Behavior of cross-links to the protein (not peptide) termini?

[eric.merkley]

Hi everyone,

I have a question about adding a new type of linkage to my xlink.ini file.  I have a sample where peptides are linked via an amide bond from an internal K residue to the protein C terminus.  The documentation suggests that I should be able to create a linker that looks like this:

name20=isopept
isopept=K ) -18.0106 -18.01528 -18.0106 -18.01528

where the syntax is:  AA1 AA2 monomass(interlinks) avg_mass(interlinks) monomass(deadend) avg_mass(dead ends).

My question is:  will this exclude or allow peptides that have formed by an internal lysine reacting with the N-terminus of the same protein?  It seems like to exclude links within the same protein, you would use

isopept=K ) -18.0106 -18.01528 -18.0106 -18.01528

and to include links within the same protein you would use

isopept=K) K) -18.0106 -18.01528 -18.0106 -18.01528


Have I got that right?

Thanks in advance,

-Eric

[Shengbo Fan]

Hi,

isopept=K ) -18.0106 -18.01528 -18.0106 -18.01528 

means that one of the peptide linked at pro N-term, and the other peptide linked at K. And that has nothing to do with the protein, it only concerns about the peptide. So this can't exclude the link in the same protein, and the version

isopept=K) K) -18.0106 -18.01528 -18.0106 -18.01528 

can neither do that. It means that K-K, )-K, K-) and )-) are all allowed, also has nothing to do with the protein.

To achieve that, I'm afraid that it can only be done after the search complete, and use the protein information to filter the result.

[eric.merkley]

Hi Shengbo,
Thanks for the reply.  You brought up another question, however.  What is the difference between ")" and "]".  The User Guide states:

Square brackets "[" and "]" denote the peptide N terminus and C terminus respectively.  Parentheses "(" and ")" denote the protein N terminus and C terminus, respectively. 

I read that and decided I needed to use ")" because I needed to use the protein C terminus.  But in your answer you stated that K ) means lysine and protein N-terminus.  According to the user's manual that should be lysine and the protein C terminus, right?  But then in the xlink.ini file, BS3 is listed as [K [K, which according to the user's manual means peptide N-terminus, which clearly isn't correct for BS3--it should be the protein N-terminus if you cross-link before digestion (the usual way!).  Is there an error in the user manual, or in the xlink.ini?  They are inconsistent with each other.

Can you please clarify the meaning of ( ) and [ ]?

Thanks,
Eric

[Shengbo Fan]

Hi,

Yes, sorry I made two mistakes in my early reply. The correct version should be:

isopept=K ) -18.0106 -18.01528 -18.0106 -18.01528 

means that one of the peptide linked at peptide C-term, and the other peptide linked at K.

The xlink.ini version is correct, BS3 should be [K. [] means protein, () means peptide. The user guide on my computer is:

"Parentheses “(“ and”)”denote the peptide N terminus and C terminus, respectively. Square brackets “[“ and“]”denote the protein N terminus and C terminus, respectively."

But I do remeber that an early version of user guide confused these two, too. Maybe an early version is used, or an early version user guide is carelessly used in the current setup kit. I will check that.