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 14N isotopes are replaced with 15N. 15N-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 15N-enriched proteins - and even a single protein - within a cell using D-SIMS. The algorithm distinguishes statistically between a low local increase in 15N 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 15N-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.

Effect of zein additive on perfume evaporation

“…In all cases, 1 H and 2 H SIMS images were analysed with Significant Isotopic Enrichment Detection (SIED) laboratory‐made software to quantify isotopic signal. SIED performs the statistical analysis of the isotopic fraction from SIMS isotopic images.…”

Mass Spectrometry Imaging of Biomolecular Information

“…Transmission geometry, resembling the classical LAMMA optical setup of the 1970s 213 , has been used for single-cell analysis with MALDI MSI to increase lateral resolution, but has been demonstrated only with non-accurate mass measurement 214 . Highest lateral resolution in the range of 50 nm has been demonstrated using dynamic SIMS of small fragment ions from isotopically labelled proteins in COS-7 cells 21,215 . The specificity of identifying the proteins was achieved using a targeted approach based on isotopic 15 N labeling of the protein of interest.…”

Single-cell imaging mass spectrometry

“…[29**] In another noteworthy application, tracking the journey of isotopically enriched lipids (free fatty acids in adipocytes), proteins (thymine DNA glycosylase (TDG) and retinoid × receptor α (RXRα) in COS-7 cells) and peptide vectors (STxB in HeLa cells) has provided insight into cellular metabolism, transport, and catabolism (Figure 2C and 2F). [30-31,32**] The sensitivity of the system allowed Delaune et al to track individual proteins within a cell. [32**]…”

“…[30-31,32**] The sensitivity of the system allowed Delaune et al to track individual proteins within a cell. [32**]…”

“…[44] C: One enriched pixel shown in red (arrow) on an MS image of 12 C 14 N indicates the position of 15 N-enriched TDG/Provectin in a single COS-7 cell after 4 h incubation; scale bar is 5 μm. [32] High resolution dynamic SIMS images of 15 N-enriched Shiga toxin B-subunit ( 15 N-STxB) protein vector in a single HeLa cell after incubation (Red: 12 C 14 N, green: 12 C 15 N, 100 nm working beam diameter). [31] D: Top, MALDI MS image of intact lipid (m/z 782) from HEK-293 cells (spatial resolution ∼2 μm) and corresponding optical image.…”