Probing glycoforms for most biologically active

[WuShock]

How would one go about, practically, probing a pool of glycoforms (isoforms) of a particular protein for the type which are most biologically active, that is to say the ones which have the greatest activity? The "brute force method," so-to-speak, would involve assaying every single one and if the potential population is very large then you can imagine how costly and cumbersome the task would be. Much insight could be gleamed from a bioinformatics approach, but I'm wondering does anyone know of a particular approach in the literature? Or does anyone have any suggestions?

[GDG]

If your activity involves affinity to a target,

• fix your target on a plate (or other substrate),
  
• add a pool of your glycoforms in solution,
  
• equilibrate, and
  
• see which glycoform binds to the substrate most.
  

[CharonY]

As GDG mentioned, one could design assays depending on their functions. In cases of enyzmatic reactions that are colorimetrically traceable you can make quick parallel assays, and if you got really a lot of different isoforms (say, over a hundred) it may make sense to make pooled assays and trace back the isoform with the highest activity.

 

But it appears that you are primarily interested in a computational approach. Here you are usually dependent on enzymes whose precise interaction with its substrate is known (or the precise mode of protein-protein interaction, or whatever). Often derived from crystallization studies. Then you can start making docking experiments, though they are often not that quick either. And of course you always need to validate it as the computational approaches normally only give suggestions, not answers.

[ivanp84]

Bioinformatics can't give you informations which isoform is more active in vivo. Depending on protein type you can make assay for testing biological activity. For example, if you investigating some lectin from some Leguminosae (or Fabaceae) and you find 5 isoforms with isofocusing and you want to find wich isoform is more active at pH = 7.00, then you can make an easy and fast assay with hemoagglutination of rabbit or human erythrocytes. (Make dilutions of erythrocytes in PBS and standard PBS dilution of separated isoforms, and then test each isoform in microtiter plate). That is the way for testing activity, but if you testing affinity, like in Ig case, then you could use pool of, for example, IgGs and a pure antigen for proper IgG. (Ig-Ag precipitation...)

None of these standard biochemical methods are "brute force method".

Cheers!