Combinatorial optimization and spatial remodeling of CYPs to control product profile.

Activating inert substrates is a challenge in nature and synthetic chemistry, but essential for creating functionally active molecules. In this work, we used a combinatorial optimization approach to assemble cytochrome P450 monooxygenases (CYPs) and reductases (CPRs) to achieve a target product profile. By creating 110 CYP-CPR pairs and iteratively screening different pairing libraries, we demonstrated a framework for establishing a CYP network that catalyzes six oxidation reactions at three different positions of a chemical scaffold. Target product titer was improved by remodeling endoplasmic reticulum (ER) size and spatially controlling the CYPs' configuration on the ER. Out of 47 potential products that could be synthesized, 86% of the products synthesized by the optimized network was our target compound quillaic acid (QA), the aglycone backbone of many pharmaceutically important saponins, and fermentation achieved QA titer 2.23 g/L.

Polarity-Specific Profiling of Metabolites in Single Cells by Probe Electrophoresis Mass Spectrometry.

Sensitive analysis of metabolites in a single cell is of fundamental significance for the better understanding of biological variability, differential susceptibility in disease therapy, and cell-to-cell heterogeneity as well. Herein, polarity-specific profiling of metabolites in a single cell was implemented by probe electrophoresis mass spectrometry (PEMS), which combined electrophoresis sampling of metabolites from a single cell and nanoelectrospray ionization-mass spectrometry (nanoESI-MS) analysis of the sampled metabolites. Enhanced extraction of either negatively or positively charged metabolites from a single cell was achieved by applying a DC voltage offset of +2.0 and -2.0 V to the probe, respectively. The experimental data demonstrated that PEMS features high throughput (≥200 peaks) and high sensitivity (≥10-times signal enhancement for [Choline + H]+, [Glutamine + H]+, [Arginine + H]+, etc.) in comparison with direct nanoESI-MS analysis. The biological effects of CdSe quantum dots (QDs) and γ-radiation on Allium cepa cells were investigated by PEMS, which revealed that CdSe QDs lead to the increase of intracellular amines while γ-radiation causes the decrease of intracellular acids. Therefore, this work provides an alternative platform to probe novel insights of cells by sensitive analysis of polarity-specific metabolites in a single cell.

Chemical Profiling of Bulk Alloys Using Micro-Electrochemical Probe Mass Spectrometry.

Micro-electrochemical probe mass spectrometry (µECP-MS) is demonstrated as a method for the direct profiling of chemical composition of bulk alloy samples without tedious sample pretreatment. The spatial distribution of Zn and Cu components of a Cu/Zn alloy sample was successively identified by scanning the electrolysis potential from -0.6 V to 0.6 V. The lateral resolution of alloy chemical profiling was ≤10 µm, and the depth resolution was ≤0.5 nm. Besides metal components, the method also allows the simultaneous detection of organic molecules on the sample surface. The limit of detection for Rhodamine B, Zn, and Cu depositions was 4.47, 9.58, and 24.25 ag per µm2, respectively. The method is particularly useful for high-throughput (<2 min per single run) quality monitoring of industrial parts and conductive materials of irregular geometries, such as alloy, microchips, solder side, etc.

Rapid detection of metal impurities on the surfaces of intact objects with irregular shapes using electrochemical mass spectrometry.

New approaches are demanded in daily life and industry for the rapid inspection of chemical impurities on various objects, particularly those with irregular shapes. Herein, an analytical strategy combining electrochemistry (EC) and mass spectrometry (MS) has been developed for the direct inspection of metal impurities on various objects which are commonly used in daily life and industry. An intact object (e.g., necklace, bearing, ring) was immersed in an electrolytic cell containing EDTA/acetonitrile/water solution at appropriate potentials to form metal ions. The formed metal ions were instantly chelated with specific ligands (e.g. ethylenediaminetetraacetic acid) and sampled for online electrospray ionization (ESI) with high-resolution MS detection. The unique feature of the method is that metal speciation information can be obtained even when just a metal impurity (e.g., Pb, Ni) is localized on a hard-to-reach tiny spot on the inner surface of objects with extremely irregular shapes. A single sample analysis requires less than 10 minutes, regardless of the object shape. The limit of detection is 0.05 ppb with sample consumption on the nanogram level. The experimental results demonstrate that the method is promising for the non-destructive quality and safety inspection of metal impurities on virtually any kinds of objects with high chemical sensitivity.

Development of sequential online extraction electrospray ionization mass spectrometry for accurate authentication of highly-similar Atractylodis Macrocephalae.

