Enhanced Competition at the Nano-Bio Interface Enables Comprehensive Characterization of Protein Corona Dynamics and Deep Coverage of Proteomes.

Introducing engineered nanoparticles (NPs) into a biofluid such as blood plasma leads to the formation of a selective and reproducible protein corona at the particle-protein interface, driven by the relationship between protein-NP affinity and protein abundance. This enables scalable systems that leverage protein-nano interactions to overcome current limitations of deep plasma proteomics in large cohorts. Here the importance of the protein to NP-surface ratio (P/NP) is demonstrated and protein corona formation dynamics are modeled, which determine the competition between proteins for binding. Tuning the P/NP ratio significantly modulates the protein corona composition, enhancing depth and precision of a fully automated NP-based deep proteomic workflow (Proteograph). By increasing the binding competition on engineered NPs, 1.2-1.7× more proteins with 1% false discovery rate are identified on the surface of each NP, and up to 3× more proteins compared to a standard plasma proteomics workflow. Moreover, the data suggest P/NP plays a significant role in determining the in vivo fate of nanomaterials in biomedical applications. Together, the study showcases the importance of P/NP as a key design element for biomaterials and nanomedicine in vivo and as a powerful tuning strategy for accurate, large-scale NP-based deep proteomic studies.

Multiomic profiling of the liver across diets and age in a diverse mouse population.

We profiled the liver transcriptome, proteome, and metabolome in 347 individuals from 58 isogenic strains of the BXD mouse population across age (7 to 24 months) and diet (low or high fat) to link molecular variations to metabolic traits. Several hundred genes are affected by diet and/or age at the transcript and protein levels. Orthologs of two aging-associated genes, St7 and Ctsd, were knocked down in C. elegans, reducing longevity in wild-type and mutant long-lived strains. The multiomics data were analyzed as segregating gene networks according to each independent variable, providing causal insight into dietary and aging effects. Candidates were cross-examined in an independent diversity outbred mouse liver dataset segregating for similar diets, with ∼80%-90% of diet-related candidate genes found in common across datasets. Together, we have developed a large multiomics resource for multivariate analysis of complex traits and demonstrate a methodology for moving from observational associations to causal connections.

Quantifying and Localizing the Mitochondrial Proteome Across Five Tissues in A Mouse Population.

We have used SWATH mass spectrometry to quantify 3648 proteins across 76 proteomes collected from genetically diverse BXD mouse strains in two fractions (mitochondria and total cell) from five tissues: liver, quadriceps, heart, brain, and brown adipose (BAT). Across tissues, expression covariation between genes' proteins and transcripts-measured in the same individuals-broadly aligned. Covariation was however far stronger in certain subsets than others: only 8% of transcripts in the lowest expression and variance quintile covaried with their protein, in contrast to 65% of transcripts in the highest quintiles. Key functional differences among the 3648 genes were also observed across tissues, with electron transport chain (ETC) genes particularly investigated. ETC complex proteins covary and form strong gene networks according to tissue, but their equivalent transcripts do not. Certain physiological consequences, such as the depletion of ATP synthase in BAT, are thus obscured in transcript data. Lastly, we compared the quantitative proteomic measurements between the total cell and mitochondrial fractions for the five tissues. The resulting enrichment score highlighted several hundred proteins which were strongly enriched in mitochondria, which included several dozen proteins were not reported in literature to be mitochondrially localized. Four of these candidates were selected for biochemical validation, where we found MTAP, SOAT2, and IMPDH2 to be localized inside the mitochondria, whereas ABCC6 was in the mitochondria-associated membrane. These findings demonstrate the synergies of a multi-omics approach to study complex metabolic processes, and this provides a resource for further discovery and analysis of proteoforms, modified proteins, and protein localization.

Correction to "Assessing Physicochemical Properties of Drug Molecules via Microsolvation Measurements with Differential Mobility Spectrometry".

[This corrects the article DOI: 10.1021/acscentsci.6b00297.].

Assessing Physicochemical Properties of Drug Molecules via Microsolvation Measurements with Differential Mobility Spectrometry.

The microsolvated state of a molecule, represented by its interactions with only a small number of solvent molecules, can play a key role in determining the observable bulk properties of the molecule. This is especially true in cases where strong local hydrogen bonding exists between the molecule and the solvent. One method that can probe the microsolvated states of charged molecules is differential mobility spectrometry (DMS), which rapidly interrogates an ion's transitions between a solvated and desolvated state in the gas phase (i.e., few solvent molecules present). However, can the results of DMS analyses of a class of molecules reveal information about the bulk physicochemical properties of those species? Our findings presented here show that DMS behaviors correlate strongly with the measured solution phase pKa and pKb values, and cell permeabilities of a set of structurally related drug molecules, even yielding high-resolution discrimination between isomeric forms of these drugs. This is due to DMS's ability to separate species based upon only subtle (yet predictable) changes in structure: the same subtle changes that can influence isomers' different bulk properties. Using 2-methylquinolin-8-ol as the core structure, we demonstrate how DMS shows promise for rapidly and sensitively probing the physicochemical properties of molecules, with particular attention paid to drug candidates at the early stage of drug development. This study serves as a foundation upon which future drug molecules of different structural classes could be examined.

