Hi,

Where did you obtain the theoretical masses of your proteins?

And did you run a marker on the 2D gel to estimate the molecular weight of proteins after 2D separation?

In general, the molecular mass estimated by 2D-GE hardly ever matches the theoretical mass, not only due to the fact that gel electrophoresis provides you with an apparent molecular weight -migration is actually determined by molecular shape and SDS coverage- but also due to the presence of post-translational modifications that can impact mass and shape (such as glycosylation).

In your case however, the difference between the theoretical and observed masses is quite big.. This could be due to proteolytic digestion of your proteins during sample storage or preparation.

Or, and this may sound silly, could it be that your theoretical molecular weight is based on a multimeric protein that falls apart into smaller subunits during reduction (assuming you ran reducing 2D-GE)? A monoclonal antibody, for example, has a theoretical molecular weight of 150 kDa, but will appear as two spots around 25 kDa (light chain) and 50 kDa (heavy chain), respectively.

Hope this helps!

Hi,

I second Dirksen view of proteolytic digestion of the protein during isolation or sample preparation. I suggest you to check using PeptideCutter (ExPASy), if these specific proteins have potential cleavage sites for some specific proteases. Generally this happens when isolating protein from cells or tissue undergoing apoptosis which is mediated by caspases or similar proteases. But in case, this patter is with all the protein spot in you gel then something has gone wrong. Use 2D protein marker to cross check your result.

Hi,

thanks for your reply. Yes, we do have a reduction and alkylation step in the protocol (DTT and IAA) and a marker for protein weight. I found for example hexamerin110 (MW 110kDa) in many of my spots and the spots indicate masses of 20-50kDa. Where can I check if there might be possible subunits? I did not find some hints on ncbi or after a short literature research.

Nice explanation @ Eef Dirksen.

Thanks, Awanish Kumar!

A quick scan of Uniprot entry D3KZF8 indeed seems to confirm that Hex110 is a monomeric protein.

Did you check the raw data of your mass spec identifications of the 20-50kDa protein spots? The sequence coverage obtained for these identifications could point you in the direct of parts of the protein that were retrieved; I'd expect that different peptides, from different parts of the sequence, are underlying the identification of Hex110 in multiple protein spots you excised.

The coverage ranges from 0,06-0,26, so its not quite clear if the matched peptides really assign to Hex110?

Can you visualize which parts of the Hex110 sequence are actually covered by the peptides identified in your experiment? A number will not say much, I'm afraid, but if you can correlate the values to parts of the sequence, that might help.

Attached you will find the results I got from the proteom factory. In the latest results ("protein") we just got a table with matching proteins without any further information. In the order before ("Hex110") we got also information about the protein coverage as you can see.