isotope labeling of proteins - (Aug/19/2008 )
Hello!
I have one question regarding isotope labeling of proteins.
I did two experiments: in one I labeled proteins with 15N and in the other I deuterated them. My question is whether this labeling may change the hydrophobicity of the proteins. I am asking this since I observed that the heavy peptides were nearly always eluting on a C18 RP column slightly after the light ones.
Maybe my question can be rephrased like this: are 15N and 2H less hydrophilic than 14N and 1H?
Hope my question is not too stupid LOL
Thx for answering!
I have one question regarding isotope labeling of proteins.
I did two experiments: in one I labeled proteins with 15N and in the other I deuterated them. My question is whether this labeling may change the hydrophobicity of the proteins. I am asking this since I observed that the heavy peptides were nearly always eluting on a C18 RP column slightly after the light ones.
Maybe my question can be rephrased like this: are 15N and 2H less hydrophilic than 14N and 1H?
Hope my question is not too stupid LOL
Thx for answering!
the chemistry should not be altered but slightly physical properties regarding the mass; elution of proteins does not only concern binding properties to C18 column but also with flow and the impulse of particles (proteins)
I have one question regarding isotope labeling of proteins.
I did two experiments: in one I labeled proteins with 15N and in the other I deuterated them. My question is whether this labeling may change the hydrophobicity of the proteins. I am asking this since I observed that the heavy peptides were nearly always eluting on a C18 RP column slightly after the light ones.
Maybe my question can be rephrased like this: are 15N and 2H less hydrophilic than 14N and 1H?
Hope my question is not too stupid LOL
Thx for answering!
the chemistry should not be altered but slightly physical properties regarding the mass; elution of proteins does not only concern binding properties to C18 column but also with flow and the impulse of particles (proteins)
Hello The Bearer!
So let me see if I understood correctly. According to you it is still possible to observe slight changes in elution between heavy and light peptides but these are not due to changes in hydrophobicity. Right? :-)
i am not an expert,
but there probably isn't much if any chemical difference between N14 and N15.
However for deuterium verse hydrogen there is a difference in chemistry. The bond between D-O is stronger (shorter ~ 3%) than a H-O bond (within a molecule). This in turn have an effect on hydrogen bonding between molecules (and H exchange). Consequently the hydrogen bonding between D2O molecules is weaker than H2O molecules (~4% longer hydrogen bonds). And in H+ ion exchange, the D+ ion more tightly bound to an electronegative atom (oxygen/sulphure/nitrogen). This phenomena has been used to extract deuterium from hydrogen sources.
But I am not sure if you will see a significant effect in your experiments.
I have one question regarding isotope labeling of proteins.
I did two experiments: in one I labeled proteins with 15N and in the other I deuterated them. My question is whether this labeling may change the hydrophobicity of the proteins. I am asking this since I observed that the heavy peptides were nearly always eluting on a C18 RP column slightly after the light ones.
Maybe my question can be rephrased like this: are 15N and 2H less hydrophilic than 14N and 1H?
Hope my question is not too stupid LOL
Thx for answering!
the chemistry should not be altered but slightly physical properties regarding the mass; elution of proteins does not only concern binding properties to C18 column but also with flow and the impulse of particles (proteins)
Hello The Bearer!
So let me see if I understood correctly. According to you it is still possible to observe slight changes in elution between heavy and light peptides but these are not due to changes in hydrophobicity. Right? :-)
exactly; if it is reproducible, you have a chance to separate differently labeled forms; I wonder if you can also separate intermediately labeld forms...