Live‐Cell Quantitative Imaging of Proteome Degradation by Stimulated Raman Scattering

Abstract

Protein degradation is a regulatory process essential to cell viability, and its dysfunction is implicated in many diseases such as aging and neurodegeneration. Here we report stimulated Raman scattering microscopy coupled with metabolic labeling of 13C-phenylalanine to visualize protein degradation in living cells with subcellular resolution. We choose the ring breathing modes of endogenous 12C-phenylalanine and incorporated 13C-phenylalanine as protein markers for the original and nascent proteome, respectively, and quantified the decay of the former through ratio maps. We demonstrated time dependent imaging of proteomic degradation in mammalian cells under steady-state condition and various perturbations including oxidative stress, cell differentiation, and huntingtin protein aggregation.

Applications of vibrational tags in biological imaging by Raman microscopy

“…This is achieved by imaging the signal decay of the ‘old’ proteome. Shen et al measured the decay of the endogenous phenylalanine peak after 13 C-phenylalanine labeling to map protein turnover under both steady state and stressed conditions 30 (Figure 2H–J). In this way, heterogeneous population was observed for perturbed protein turnover in a model for neurogenerative disease.…”

“…Scale bar: 10 μm. (H) SRS ratiometric imaging of 12 C- and 13 C-phenylalanine can be used to quantify protein turnover in HeLa cells 30 . Scale bar: 20 μm.…”

Coherent Raman Scattering Microscopy in Biology and Medicine

“…Deuterated compounds have also helped visualize important metabolites in cells, such as protein and choline (112, 116, 117). Shen et al (115) studied protein degradation in cells with carbon-isotope-labeled SRS imaging. In their work, endogenous and metabolically incorporated phenylalanine were marked by, respectively, carbon-12 ( 12 C) and carbon-13 ( 13 C).…”

“…Abbreviations: Daf-2 , L2: daf-2 mutant C. elegans at L2 stage; LROs: lysosome-related organelles; SRS: stimulated Raman scattering; WT N2, L2: wild-type N2 C. elegans at the L2 stage. Panels a , b , c , and d are adapted with permission from References 78, 115, 120, and 121, respectively.…”

Beyond the borders — Biomedical applications of non-linear Raman microscopy

“…These are much smaller if, e.g., 13 C is exchanged against 12 C. Nevertheless, the viability of 13 C labeling for SRS microscopic imaging was demonstrated. In one experiment, proteome degradation was monitored by using 13 C marked phenylalanine and subsequent ratiometric imaging [100]. A second experiment finally combined alkyne labeling with 13 C isotopic exchange.…”