[Re(CO)3]+ labelling of a novel cysteine/hexahistidine tag: Insights into binding mode by liquid chromatography-mass spectrometry

We recently described a novel amino acid sequence, KCKLAAALEHHHHHH, for site-specific radiolabelling of proteins with [99mTc(CO)3(OH2)3]+ or [Re(CO)3(OH2)3]+ with improved efficiency compared to conventional hexahistidine tags (His-tag). C2AH, a modification of the protein C2A (the phosphatidylserine (PS)-binding domain of rat synaptotagmin I) engineered to contain this novel C-terminal tag, was produced. Rhenium tricarbonyl conjugates of C2AH were analysed post tryptic digest by liquid chromatography-electrospray mass spectrometry (LC-MS), giving rise to a peak with the molecular weight corresponding to M+ = [Re(CO)3 + CK + LAAALEHHHHHH]+. This species arises as a result of trypsin cleavage on the C-terminus of both the lysine (Lys) residues on either side of the Cys while both fragments still remain bound to the rhenium. This confirmed that cysteine (Cys) was directly involved in the coordination of the rhenium tricarbonyl. To demonstrate the superiority of the cysteine containing His-tag sequences for binding [Re(CO)3]+, two peptides CKLAAALEHHHHHH and LAAALEHHHHHH were synthesised. In a competition experiment the mixed peptides were incubated with one molar equivalent of [Re(CO)3(H2O)3]+, and LC-ESMS demonstrated that 92% and 9% of CKLAAALEHHHHHH and LAAALEHHHHHH respectively were co-ordinated by one [Re(CO)3]+.

Rhenium tricarbonyl binds site specifically to the novel histag sequence (CKLAAALEHHHHHH) via the Cys and histag. Competition assays demonstrate that rhenium tricarbonyl preferentially binds to the Cys-Histag. Such sequences incorporated into recombinant proteins will allow for high throughput screening and site specific labelling of potential recombinant protein based radiopharmaceuticals.Figure optionsDownload as PowerPoint slideHighlights
► Rhenium tricarbonyl coordinates via histag and adjacent cysteine
► Rhenium tricarbonyl preferentially binds to Cys-Histag as compared to histag alone
► Electrospray ionisation mass spectrometry can be used to study inorganic complexes