How can I locate the site of PEGylation using this mass spectra? I have carried out peptide mass fingerprinting for the m/z ratios obtained for both the molecules, but not able to deduce a strong conclusion.
Thank you Dr. Pach. Would it helpful if I provide the m/z ratios corresponding to each mass spectra? Please find the file attached.
Dear Pooja Nanda,
It is pretty clear that there is no traces of PEG in these mass spectra.
Otherwise I agree with Mr. Päch´s suggestions and comments.
Regards
Robert Luxenhofer
Thank you Dr. Luxenhofer and Dr. Pach.
I have used a protein called uricase and PEGylated it at the thiol group of its cysteine residue. The SDS-PAGE results indicate that following PEGylation with 5 kDa mPEG-derivative, there was an increase in the molecular weight of uricase from 33 kDa to 43 kDa (per subunit). Please find the gel doc image of native and PEGylated uricase as an image attachment.
I subjected the excised native and PEGylated uricase bands from the SDS-PAGE gel separately to tryptic digestion for fragmentation into smaller peptides. This was followed by generation of mass spectra for the digested samples using the routine procedure of MALDI-MS.
Upon comparison of mass spectra of both the molecules, the position of the missing amino acid on a certain peptide (specifically cysteine residue where PEGylation occurred in this case) can be located. The m/z ratios thus obtained, can then be subjected to peptide mass fingerprinting for confirmation of results.
There is slight difference in both the mass spectra obtained, but the peptide mass fingerprinting results of both the molecules aren't conclusive. As a result of this, I am unable to figure out/ confirm the location of PEGylated amino acid to properly conclude my results.
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