I have a small fluorescent molecule (MW ~1000) containing a terminal alkyne. I want to conjugate it hopefully by the reaction between thiol on BSA surface and the alkyne of my molecule. There may be a problem that the unreacted molecules may be non-covalently trapped inside BSA so UV-vis absorption or emission spectrum cannot be used to confidently confirm if the alkyne is reacted or not. What method should I use to confidently confirm the reaction?
Mass spectrometry may not be sufficient as BSA is 66kDa while my dye is 1000Da.
Whole protein mass spectrometry using electrospray ionization, connected to a reverse-phase LC, is an excellent method for determining whether your label is covalently attached. Compare the mass of unreacted BSA to the mass of labeled BSA. A mass shift of 1000 Da can easily be detected. Noncovalently associated label will not be detected.
Adam, but i dont think my small molecule can affect the BSA mobility/affinity on the reverse-phase LC. How can it be possible to separate the label & unlabeled BSA?
Why not try the thiol reactive derivative of the fluorescent molecule, such as I-acetamide/N-maleimide derivatives iof the fluorescent molecule.
It is not necessary to separate the labeled and unlabeled proteins by LC before measuring the masses. The main purpose of the LC in this case is to separate the protein from the salts in the buffer and put it into the right solvent for electrospray ionization.
Lee,
1kDa is too low compared to 66 kDa to affect the retention time in HPLC. You will not see the separation of peaks. ESI mass spec could find the relative abundance of the labelled protein in the mixture.
You could try different labelling strategies but the issue would be the same. Labelling reactions are differently efficient for different proteins. Definitely the labelling mixture would contain unreacted protein, which could even interact with the labelled dye. Possibly the only solution for such condition is dilution, where free protein concentration would be too low to be detected. At this condition only single molecule experiments could filter the labelled protein from the non labelled protein. Bulk experiments will not do it. As dilution will deteriorate the signal to noise ratio in bulk.
My logical part tells me to separate the labelled from the non labelled fraction but I am learning day by day that it's almost impossible for big proteins.
Thanks
dialize your protien with vast excess of water and then run a MALDI this is more than sufficient to analyse BSA. We regularly functionalize BSA with our choice of an average 7,10, 30 alkyne linkers depending on our intended use. We regularly use of the the copper free click reagents (MW approx of each in the 260 region) and we can see the distribution shift clearly and measure the change using MALDI
hope this helps
J
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