biotinylated search (#371)

External Email - Use Caution

Dear all,

I have just a curiosity, Recently I’ve performed histone pulldown, And I would like to analyse my sample using LC-MS/MS. The sequence are: Histone H3K9 Peptide - biotinylated ARTKQTARKSTGGKAPRKQLA - GGYK(Biotin) - NH2 Histone H3K9me3 Peptide - biotinylated ARTKQTAR-Kme3- STGGKAPRKQLA - GGYK (Biotin) - NH2 A synthetic peptide derived from Histone H3.1, amino acids 1-21, with an added lysine residue containing a biotin moiety and a C-terminal amide group (CONH2). C-term biotinylated Lys residue is separated with two Gly (provide short spacer) and one Tyr (provides accurate UV quantification) from the rest of the peptide sequence.

Which would be the best way to search the data using fragpipe, Variable modification, or fixed modification or is there any specific functionality at PTMs level bar? Thanks, Regards

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giorgiolivi commented 3 years ago

thanks a lot for your quick reply. In my case I was just curious to see if the histone pulldown interactome. (in this case I will use the regular search). Since the histone fragment has plenty of K or R , I do believe it is a massive challenge to reconstruct the original peptide, for the software. I was just inquiring if at least I could see the Biothylated fragment (GGYK (Biotin) - NH2) using fragpipe. Thanks again.

On Tue, 18 May 2021 at 13:34, Alexey Nesvizhskii @.***> wrote:

I am not sure I fully understand. If you just care about the interactors of your bait peptides, then do a normal search. Are you asking what is the best way to identify the bait peptides themselves? I do not know. In addition to the modifications on K, they have a lot of trypsin cleavage sites. Are you digesting samples from the pulldowns with trypsin? We do not have any special functionality. How would you search with other tools? Alexey

From: giorgiolivi @.> Sent: Tuesday, May 18, 2021 4:23 AM To: Nesvilab/FragPipe @.> Cc: Subscribed @.***> Subject: [Nesvilab/FragPipe] biotinylated search (#371)

External Email - Use Caution

Dear all,

I have just a curiosity, Recently I’ve performed histone pulldown, And I would like to analyse my sample using LC-MS/MS. The sequence are: Histone H3K9 Peptide - biotinylated ARTKQTARKSTGGKAPRKQLA - GGYK(Biotin) - NH2 Histone H3K9me3 Peptide - biotinylated ARTKQTAR-Kme3- STGGKAPRKQLA - GGYK (Biotin) - NH2 A synthetic peptide derived from Histone H3.1, amino acids 1-21, with an added lysine residue containing a biotin moiety and a C-terminal amide group (CONH2). C-term biotinylated Lys residue is separated with two Gly (provide short spacer) and one Tyr (provides accurate UV quantification) from the rest of the peptide sequence.

Which would be the best way to search the data using fragpipe, Variable modification, or fixed modification or is there any specific functionality at PTMs level bar? Thanks, Regards

— You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub< https://github.com/Nesvilab/FragPipe/issues/371>, or unsubscribe< https://github.com/notifications/unsubscribe-auth/AIIMM6657I4OTIPKTVAQTLDTOIPXZANCNFSM45CA3C6Q>.

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anesvi commented 3 years ago

GGYK (Biotin) - NH2 fragment is too short to be identified from MS/MS in a normal search. We cannot easily help with that, unfortunately, in our tools

From: giorgiolivi @.> Sent: Tuesday, May 18, 2021 9:45 AM To: Nesvilab/FragPipe @.> Cc: Nesvizhskii, Alexey @.>; Comment @.> Subject: Re: [Nesvilab/FragPipe] biotinylated search (#371)

External Email - Use Caution thanks a lot for your quick reply. In my case I was just curious to see if the histone pulldown interactome. (in this case I will use the regular search). Since the histone fragment has plenty of K or R , I do believe it is a massive challenge to reconstruct the original peptide, for the software. I was just inquiring if at least I could see the Biothylated fragment (GGYK (Biotin) - NH2) using fragpipe. Thanks again.

