I have expressed a human glycoprotein in plant ER through a signal sequence and it also contains ER retention signal at the C-terminus. Thus, if this protein contains an N-glycan moiety, it should be the high mannose type. To confirm the high mannose type glycan, I want to do MALDI-TOF mass spectrometry after purification of this protein. So, What is the minimum concentration of glycoproteins required for this analysis? Any suggestions would be greatly help!
Hi,
What is going to be your strategy: are you planning to look at the intact protein, at proteolytic peptides, or at enzymatically released glycans in order to determine the type of glycosylation on your protein?
What is the molecular weight of your protein of interest?
Do you expect it to be fully glycosylated? On a single asparagine residue?
What kind of MALDI-TOF are you planning to use?
I guess that regardless of the route you would like to take, you will probably need at least about 10 µg of protein (I guess concentration is not so important, it's more about the absolute amount of protein you have available).
Good luck!
Thank you Dirksen!
I want to determine the enzymatically released glycans pattern from my protein and I am expecting high mannose type glycan moiety on it.
The theoretical molecular weight of the protein is 20 kDa monomer and It has 6 N-glycosylation sites, showing an apparent molecular weight of 32 kDa to 62 kDa (multiple proteins bands) on western blot using specific antibody. However, from the western blotting pattern, we can assumed that this protein is not completely glycosylated in plant ER and may be some proteins are partially and non-glycosylation as well.
What kind of MALDI-TOF would be appropriate in this case? Any suggestions?
Hi,
The type of MALDI you're using is probably not so important, other than that newer machines could be more sensitive than the older ones.
Will you be labeling your glycans after enzymatic release, for example with Rapifluor? This will likely enhance your signal intensity in MS when compared to analysis of unlabeled glycans.
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