Hi Eduard

I am curious to know why you say that changing the availability of MAL terminals will have no effect on your grafting ratio?

For the most part, the level of successful grafting will be dictated by the amounts of either your MAL ligands or the peptide you are binding. Preferably an excess of peptide will ensure complete binding up until the bound peptide is sterically hindering the binding of any more peptide..

Another issue that could result in a poor binding efficiency is that when your methoxy-PEG coverage is very dense and the MAL-PEGs are the same length it can be difficult for the peptide to access the binding site. Possibly investigate using a slightly longer MAL-PEG to aid accessibility. 

Depending on what the bound peptide is and its desired purpose, the optimal binding percentage for you will usually have to be determined experimentally.

Hope this help,

Alan

Hi Eduard

I am curious to know why you say that changing the availability of MAL terminals will have no effect on your grafting ratio?

For the most part, the level of successful grafting will be dictated by the amounts of either your MAL ligands or the peptide you are binding. Preferably an excess of peptide will ensure complete binding up until the bound peptide is sterically hindering the binding of any more peptide..

Another issue that could result in a poor binding efficiency is that when your methoxy-PEG coverage is very dense and the MAL-PEGs are the same length it can be difficult for the peptide to access the binding site. Possibly investigate using a slightly longer MAL-PEG to aid accessibility. 

Depending on what the bound peptide is and its desired purpose, the optimal binding percentage for you will usually have to be determined experimentally.

Hope this help,

Alan

Hi, Eduard,

I have experience working with PEGs. I have to agree with Alan Hibbitts. Changing the MAL:methoxy ratio might have an effect on your conjugation. In addition, changing the ratio of the peptide thiol relative to MAL might have an effect on your conjugation. With a sufficiently large peptide, steric hindrance will be a problem that will limit the degree of conjugation you get.

Alan Hibbitts is also correct that making the methoxy-PEG shorter than the MAL-PEG will improve your reaction. If the methoxy-PEG is used simply to eliminate non-specific binding, then you can use a relatively short PEG and improve your conjugation efficiency.

Reaction pH is also important. Although the thiol-maleimide reaction is a form of click chemistry, the maleimide reactivity improves (up to a point) with higher pH. We perform bioconjugation reactions in our lab, and we keep the pH around 6.5 in phosphate buffer, because the maleimide reacts with free amines starting at about pH 7, and at pH 7.5 and above, the rate of maleimide reaction is significant. We use 2-5 mM EDTA in our phosphate reaction buffer to prevent oxidation of the free thiols to which we are trying to conjugate.

Above all, you have to optimize your conjugation conditions experimentally thinking through the points given here.

I hope this helps.

Thank you for the responses gentlemen.

In regard to the grafting ratio it was suggested to me that, given a 10% molar excess of peptide, the proportion of available MAL endcaps successfully conjugated would be approximately 50% regardless of how many MAL endcaps there were in the first place.

I can definitely see the increase of steric hindrance of peptide that comes with an increase in the MAL coverage density (say, 10% to 30%) leading to a decrease in the grafting efficiency. However, given that the MAL coverage density remains relatively low and that the peptide is small (~2000 Da), what phenomenon would cause this change in graft efficiency?

As far as using shorter PLGA-PEGm chains that is an excellent idea I will definitely try!

Lastly, about avoiding disulfide bridging, I have been taking care to use my peptide (transferrin) within one minute of making the peptide stock (dry protein in DMSO) thinking this would keep most of the thiols reduced. Am I mistaken in this assumption?

Thank you so much for the help!

EF

Hi, Eduard,

In our lab, we typically get >80% efficiency when labeling free thiols on peptides and proteins with maleimide-functionalized PEG linkers. We're going at the problem in the opposite direction from you; we are running the MAL in excess relative to the free thiols, but 80% efficiency is still much better than 50% efficiency. Our results are consistent with published results, for example: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3086356/

I don't know for sure that steric hindrance is an issue for you, but PEG has a very large hydrodynamic volume relative to its molecular weight. This large hydrodynamic volume excludes proteins. See, Kap Lim and James N. Herron, "Molecular Simulation of Protein-PEG Interaction," in Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications. J. Milton Harris, ed. Plenum Press, New York, copyright 1992, page 53. This could possibly happen to your peptide also, even though, at 2000 Daltons, it is relatively small.

Regarding disulfide formation, in aqueous solution, your assumption would be correct that using peptide within one minute of making the stock solution would be okay. I don't know if that holds true for solutions in DMSO; my guess -- and it's only a guess; I have not researched it -- is that one minute is still fine. Also, do you take care to exclude the possibility of air oxidation of thiols?

Hope this helps.

Hi Robert,

Thank you for the detailed response!

I think you are correct that the 2000 Da peptide would not cause much steric hindrance, however, I will soon be conjugating antibodies instead of peptides so that Lim/Herron paper will definitely come in handy.

Using excess MAL is an interesting idea which I will need to try. Just to be clear then, when you talk about conjugation efficiency in your case you mean 80% of peptide/protein you started with has been conjugated, as opposed to 80% of MAL endcaps being conjugated? 

And yes, I purge the reaction vial with nitrogen to avoid oxidation. The gas is bubbled through for around a minute for a 20 ml vial.

Thanks so much!

EF

Hi, Eduard,

Yes, I mean that >80% of the peptide/protein is conjugated. Very often we can push that well past 90%.

I wish you great success in your research!

Ok great! I am making these changes already.

Thank you both for the help and good luck in your research!

EF