On Tue, 18 May 2021 at 13:34, Alexey Nesvizhskii @.<mailto:@.>> wrote:

I am not sure I fully understand. If you just care about the interactors of your bait peptides, then do a normal search. Are you asking what is the best way to identify the bait peptides themselves? I do not know. In addition to the modifications on K, they have a lot of trypsin cleavage sites. Are you digesting samples from the pulldowns with trypsin? We do not have any special functionality. How would you search with other tools? Alexey

From: giorgiolivi @.<mailto:@.>> Sent: Tuesday, May 18, 2021 4:23 AM To: Nesvilab/FragPipe @.<mailto:@.>> Cc: Subscribed @.<mailto:@.>> Subject: [Nesvilab/FragPipe] biotinylated search (#371)

External Email - Use Caution

Dear all,

I have just a curiosity, Recently I’ve performed histone pulldown, And I would like to analyse my sample using LC-MS/MS. The sequence are: Histone H3K9 Peptide - biotinylated ARTKQTARKSTGGKAPRKQLA - GGYK(Biotin) - NH2 Histone H3K9me3 Peptide - biotinylated ARTKQTAR-Kme3- STGGKAPRKQLA - GGYK (Biotin) - NH2 A synthetic peptide derived from Histone H3.1, amino acids 1-21, with an added lysine residue containing a biotin moiety and a C-terminal amide group (CONH2). C-term biotinylated Lys residue is separated with two Gly (provide short spacer) and one Tyr (provides accurate UV quantification) from the rest of the peptide sequence.

Which would be the best way to search the data using fragpipe, Variable modification, or fixed modification or is there any specific functionality at PTMs level bar? Thanks, Regards

— You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub< https://github.com/Nesvilab/FragPipe/issues/371>, or unsubscribe< https://github.com/notifications/unsubscribe-auth/AIIMM6657I4OTIPKTVAQTLDTOIPXZANCNFSM45CA3C6Q>.

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giorgiolivi commented 3 years ago

Thanks again, and thanks also to the lovely tutorial of fragpipe shown last week at the May Institute. Regards.

On Tue, 18 May 2021 at 13:49, Alexey Nesvizhskii @.***> wrote:

GGYK (Biotin) - NH2 fragment is too short to be identified from MS/MS in a normal search. We cannot easily help with that, unfortunately, in our tools

From: giorgiolivi @.> Sent: Tuesday, May 18, 2021 9:45 AM To: Nesvilab/FragPipe @.> Cc: Nesvizhskii, Alexey @.>; Comment @.> Subject: Re: [Nesvilab/FragPipe] biotinylated search (#371)

External Email - Use Caution thanks a lot for your quick reply. In my case I was just curious to see if the histone pulldown interactome. (in this case I will use the regular search). Since the histone fragment has plenty of K or R , I do believe it is a massive challenge to reconstruct the original peptide, for the software. I was just inquiring if at least I could see the Biothylated fragment (GGYK (Biotin) - NH2) using fragpipe. Thanks again.

On Tue, 18 May 2021 at 13:34, Alexey Nesvizhskii @.<mailto:@.>>

wrote:

I am not sure I fully understand. If you just care about the interactors of your bait peptides, then do a normal search. Are you asking what is the best way to identify the bait peptides themselves? I do not know. In addition to the modifications on K, they have a lot of trypsin cleavage sites. Are you digesting samples from the pulldowns with trypsin? We do not have any special functionality. How would you search with other tools? Alexey

From: giorgiolivi @.<mailto:@.>> Sent: Tuesday, May 18, 2021 4:23 AM To: Nesvilab/FragPipe @.<mailto:@.>> Cc: Subscribed @.<mailto:@.>> Subject: [Nesvilab/FragPipe] biotinylated search (#371)

External Email - Use Caution

Dear all,

I have just a curiosity, Recently I’ve performed histone pulldown, And I would like to analyse my sample using LC-MS/MS. The sequence are: Histone H3K9 Peptide - biotinylated ARTKQTARKSTGGKAPRKQLA - GGYK(Biotin) - NH2 Histone H3K9me3 Peptide - biotinylated ARTKQTAR-Kme3- STGGKAPRKQLA - GGYK (Biotin) - NH2 A synthetic peptide derived from Histone H3.1, amino acids 1-21, with an added lysine residue containing a biotin moiety and a C-terminal amide group (CONH2). C-term biotinylated Lys residue is separated with two Gly (provide short spacer) and one Tyr (provides accurate UV quantification) from the rest of the peptide sequence.

Which would be the best way to search the data using fragpipe, Variable modification, or fixed modification or is there any specific functionality at PTMs level bar? Thanks, Regards

— You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub< https://github.com/Nesvilab/FragPipe/issues/371>, or unsubscribe<

https://github.com/notifications/unsubscribe-auth/AIIMM6657I4OTIPKTVAQTLDTOIPXZANCNFSM45CA3C6Q>.

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