The protein was successfully cloned in pET19b, over expressed in E. coli BL21plys DE3 and purified in NI-NTA column using denaturating conditions. It is a bacterial surface protein can we detect PTM.
Mass spectrometry will tell you whether the protein mass is different from the predicted mass due to a post-translational modification.
First thing I would do is subject the full length protein to LC-MS. Make sure that your protein does not contain any glycerol or detergent or too much salt since all of these will act as ion suppressor. If you find that your protein as a mass corresponding to a PTM, then I would trypsinize it and apply the peptides to LC-MS. This will eventually tell you if you have good peptide coverage on which specific amino acid the PTM is.
Hope this helps,
Marc
Hi,
could someone tell how much initial protein is required for PTM analysis through mass spectrometry? I am trying to analyze a purified tagged protein for PTM.
Thanks
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