I have expressed my peptide (2.9 KDa) as a fusion protein to a 22 KDa protein in e coli (bl21 de3), and then cleave it off in vitro after Ni-NTA purification. I have then purified further, and obtained pure peptide of the exact expected mass (many times).
Recently, I tried expressing the protein, purifying and characterizing though this time I only saw one product of exactly 108 mass units heavier than expected. I re-expressed and purified again with fresh materials, and still I see the M+108 product only.
This is strange because I have been using the same plasmid DNA miniprep for expression, the same batch of BL21 DE3 competent cells, exactly the same expression conditions, the same Ni-NTA column, and the same cleavage conditions and materials. I have a small amount of the previous successful peptide preparation left and run the mass spec analysis in parallel to ensure the instruments are functioning properly, matrix (MALDI) and buffer (LCMS) are okay, and they are. I see the peptide prepared last month at exactly the correct mass and retention time (LCMS) and the new batch at M+108 and a different retention time.
The problem doesn't seem to be unique to this peptide sequence. I have expressed another peptide as a 'control' (same method but a different primary sequence) and also see only the M+108 mass peak.
if you have any insight into the issue, I would certainly appreciate it.
this is interesting. there is a paper describing artefact that leads to the addition of 108 Da to cysteine http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911956/
since your peptide size is small and you know the sequence, I would advise you to run MS/MS and see where this modification occurs.
Cheers,
David.
Thanks David, I actually found this paper early in my search and preformed MS/MS. unfortunately this did not lead to the identification of the possible modification. Though the authors mention that this modification could be the result of SDS-PAGE and/or gel-staining/destaining steps, which I am not using on these samples.
Still, your help is appreciated!
Hi Andrew - did you manage to identify all of amino acids in the peptide? did you localize the mass shift? or is it amorphous?
i was not able to localize the shift, though i was only able to identify about half the B and Y ions expected.
Hi Andrew,
As you might be aware adducts leading to mass increment depends on the nucleophilic sites.. If you have lysine, histidine, arginine etc.. you can expect MDA or MDA like adducts (+108 da) Malonaldehyde schiff base adduct (products of oxidation).....
The only way to sort out is to acquire good quality MS/MS spectra and try to annotate the spectra either through De novo sequencing softwares (PEAKS) or through manual annotation... You may also consider using error tolerant searches for your MS/MS spectra.
All the best
Saravanan K
Hi Andrew,
if you can't localize the mass shift in the spectrum (do you allow for neutral losses?), it may be an adduct. you can use low-energy ISD to shake off any "dirt" and see if you still see the mass shift.
Cheers,
David.
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