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.

Development of a miniature time-of-flight mass spectrometer coupled with an improved substrate-enhanced laser-induced acoustic desorption source (SE-LIAD/TOF-MS).

A novel, compact and sensitive SE-LIAD/TOF-MS has been described. It facilitates fast sample preparation, and a full mass spectrum is acquired efficiently and sensitively. More importantly, it features the detection of non-acidic and non-basic or non-polar species, which is not suitable for determination by ESI and MALDI techniques. In this technique, standard samples, carbazole and melamine, are prepared on a Ti foil with a quartz plate attached to the backside of the Ti foil to perform a laser-induced acoustic desorption experiment (SE-LIAD) coupled to TOF-MS for analysis. Enhanced signals are observed with about 5.6 to 13.8 times higher than that obtained in the standard LIAD method, dependent on different ionization techniques. Compared to the EI spectra, the PI spectra for both species show intact and sharp molecular peaks. The limits of detection (LOD) of melamine were evaluated experimentally in the range from ∼2-6 pg (EI/MS mode) to ∼0.3-0.5 ng (VUV-SPI/MS mode). Thus, the method in this study exhibits rapid qualitative and quantitative analysis with good sensitivity, being free of the complex matrix influences.

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.

Targeted cross-linker delivery for the in situ mapping of protein conformations and interactions in mitochondria.

Current methods for intracellular protein analysis mostly require the separation of specific organelles or changes to the intracellular environment. However, the functions of proteins are determined by their native microenvironment as they usually form complexes with ions, nucleic acids, and other proteins. Here, we show a method for in situ cross-linking and analysis of mitochondrial proteins in living cells. By using the poly(lactic-co-glycolic acid) (PLGA) nanoparticles functionalized with dimethyldioctadecylammonium bromide (DDAB) to deliver protein cross-linkers into mitochondria, we subsequently analyze the cross-linked proteins using mass spectrometry. With this method, we identify a total of 74 pairs of protein-protein interactions that do not exist in the STRING database. Interestingly, our data on mitochondrial respiratory chain proteins ( ~ 94%) are also consistent with the experimental or predicted structural analysis of these proteins. Thus, we provide a promising technology platform for in situ defining protein analysis in cellular organelles under their native microenvironment.

The photoelectron-imaging spectroscopic study and chemical bonding analysis of VO2 -, NbO2 - and TaO2.

The transition-metal di-oxides, namely VO2 -, NbO2 - and TaO2 - have been studied using photoelectron velocity map imaging (PE-VMI) in combination with theoretical calculations. The adiabatic electron affinities of VO2 -, NbO2 - and TaO2 - are confirmed to be 2.029(8), 1.901(10) and 2.415(8) eV, respectively. By combining Franck-Condon (FC) simulation with theoretical calculations, the vibrational feature related to Nb-O and Ta-O stretching modes for the ground state has been unveiled. The photoelectron angular distribution (PAD) for VO2 -, NbO2 - and TaO2 - is correlated to the photo-detachment of the highest occupied molecular orbitals (HOMOs), which primarily gets involved in s- and d-orbitals of the V, Nb and Ta atoms. A variety of theoretical calculations have been used to analyze the chemical bonding features of VO2 -1/0, NbO2 -1/0 and TaO2 -1/0, which show that the strong M-O (M = V, Nb and Ta) bond is mainly characterized as ionicity.

Epitope Imprinting Technology: Progress, Applications, and Perspectives toward Artificial Antibodies.

Epitope imprinting is a promising tool to generate antibody-like specific recognition sites. Recently, because of the ease of obtaining templates, the flexibility in selecting monomers, their resistance to harsh environments, and the high specificity toward targets, epitope-imprinted materials have attracted much attention in various fields, such as bioanalysis, clinical therapy, and pharmacy. Here, the discussion is focused on the current representative epitope imprinting technologies, including epitope bulk imprinting and epitope surface imprinting. Moreover, the application of epitope-imprinted materials to the recognition of peptides, proteins, and cells is reviewed. Finally, the remaining challenges arising from the intrinsic properties of epitope imprinting are discussed, and future development in the field is prospected.

Structural Dynamics of Dimethyl Sulfoxide Aqueous Solutions Investigated by Ultrafast Infrared Spectroscopy: Using Thiocyanate Anion as a Local Vibrational Probe.

The microscopic structure of dimethyl sulfoxide (DMSO) aqueous solutions was investigated by Fourier transform infrared (FTIR) spectroscopy and ultrafast IR spectroscopy. The structural dynamics of the binary mixtures were reflected by using thiocyanate anion (SCN-) as a local vibrational probe. FTIR spectra of SCN- anion showed that the hydrogen bond networks of water are affected by the presence of DMSO molecules, and the peak position and bandwidth of SCN- anions are red shifted and narrowed accordingly because of the weak hydration in the binary mixture. The vibrational lifetime of the SCN- anion showed almost linear enhancement with the increase of DMSO, which can be explained by the weak interaction between SCN- and the hydrophobic groups in the DMSO molecule. However, the rotational dynamics of SCN- are slowing down significantly and showed a maximum response at XDMSO (mole fraction) of 0.35, which is mainly caused by the confinement of SCN- anions positioned in the vicinity of the complex structure formed between DMSO and water molecules. The concentration-dependent rotational dynamics of water molecules and SCN- anions are having similar behavior, indicating that the complex structure can be formed between water and DMSO molecules because of the strong interaction. The result also demonstrates that the structural inhomogeneity in aqueous solution can be unraveled by monitoring the vibrational relaxation dynamics of SCN- anion serving as the local vibrational probe.