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GENERATION OF TRYPTIC PEPTIDE

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GENERATION OF TRYPTIC PEPTIDE vanyabangera  2024-01-28 06:01
 

Hello Team,

I have generated tryptic peptides using skyline.

Could you please confirm whether the method I followed is correct or not?

a) I have edited peptide settings with and without (none) background proteome
b) Edit>import>FASTA>and paste by peptide sequence from the FASTA file

I would require clarifications on background proteome and edit>import>FASTA options. At first, I had the whole FASTA file of an organism as background proteome and pasted the whole FASTA file of an organism in edit>import>FASTA options. It gave me some tryptic peptides.
Secondly, I gave none as background proteome and imported a peptide sequence of interest on edit options. It gave me some tryptic peptides

But different results. why is that so? What exactly do background proteome and edit>import>FASTA options do in this case?
 
 
Nick Shulman responded:  2024-01-28 06:16
Your choice of background proteome would not normally affect which peptides ended up in the Targets tree unless you also told Skyline to enforce uniqueness using one of the uniqueness options at "Settings > Peptide Settings > Digestion".

I am not sure I understand your question but if you send us a couple of Skyline documents I could probably tell you why they are different.

In Skyline you can use the menu item:
File > Share to create a .zip file containing your Skyline document and supporting files including background proteome (.protdb) and potentially audit log (.skyl) files.

Files which are less than 50MB can be attached to this support request. You can upload larger files here:
https://skyline.ms/files.url

--Nick
 
vanyabangera responded:  2024-01-28 21:38

Hello Nick,

Thank you for your response.

As per the instructions, I have uploaded the skyline zip files here https://skyline.ms/project/home/support/file sharing/begin.view?. However, I could not give a better explanation which might find you difficult in tracking my query. So I decided to share the files in continuation with our conversation through this.

Pardon me if I am uploading multiple files. The uploaded files are as follows:

1)GP_15_400_w_o_backgroundproteome_peptideofinterest: I have not given background proteome and peptide sequence (FASTA) of interest given in edit>import>FASTA option

2)GP_15_400_withbackgroundproteome_allFASTAsequence: I have given background proteome (Uniprot_GP_15_400 protein FASTA sequence) and Uniprot sequence of GP_15_400 FASTA sequence given in edit>import>FASTA option
 
Nick Shulman responded:  2024-01-29 07:54
It sounds like your question is why is Skyline giving you so few tryptic peptides for your protein of interest.

The protein sequence in your "without" document was:
LTDAQKDEIRKMKEEGKPKIDMQKKIFDYYENLTGDGKKEAGEKLRGGCRELLRQIVGDEKMAELKQMKESGLGQEELIAKVDEMLGHITDEAKKQKIHEYGPSCRKIYEDRYKRDNHEHSLDDYFRTHLS
and the only peptides that Skyline has given you are:
QIVGDEK
ESGLGQEELIAK

The reason that these are the only peptide sequences that Skyline gave you has to do with the options that have been selected at "Settings > Peptide Settings > Filter".
The checkboxes have been checked next to "Cys", "Met", "His", "NXT/NXS" and "RP/KP" in the "Exclude peptides containing:" list box.
This means that Skyline will not give you peptides containing those amino acids or sequences.

One thing that you could do is uncheck those checkboxes and then Skyline would give you more peptides.
A different thing that you could do is right-click on your protein in the Targets tree and choose "Pick children". In the child picker window that appears, you could click the filter button to unselect it and then Skyline would allow you to choose peptides that would normally be excluded by the peptide filter settings.
-- Nick
 
vanyabangera responded:  2024-01-30 20:25
Thank you for your response