Targeted Interleukin-10 Nanotherapeutics Developed with a Microfluidic Chip Enhance Resolution of Inflammation in Advanced Atherosclerosis.

Inflammation is an essential protective biological response involving a coordinated cascade of signals between cytokines and immune signaling molecules that facilitate return to tissue homeostasis after acute injury or infection. However, inflammation is not effectively resolved in chronic inflammatory diseases such as atherosclerosis and can lead to tissue damage and exacerbation of the underlying condition. Therapeutics that dampen inflammation and enhance resolution are currently of considerable interest, in particular those that temper inflammation with minimal host collateral damage. Here we present the development and efficacy investigations of controlled-release polymeric nanoparticles incorporating the anti-inflammatory cytokine interleukin 10 (IL-10) for targeted delivery to atherosclerotic plaques. Nanoparticles were nanoengineered via self-assembly of biodegradable polyester polymers by nanoprecipitation using a rapid micromixer chip capable of producing nanoparticles with retained IL-10 bioactivity post-exposure to organic solvent. A systematic combinatorial approach was taken to screen nanoparticles, resulting in an optimal bioactive formulation from in vitro and ex vivo studies. The most potent nanoparticle termed Col-IV IL-10 NP22 significantly tempered acute inflammation in a self-limited peritonitis model and was shown to be more potent than native IL-10. Furthermore, the Col-IV IL-10 nanoparticles prevented vulnerable plaque formation by increasing fibrous cap thickness and decreasing necrotic cores in advanced lesions of high fat-fed LDLr(-/-) mice. These results demonstrate the efficacy and pro-resolving potential of this engineered nanoparticle for controlled delivery of the potent IL-10 cytokine for the treatment of atherosclerosis.

The structures and properties of proton- and alkali-bound cysteine dimers.

The proton-, lithium-, and sodium-bound cysteine dimers have been investigated in a joint computational and experimental infrared multiple photon dissociation (IRMPD) study. IRMPD spectra in the 1000-2000 cm(-1) region show that protonation is localized on an amine group, and that intermolecular hydrogen bonding occurs between the protonated amine and the carbonyl oxygen of the neutral Cys moiety. Alkali-bound dimers adopt structures reminiscent of those observed for the monomeric Cys·Li(+) and Cys·Na(+) species. Calculations of the heavier Cys2·M(+) (M = K, Rb or Cs) species suggest that these are significantly less strongly bound than the lighter (M = H, Li, or Na) dimers.

Assessing the impact of anion-π effects on phenylalanine ion structures using IRMPD spectroscopy.

The gas-phase structures of two halide-bound phenylalanine anions (PheX(-), X = Cl(-) or Br(-)) and five fluorinated derivatives have been identified using infrared multiple photon dissociation (IRMPD) spectroscopy. The addition of electron-withdrawing groups to the aromatic ring creates a π-acidic system that additionally stabilizes the halide above the ring face. Detailed ion structures were determined by comparing the IRMPD spectra with harmonic and anharmonic infrared spectra computed using B3LYP/6-311++G(d,p) as well as with 298 K enthalpies and Gibbs energies determined by the MP2(full)/6-311++G(2d,2p)//B3LYP/6-311++G(d,p) and MP2(full)/aug-cc-pVTZ//B3LYP/6-311++G(d,p) methods. PheX(-) structures were found to be dependent on both the nature of the anion and the extent of ring fluorination. Canonical isomers were established to be the dominant structures in every case, but halide addition significantly narrowed the energy gap with zwitterionic potential energy surfaces. This enabled zwitterions to appear as minor contributors to the gas-phase populations of Phe35F2Cl(-) and PheF5Br(-).

Persistent Intramolecular C-H···X (X = O or S) Hydrogen-Bonding in Benzyl Meldrum's Acid Derivatives.

C-H···X (where X = O or S) intramolecular hydrogen bonding is investigated in three benzyl Meldrum's acid derivatives using a combination of solution phase NMR spectroscopy, gas phase infrared multiple photon dissociation spectroscopy, and density functional theory calculations. In one compound, an abnormally large C-H···S hydrogen bond energy of 30.4 kJ mol-1 is calculated with a natural bond orbital analysis. Intramolecular C-H···O hydrogen bonding is found to persist in the gas phase. Gibbs energy decomposition pathways are calculated.