Hello, I am looking into Ubqitination modification. Actually, I am new to this field. We have analyzed Pure Ubiquitin protein using LC-MS. As per my understanding, Pure ubiquitinated protein should not be ubiquitinated (please correct me here). While studying MS/MS spectra, I could see the +114.1 Da mass difference on most of the lysine ubiquitinated (K-GG, +114.1). I also observe that mass of Asparagine amino acid is similar to K-GG. Could it interfere?. We are using Proteome Discoverer Software. Is there other modification that can be misinterpreted as K-GG (+114.1) apart from NEDD8?
Thanks for your help.
Hello Chhaya Patole ,
In response to your post:
1)
"...we have analyzed Pure Ubiquitin protein using LC-MS. As per my understanding, Pure ubiquitinated protein should not be ubiquitinated..."
Do you mean recombinant ubiquitin? If so, it should not have self-ubiquitinated.
2)
"I also observe that mass of Asparagine amino acid is similar to K-GG. Could it interfere?..."
It should not as the amino acid sequence will be searched against the protein sequence whereas modifications will be set in the variable modification section of Proteome Discoverer.
3)
"...Is there other modification that can be misinterpreted as K-GG (+114.1) apart from NEDD8?..."
Yes - you should be aware that if you reduce disulfide cysteine bonds with a reducing agent (DTT/TCEP etc) then alkylate with an acetamide-containing reagent (IAM/CAM etc), a double addition of acetamide will give the same +114 as GG.
The information below can be found at Unimod (http://www.unimod.org/modifications_list.php)
Accession: 121
PSI MS name: GG
Interim name: GlyGly
Description: ubiquitinylation residue
Monoisotopic mass: 114.042927
Composition: H(6) C(4) N(2) O(2)
Accession: 1290
Interim name: Dicarbamidomethyl
Description: Double Carbamidomethylation
Monoisotopic mass: 114.042927
Composition: H(6) C(4) N(2) O(2)
Good luck.
Peter
Thanks Peter for the information. Really, helpful. It is recombinant ubiquitin protein. One question, how to differentiate between Dicarabamidomethyl and K-GG modifications ?
Hello Chhaya Patole
"One question, how to differentiate between Dicarabamidomethyl and K-GG modifications?..."
You will need to manually review the raw spectra for the identified peptide(s).
One potential work around can be the use of chloracetamide instead of iodoacetamide as described in this paper (https://www.nature.com/articles/nmeth0608-459)
Best,
Peter
Update on the above experiments, we digested the pure ubiquitin without Iodoacetamide, and only with trypsin enzyme as it doesn't contain cysteine. Still we could see most of the Lysine modified with the 114 Da mass.
We also tried running the ubiquitin protein on gel and carrying out in-gel digestion without Iodoacetamide and Diothiotheritol. Here also we could see 114 Da modification on all the lysine .
Anyone can suggest reason for this pattern or a solution.
Thanks
Chhaya
Hello Chhaya Patole ,
Perhaps the addition of 114 Da is auto-ubiquitination if you have ruled out any extraneous source?
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