Sub-cellular localisation of a 15N-labelled peptide vector using NanoSIMS imaging

Dynamic SIMS imaging is proposed to map sub-cellular distributions of isotopically labelled, exogenous compounds. NanoSIMS imaging allows the characterisation of the intracellular transport pathways of exogenous molecules, including peptide vectors employed in innovative therapies, using stable isotopes as molecular markers to detect the compound of interest. Shiga toxin B-subunit (STxB) was chosen as a representative peptide vector. The recombinant protein (15N-STxB) was synthesised in Escherichia coli using 15NH4Cl as sole nitrogen source resulting in 15N enrichment in the molecule. Using the NanoSIMS 50 ion microprobe (Cameca), different ion species (12C14N , 12C15N , 31P ) originating from the same sputtered micro volume were simultaneously detected. High mass resolving power enabled the discrimination of 12C15N from its polyatomic isobars of mass 27. We imaged the membrane binding and internalisation of 15N-STxB in HeLa cells at spatial resolutions of less than 100 nm. Thus, the use of rare stable isotopes like 15N with dynamic SIMS imaging permits sub-cellular detection of isotopically labelled, exogenous molecules and imaging of their transport pathways at high mass and spatial resolution. Application of stable isotopes as markers can replace the large and chemically complex tags used for fluorescence microscopy, without altering the chemical and physical properties of the molecule.