Detection of Glycosylations-Mass spectrometry

5 posts / 0 new
Last post
calandino77
calandino77's picture
Detection of Glycosylations-Mass spectrometry

Hello everybody,
I would like to ask you if someone of you know if it is possible to use mass spectrometry (or something similar) to see the number of glycosylations of a glycoprotein. I am trying to explain a little more.
I work with a protein that has 6 natural N-glycans, with directed-site mutagenesis I introduce an extra N-glycosylation; with western blotting I can see a litlle shift respect to the wild type, but I would like to confirm with another more accurate technique that I have already add this extra N-glycan.
Does anyone have any suggestions? I was thinking about mass spectrometry, just resolve the wild type and the mutant separately and see the difference in their MW, but I am not sure if it is possible.
Thanks in advance.

Chin Fen Teo
Chin Fen Teo's picture
 Hi calandino77,

 Hi calandino77,

This is a very interesting question, and I believe it is doable... Let's see. Below is the strategy that first came across my mind.

First, are you familiar with the N-glycosylation site mapping technique in which PNGaseF digestion is performed on tryptic peptides in the presence of O18 water? You can used this method to labeled your N-glycosylation sites on both your wild type and mutant proteins- If all the natural N-glycosylation sites are occupied, you will obtain 6 labeled Asp in your wild type protein and 7 on your mutant protein. Then when you perform a LC-MS/MS-spec analysis and look at your MS-spec sequence fingerprint, you should be able to find that one extra tryptic peptide that contains the new N-glycosylation site in the mutant that is different from the spectra you get from the wild type sample.

Hope this helps, and good luck.

calandino77
calandino77's picture
Hi Pipurri,

Hi Pipurri,
First of all, thank you so much for your time and help.
I am trying now a different method using a kit for deglycosylation in reducing and native conditions (because I would like to measure the activity of my protein as well), then I run a western blotting and I hope to see different bands that correspond to the different levels of glycosylation of my protein, from 6 N-glycans (or 7 in my mutants) to 0 N-glycans. I can see several bands but unfortunatelly not all that I would like.
Your suggestion sounds very good, but I am not very familiar with this technique of PNGaseF digestion on tryptic peptides in the presence of O18 water. Which is the procedure? it does not seem to be very difficult, but time consuming, Do I need to use a lot of protein? Do you know any protocol for this purpose? Sorry for asking you so many questions, and thanks a lot again for your help.

Chin Fen Teo
Chin Fen Teo's picture
 Hi calandino7,

 Hi calandino7,

Due to the microheterogenuity commonly found on glycosylation, I am not surprise that your de-glycosylation/western blotting approach turned out unsatisfactory. That said, MS-spec approach can provide a much more convincing result.... 

You can find the method for PNGaseF/O18 labeling in the following selected papers (provided as PubMed Index) that I came across. No, you don't need to use a lot of protein. 1 to 100 ug should be more than enough. (Every sample prep prior to MS-spec is time-consuming... No short cut there.... Learnt it the hard way.)

PMID: 15084671
PMID: 19072240
PMID: 17406563

Good luck.

calandino77
calandino77's picture
Hi Pipurri,

Hi Pipurri,
Thanks a lot for your help. I really apreciate it. I will try this method and I will let you know how the results are going.

Greetings