I have a protein that fractionates with the outer membrane sample of Gram-negative bacterial cells.
SignalP4.1 predicts that the native protein is processed between amino acids 19(Ala)—20(Cys).
LipoP1.0 predicts that there is indeed a lipid moiety attached to the +2 position, i.e. residue 21 (Glu)
What would be the most straight-forward way in which to experimentally determine the specific amino acid on the protein that is attached to the lipid?
Is there a way in which the lipid moiety can be removed to facilitate Edman degradation (i.e. N-terminal sequencing)?
Any insights or suggestions would be happily entertained.
Cheers,
Salim
Hi,
Do you have access to a mass spec facility with an ETD equipped instrument?
If you digest the protein and analyse the peptides with this technique you can unanimously determine where the modification is.
Alternatively, you can digest the protein, create two aliquots of the resulting peptide and treat one of them in order to cleave the lipid residue.
The you compare the two. You should have one peptides with a modified MW equal to the cleaved lipid residue.
A last possibility is native Mass Spectrometry (see link)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536499/
Hi Salim, the most convincing experiment would be if you can mutate the Glu and show that the resulting mutant does no longer fractionate with membranes.
If you want to do this through chemistry, then Fabrizio has the best approach.
In Gram-negative bacteria, OM lipoproteins have lipid attached always at Cys (+1).
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