50nm-scale localization of single unmodified, isotopically enriched, proteins in cells.

ABSTRACT: Imaging single proteins within cells is challenging if the possibility of artefacts due to tagging or to recognition by antibodies is to be avoided. It is generally believed that the biological properties of proteins remain unaltered when (14)N isotopes are replaced with (15)N. (15)N-enriched proteins can be localised by dynamic Secondary Ion Mass Spectrometry (D-SIMS). We describe here a novel imaging analysis algorithm to detect a few (15)N-enriched proteins--and even a single protein--within a cell using D-SIMS. The algorithm distinguishes statistically between a low local increase in (15)N isotopic fraction due to an enriched protein and a stochastic increase due to the background. To determine the number of enriched proteins responsible for the increase in the isotopic fraction, we use sequential D-SIMS images in which we compare the measured isotopic fractions to those expected if 1, 2 or more enriched proteins are present. The number of enriched proteins is the one that gives the best fit between the measured and the expected values. We used our method to localise (15)N-enriched thymine DNA glycosylase (TDG) and retinoid X receptor ? (RXR?) proteins delivered to COS-7 cells. We show that both a single TDG and a single RXR? can be detected. After 4 h incubation, both proteins were found mainly in the nucleus; RXR? as a monomer or dimer and TDG only as a monomer. After 7 h, RXR? was found in the nucleus as a monomer, dimer or tetramer, whilst TDG was no longer in the nucleus and instead formed clusters in the cytoplasm. After 24 h, RXR? formed clusters in the cytoplasm, and TDG was no longer detectable. In conclusion, single unmodified proteins in cells can be counted and localised with 50 nm resolution by combining D-SIMS with our method of analysis.

SUBMITTER: Delaune A 

PROVIDER: S-EPMC3576336 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature