Hi all, I am having difficulty in identifying post-translational modification in the protein I am working with. So any suggestions will be highly appreciated. I am working with an enzyme that belongs to oxidoreductase family and it uses a cofactor (NADH or NAD+) to convert a substrate into a product. Mass spec of the enzyme shows the addition of 742Da to the expected molecular weight (52214 Da). Initially I suspected that the extra molecular weight is due to an adduct formation of NADPH with the enzyme through a cysteine residue. There are two cysteines and LC-MS/MS shows that both the cysteines are free and capable of alkylation. That means the modification is not at cysteine but at a different residue. I am wondering what are the possible ways with which I can identify this modification and what residues can make adduct with this cofactor?
Hi,
Depending on the conditions (native or denaturing) you've applied to generate the MS data showing the mass increase, you could be looking at a non-covalent interaction of NADPH with your protein; have you checked whether the modification is actually covalent?
If so, you could consider performing a proteolytic (e.g. trypsin) digest of your protein and compare the resulting set of peptides coming from the protein with a suspected modification to that coming from your protein without the suspected modification to pinpoint in what part of the sequence of your protein this modification might be occurring.
For that, you will need an LC-MS/MS system that allows you to perform tandem ('MS/MS') fragmentation of peptide masses.
Hope this helps!
Hi Jhanvi, Eef is right (as usual) , that you might have too much ambiguity unless you do an enzymatic digestion and MS/MS; And you could then also try a 'wildcard' search for specific masses, or on specific residues which will home in on the modification and/ or location. That will narrow down the possibilities, which is why we created that 'wildcard' capability.
Hi,
Were you able to generate digests of both modified and unmodified protein? That could already provide some insight into what sequence(s) are affected, using the unmodified digest data.
Ideally, that information can then be confirmed by actual MS/MS data, even of these are incomplete or sub-optimal.
Hi Jhanvi, well your task might be easier than you think... if you have data in hand you can simply ask software to narrow down the options for you. I know your institution already has a copy of Byonic which would rank the matches according to the b/y (and ../c/z for ETD) ions and annotate them with the proposed match from all of the options available in a long list. You could also try restricting the mass you saw at 742Da to certain residues, and see if that matches directly. And also to prevent a giant list, so I think this will end up being just a few minutes' work. And if you want to look at the different locations - if there is more than one - and tabulate that, we can automate that for you too. If you think there really is poor MS/MS for some critical peptides that might have the modification/ adduct [?] then you can simply add in the theoretical MS1 in-silico too from an automated checkbox (that's Byologic, which can add in XICs and use MS1 only as well as the MSMS info you have). If your core facility does not have time to let you use the software, you can always contact Brent on our website who can let you try a free demo copy. Beats doing it by hand if you ask me...
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