Crosslinker Nanocarriers Delivery to Chloroplasts for In Vivo Mapping of Photosynthetic Membrane Protein Complexes in Living Chlamydomonas reinhardtii Cells.

Photosynthesis, as the core of solar energy biotransformation, is driven by photosynthetic membrane protein complexes in plants and algae. Current methods for intracellular photosynthetic membrane protein complex analysis mostly require the separation of specific chloroplasts or the change of the intracellular environment, which causes the missing of real-time and on-site information. Thus, we explored a method for in vivo crosslinking and mapping of photosynthetic membrane protein complexes in the chloroplasts of living Chlamydomonas reinhardtii (C. reinhardtii) cells under cultural conditions. Poly(lactic-co-glycolic acid) (PLGA) and poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles were fabricated to deliver bis(succinimidyl)propargyl with a nitro compound (BSPNO) into the chloroplasts to crosslink photosynthetic membrane protein complexes. After the in vivo crosslinked protein complexes were extracted and digested, mass spectrometry was employed to detect lysine-specific crosslinked peptides for further elucidating the protein conformations and interactions. With this method, the weak interactions between extrinsic proteins in the luminal side (PsbL and PsbH) and the core subunits (CP47 and CP43) in photosynthetic protein complexes were directly captured in living cells. Additionally, the previously uncharacterized protein (Cre07.g335700) was bound to the light-harvesting proteins, which was related to the biosynthesis of light-harvesting antennae. These results indicated that in vivo analysis of photosynthetic protein complexes based on crosslinker nanocarriers was expected to not only figure out the difficulty in the study of photosynthetic protein complexes in living cells but also provide an approach to explore transient and weak interactions and the function of uncharacterized proteins.

Tracking the effects of PLGA-based nanoparticles on protein expression in living cells through quantitative proteomics.

Mass spectrometry (MS)-based proteomics can identify and quantify the differential abundance of expressed proteins in parallel, and bottom-up proteomic approaches are even approaching comprehensive coverage of the complex eukaryotic proteome. Protein-nanoparticle (NP) interactions have been extensively studied owing to their importance in biological applications and nanotoxicology. However, the proteome-level effects of NPs on cells have received little attention, although changes in protein abundance can reflect the direct effects of nanocarriers on protein expression. Herein, we investigated the effect of PLGA-based NPs on protein expression in HepG2 cells using a label-free quantitative proteomics approach with data independent acquisition (DIA). The percentage of two-fold change in the protein expression of cells treated with PLGA-based NPs was less than 10.15% during a 6 hour observation period. Among the changed proteins, we found that dynamic proteins involved in cell division, localization, and transport are more likely to be more susceptible to PLGA-based NPs.