RESUMEN
Secondary ion mass spectrometry (SIMS) is generally used in imaging the isotopic composition of various materials. It is becoming increasingly popular in biology, especially for investigations of cellular metabolism. However, individual proteins are difficult to identify in SIMS, which limits the ability of this technology to study individual compartments or protein complexes. We present a method for specific protein isotopic and fluorescence labeling (SPILL), based on a novel click reaction with isotopic probes. Using this method, we added (19) F-enriched labels to different proteins, and visualized them by NanoSIMS and fluorescence microscopy. The (19) F signal allowed the precise visualization of the protein of interest, with minimal background, and enabled correlative studies of protein distribution and cellular metabolism or composition. SPILL can be applied to biological systems suitable for click chemistry, which include most cell-culture systems, as well as small model organisms.
Asunto(s)
Nanotecnología , Proteínas/genética , Espectrometría de Masa de Ion Secundario , Animales , Línea Celular , Química Clic , Cricetinae , Colorantes Fluorescentes/química , Radioisótopos de Flúor , Microscopía Fluorescente , Estructura Molecular , Proteínas/química , Proteínas/metabolismoRESUMEN
The advent of super-resolution microscopy (nanoscopy) has set high standards for fluorescence tagging. Fluorescent proteins (FPs) are convenient tags in conventional imaging, but their use in nanoscopy has been questioned due to their relatively large size and propensity to form multimers. Here, we compared the nanoscale organization of proteins with or without FP tags by introducing the unnatural amino acid propargyl-L-lysine (PRK) in 26 proteins known to form multimolecular arrangements and into their FP-tagged variants. We revealed the proteins by coupling synthetic fluorophores to PRK via click chemistry and visualized them using ground-state depletion microscopy followed by individual molecule return, as well as stimulated emission depletion microscopy. The arrangements formed by the FP-tagged and nontagged proteins were similar. Mild, but statistically significant differences were observed for only six proteins (23% of all proteins tested). This suggests that FP-based nanoscopy is generally reliable. Unnatural amino acids should be a reliable alternative for the few proteins that are sensitive to FP tagging.
Asunto(s)
Aminoácidos/química , Química Clic/métodos , Colorantes Fluorescentes/química , Proteínas Luminiscentes/química , Microscopía Fluorescente/métodos , TransfecciónRESUMEN
Imaging techniques should differentiate between specific signals, from the biomolecules of interest, and non-specific signals, from the background. We present a probe containing (15)N and (14)N isotopes in approximately equal proportion, for secondary ion mass spectrometry imaging. This probe designed for a precise biomolecule analysis is insensitive to background signals.