Where is my protein labelled?

There are many applications for which labelling of proteins is commonly used, for example tracking of the protein of interest within a complex biological system, and even in vivo.  However, the specific residues labelled and the extent of the labelling may have an effect on the activity of the protein.

We used our bottom-up DDA proteomics platform technology, ProQuant^® , to locate and quantify the sites and extent of iodine labelling in a purified protein. In this case, the protein was designed to be processed following administration, and the location of the iodine would have a marked impact on the interpretation of the results.

We detected no labelling on histidine residues, confirming the preferential labelling of tyrosine residues.  However, across the different tyrosine residues (all of which had some labelling detected), there were differences in the extent of the iodination detected.  The exceptional precision of our patented ProQuant^® DDA platform technology in the measurement of the abundance of individual peptides enabled us to detect and interpret even small changes in labelling.

Furthermore, in addition to this simple analysis we also examined those peptides originally unidentified by the bioinformatics software but which showed a dose-dependent increase in signal following labelling. Using a series of custom scripts we delved into the MS2 fragmentation patterns and identified not only di-iodination of some of the tyrosine residues but even occasions where presumably the generation of the probe was in error because some of the tyrosines were found to be labelled with bromine!

Overall, as well as the reassurance that the labelling protocol had been successful, it was possible to confirm which part of the molecule would be likely to be detected in in vivo experiments after cleavage and processing and the labelling was unlikely to affect the biological activity of the molecule.