title: 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 α (RXRα) proteins delivered to COS-7 cells. We show that both a single TDG and a single RXRα can be detected. After 4 h incubation, both proteins were found mainly in the nucleus; RXRα as a monomer or dimer and TDG only as a monomer. After 7 h, RXRα 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, RXRα 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.
authors: Laboratoire MERCI EA3829, équipe AMMIS, Faculté des Sciences de l'Université de Rouen, Mont Saint Aignan, France. anthony.delaune@univ-rouen.fr
authors: Anthony
authors: A
authors: Delaune
authors: Armelle
authors: A
authors: Cabin-Flaman
authors: Guillaume
authors: G
authors: Legent
authors: David
authors: D
authors: Gibouin
authors: Caroline
authors: C
authors: Smet-Nocca
authors: Fabrice
authors: F
authors: Lefebvre
authors: Arndt
authors: A
authors: Benecke
authors: Marc
authors: M
authors: Vasse
authors: Camille
authors: C
authors: Ripoll
mesh_terms: D000465:Algorithms; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D003627:Data Interpretation, Statistical; D007091:Image Processing, Computer-Assisted; D009587:Nitrogen Isotopes / Q000737:chemistry; D021381:Protein Transport; D047490:Retinoid X Receptor alpha / Q000737:chemistry / Q000378:metabolism; D059010:Single-Cell Analysis; D044502:Thymine DNA Glycosylase / Q000737:chemistry / Q000378:metabolism
chemical_list: D009587:Nitrogen Isotopes; D047490:Retinoid X Receptor alpha; D044502:Thymine DNA Glycosylase