The accurate and rapid authentication techniques and strategies for highly-similar foods are still lacking. Herein, a novel sequential online extraction electrospray ionization mass spectrometry (S-oEESI-MS) was developed to achieve spatio-temporally resolved ionization and comprehensive characterization of complex foods with multi-components (high, medium, and low polarity substances). Meanwhile, a characteristic marker screening method and an integrated research strategy based on MS fingerprinting, characteristic marker and chemometrics modeling were established, which are especially suitable for the accurate and rapid authentication of highly-similar foods that are difficult to be authenticated by traditional techniques (e.g., LC-MS). Thirty-two batches of highly-similar Atractylodis macrocephalae rhizome from four different origins were used as model samples. As a result, S-oEESI-MS enabled a more comprehensive MS characterization of substance profiles in complex plant samples in 1.0 min. Further, 22 characteristic markers of Atractylodis macrocephalae were ingeniously screened out and combined with multivariate statistical analysis model, the accurate authentication of highly-similar Atractylodis macrocephalae was realized. This study presents a comprehensive strategy for accurate authentication and origin analysis of highly-similar foods, which has potentially significant applications for ensuring food quality and safety.

Enrichment of Surface-Active Compounds in Bursting Bubble Aerosols.

The pronounced enrichment of surface-active compounds in the aerosols produced by bubble bursting plays a central role in the chemical transfer from the ocean into the atmosphere and has an important impact on the global Earth's climate. However, the mechanism of chemical enrichment in bursting bubble aerosols remains poorly understood and controversial due to the high complexity and diversity of experimental behaviors. Contrary to the common belief, here we show that the major share of surfactants in the jet droplets produced by individually bursting bubbles at a calm solution surface is released directly from the bubble surface rather than from the solution surface or subsurface microlayer. We reveal that surfactants are accumulated at the surface of a rising bubble in solution following three successive stages with strongly distinct adsorption profiles: linear kinetic, mixed kinetic, and equilibrium. The magnitude of surfactant enrichment in the aerosol is directly determined by which adsorption mode is in control by the moment of the bubble bursting at solution surface. Our mechanistic description explains the diversity of experimental observations regarding the surfactant enrichment in aerosol droplets and lays the ground for understanding the more complex behaviors associated with collective effects at the solution surface (e.g., breaking waves).

Deciphering the chemical origin of the semen-like floral scents in three angiosperm plants.

The chemical origin and biological role of distinct semen-like odor occasionally found in some flowers are very curious but remain scarcely studied. Here, we used direct ambient corona discharge ionization mass spectrometry (MS) to study the volatile chemical composition behind the semen-like odor emitted by the fresh flowers of Photinia serrulata, Castanopsis sclerophylla and Stemona japonica without any chemical pretreatment. Chemical identification was performed using high-resolution MS analysis in combination with tandem MS analysis and whenever possible was confirmed by the analysis of standard reference compounds. A total of 19 compounds, mostly belonging to nitrogenous volatiles, were identified in P. serrulata, C. sclerophylla, and S. japonica flowers, 1-pyrroline, 1-piperideine, 2-pyrrolidone, and phenethylamine being common in all the three studied species. Several lines of evidence indicate that the major component responsible for the semen-like odor is most likely 1-pyrroline. 1-Pyrroline is most probably formed via the oxidative deamination of putrescine, as indicated by the observation of signal from 4-amino-butanal intermediate. Flower visitation observations suggest that the released volatiles serve to attract dipterans, including Syrphidae, Calliphoridae, and Muscidae. On the analytical side, the comparison of our results to earlier studies also indicate that compared to the traditional GC-MS approach the direct corona discharge ionization mass spectrometry provides more sensitive detection of VOCs with high proton affinity, in particular volatile amines, and therefore can be used to complement traditional GC-MS approach for the highest chemical coverage of VOC analysis.

On the chemistry of 1-pyrroline in solution and in the gas phase.

1-Pyrroline has a highly characteristic odor, which is employed by living organisms for chemical signaling and other purposes, but the mechanism whereby this odor is formed remains poorly understood. Here we used a combination of ambient mass spectrometry (AMS) and nuclear magnetic resonance (NMR) spectroscopy to experimentally address the mechanistic aspects of 1-pyrroline volatility and other controversies regarding the chemistry of this compound. Our results indicate that in solution the volatility of the monomer species is significantly higher than that of the trimer species, and 1-pyrroline is evaporated mainly in its monomer state. Neat 1-pyrroline is essentially the pure trimer and displays ca. 100-fold lower evaporation rate than the monomer state in solution. In the gas-phase the trimer species is irreversibly decomposed into monomer species. Under equilibrium conditions the vapor of 1-pyrroline entirely consists of monomer species. The evaporation rate of 1-pyrroline in water has a step-wise dependence on the solution pH, the abrupt increase in volatility (>1,000-fold) occurring around the pKa value of 1-pyrroline (6.8). The pronounced step-wise dependence of 1-pyrroline volatility around neutral pH may also be an important evolutionary factor allowing living systems to regulate the odor strength from very weak to very strong with minimal efforts.