Hi All,
I have purified a His-tagged recombinant human protein and performed Q-TOF for determining molecular mass. However, I get two peaks all time with mass difference of 131 Da (deconvoluted mass of the protien attached). Although we thought it might be a due to deletion of N-terminal methionine, the peak ratio was low for the intact protein. What might be the reason for the second peak? has anyone encountered such problem? Kindly give suggestion.
Thank you.
It looks like your protein may have some partial post translational modifications of the polypeptide chain like phosphorylation or sulphation. That would explain the differences in the molecular weight that you observe. Usually, this type of protein modification may be revealed in SDS-PAGE. You should see two bands with slight difference in their molecular weight. If so, you could try to use specific antibodies to detect those modified proteins in western blot.
I guess the native protein sequence has been affected during N-terminal tags removal.
I agree with Markus. E. Coli systems tend to remove N-termini methionine.
Thanks Viktor and Markus for the suggestion. Viktor, there is no additional bands in purified protein suggesting for phosphorylation or sulphation. I agree Markus that it might be because of excision of N-terminal methionine. This fits well for the molecular mass difference. We also conclude it might be because of loss of methionine. Thank you for your suggestions.
It is common for us also to see His-tagged purified recombinant proteins with and without the Methionine as first amino acid during N-terminal Edman sequencing experiments..
Best, Hediye
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