grandrea
commented
1 year ago Hey,
As far as I know, those are additive i.e. you are defining a modification on linear peptides only called bs3nh with a deltamass of 155.094619105 AND a modification on all peptides with an NH2 group that has a mass of crosslinker mass+17.026549105. The priority will be given to the one that matches better (but here @lutzfischer is the one that should answer!).
If you want to simplify the search, use only
modification:linear::SYMBOLEXT:bs3nh;MODIFIED:K,S,T,Y;DELTAMASS:155.094619105and remove the MODIFICATIONS lines from the BS3 defitinition.
If instead you want to consider more possibilities (mods on both linear and crosslinked peptides), use only
modification:variable::SYMBOLEXT:bs3nh;MODIFIED:K,S,T,Y;DELTAMASS:155.094619105
What you are referring to is the fact that the same syntax can be put inside the crosslinker definition instead (https://github.com/Rappsilber-Laboratory/XiSearch#crosslinker-settings) Here with modifications on linear peptides only
crosslinker:SymetricSingleAminoAcidRestrictedCrossLinker:Name:BS3;MASS:138.06807;LINKEDAMINOACIDS:K(0),S(0.2),T(0.2),Y(0.2),nterm(0);LINEARMODIFICATIONS:NH2,17.026549105here with modifications on all peptides
crosslinker:SymetricSingleAminoAcidRestrictedCrossLinker:Name:BS3;MASS:138.06807;LINKEDAMINOACIDS:K(0),S(0.2),T(0.2),Y(0.2),nterm(0);MODIFICATIONS:NH2,17.026549105to define Tris as the quencher, you can define a modification for it, again as a separate modification or inside the crosslinker definition (please double check the bs3-tris mass!!)
modification:linear::SYMBOLEXT:bs3tris;MODIFIED:K,S,T,Y;DELTAMASS:259.141973or
modification:variable::SYMBOLEXT:bs3tris;MODIFIED:K,S,T,Y;DELTAMASS:259.141973And the same definitions can be given inside the crosslinker as well with the same syntax as above.
cxdummies
lutzfischer
Dear Lutz
If I understood correctly, in the default setting, the following line defines the search for BS3amidated linear peptide
modification:linear::SYMBOLEXT:bs3nh;MODIFIED:K,S,T,Y;DELTAMASS:155.094619105
In the defaut BS3 crosslinker setting, there is also the MODIFICATIONS:NH2,17.026549105
From which one of these two does xiSEARCH use to find the linear modification? Does one take precedence over the other? If BS3 is quenched by an amine-based reagent other than ammonia, e.g. Tris, where should the modifiation be defined to find the corresponding monolinks?
In the Linear Peitides result table from a search using the default BS3 setting, one can find both bs3nh and bs3nh2 in the peptide sequences. bs3nh2 is not in the search setting. Could you explain what it is and why it shows up in the sequences?
Many thanks!