Isotopic Labeling Experiments Research Articles

A chemo-selective enrichment strategy to achieve in-depth coverage of methyllysine proteome.

Proteomics is a powerful method to comprehensively understand cellular posttranslational modifications (PTMs). Due to low abundance, tryptic peptides with PTMs are usually enriched for enhanced coverage by LC-MS/MS. Affinity chromatography for phosphoproteomes by metal-oxide and pan-specific antibodies for lysine acetylome allow identification of tens of thousands of modification sites. Lysine methylation is a significant PTM, however, only hundreds of methylation sites were identified from available approaches. Here we report an aryl diazonium-based chemoselective strategy that enables enrichment of monomethyllysine (Kme1) peptides via covalent bond with extraordinary sensitivity. We identified more than ten thousand Kme1 peptides from diverse cell lines and mouse tissues, that implied wide lysine methylation impact on cellular processes. In addition, we found a significant amount of methyl marks that were not S-adenosyl methionine (SAM)-dependent by isotope labeling experiments. And therefore, this method paves a way to broad application in lysine methylation research and new biology discovery.

Demethylation C–C coupling reaction facilitated by the repulsive Coulomb force between two cations

Carbon chain elongation (CCE) is normally carried out using either chemical catalysts or bioenzymes. Herein we demonstrate a catalyst-free approach to promote demethylation C–C coupling reactions for advanced CCE constructed with functional groups under ambient conditions. Accelerated by the electric field, two organic cations containing a methyl group (e.g., ketones, acids, and aldehydes) approach each other with such proximity that the energy of the repulsive Coulomb interaction between these two cations exceeds the bond energy of the methyl group. This results in the elimination of a methyl cation and the coupling of the residual carbonyl carbon groups. As confirmed by high-resolution mass spectrometry and isotope-labeling experiments, the C–C coupling reactions (yields up to 76.5%) were commonly observed in the gas phase or liquid phase, for which the mechanism was further studied using molecular dynamics simulations and stationary-point calculations, revealing deep insights and perspectives of chemistry.

Atomic-Level Asymmetric Tuning of the Co1-N3P1 Catalyst for Highly Efficient N-Alkylation of Amines with Alcohols.

Despite the extensive development of non-noble metals for the N-alkylation of amines with alcohols, the exploitation of catalysts with high selectivity, activity, and stability still faces challenges. The controllable modification of single-atom sites through asymmetric coordination with a second heteroatom offers new opportunities for enhancing the intrinsic activity of transition metal single-atom catalysts. Here, we prepared the asymmetric N/P hybrid coordination of single-atom Co1-N3P1 by absorbing the Co-P complex on ZIF-8 using a concise impregnation-pyrolysis process. The catalyst exhibits ultrahigh activity and selectivity in the N-alkylation of aniline and benzyl alcohol, achieving a turnover number (TON) value of 3480 and a turnover frequency (TOF) value of 174-h. The TON value is 1 order of magnitude higher than the reported catalysts and even 37-fold higher than that of the homogeneous catalyst CoCl2(PPh3)2. Furthermore, the catalyst maintains its high activity and selectivity even after 6 cycles of usage. Controlling experiments and isotope labeling experiments confirm that in the asymmetric Co1-N3P1 system, the N-alkylation of aniline with benzyl alcohol proceeds via a transfer hydrogenation mechanism involving the monohydride route. Theoretical calculations prove that the superior activity of asymmetric Co1-N3P1 is attributed to the higher d-band energy level of Co sites, which leads to a more stable four-membered ring transition state and a lower reaction energy barrier compared to symmetrical Co1-N4.

Spectroscopic Characterization and Photochemistry of the Atmospherically Relevant Methanesulfenic Acid.

Methanesulfenic acid, CH3SOH, is a fleeting intermediate in the ·OH-initiated oxidation reactions of dimethyl sulfide (DMS) and dimethyl disulfide (DMDS) in the atmosphere. Herein, we report the characterization and photochemistry of CH3SOH in Ar- and N2-matrices at 10 K. The characterization of CH3SOH with matrix-isolation IR and UV-vis spectroscopy is supported by D and 13C isotope labeling experiments and quantum chemical calculations. In line with the observed absorption at 260 nm for CH3SOH, its photolysis at 254 nm leads to dissociation by yielding the novel water complex H2CS···H2O, which exhibits a five-membered ring structure with intermolecular S···HO and CH···O hydrogen bonding interactions. Upon further irradiation at 193 nm, the H2CS···H2O complex undergoes dehydrogenation to form CS···H2O, which can further convert to HC(O)SH under irradiation at 254 nm. When the photolysis of CH3SOH was performed in an O2-doped Ar-matrix, methanesulfonic acid (MSA, CH3SO3H) was obtained as the oxidation product.

Spectroscopic Characterization and Photochemistry of the Atmospherically Relevant Methanesulfenic Acid.

Methanesulfenic acid, CH3SOH, is a fleeting intermediate in the ·OH-initiated oxidation reactions of dimethyl sulfide (DMS) and dimethyl disulfide (DMDS) in the atmosphere. Herein, we report the characterization and photochemistry of CH3SOH in Ar- and N2-matrices at 10 K. The characterization of CH3SOH with matrix-isolation IR and UV-vis spectroscopy is supported by D and 13C isotope labeling experiments and quantum chemical calculations. In line with the observed absorption at 260 nm for CH3SOH, its photolysis at 254 nm leads to dissociation by yielding the novel water complex H2CS···H2O, which exhibits a five-membered ring structure with intermolecular S···HO and CH···O hydrogen bonding interactions. Upon further irradiation at 193 nm, the H2CS···H2O complex undergoes dehydrogenation to form CS···H2O, which can further convert to HC(O)SH under irradiation at 254 nm. When the photolysis of CH3SOH was performed in an O2-doped Ar-matrix, methanesulfonic acid (MSA, CH3SO3H) was obtained as the oxidation product.

Production of succinate with two CO2 fixation reactions from fatty acids in Cupriavidus necator H16

BackgroundBiotransformation of CO2 into high-value-added carbon-based products is a promising process for reducing greenhouse gas emissions. To realize the green transformation of CO2, we use fatty acids as carbon source to drive CO2 fixation to produce succinate through a portion of the 3-hydroxypropionate (3HP) cycle in Cupriavidus necator H16.ResultsThis work can achieve the production of a single succinate molecule from one acetyl-CoA molecule and two CO2 molecules. It was verified using an isotope labeling experiment utilizing NaH13CO3. This implies that 50% of the carbon atoms present in succinate are derived from CO2, resulting in a twofold increase in efficiency compared to prior methods of succinate biosynthesis that relied on the carboxylation of phosphoenolpyruvate or pyruvate. Meanwhile, using fatty acid as a carbon source has a higher theoretical yield than other feedstocks and also avoids carbon loss during acetyl-CoA and succinate production. To further optimize succinate production, different approaches including the optimization of ATP and NADPH supply, optimization of metabolic burden, and optimization of carbon sources were used. The resulting strain was capable of producing succinate to a level of 3.6 g/L, an increase of 159% from the starting strain.ConclusionsThis investigation established a new method for the production of succinate by the implementation of two CO2 fixation reactions and demonstrated the feasibility of ATP, NADPH, and metabolic burden regulation strategies in biological carbon fixation.

Carbamoylphosphinidene: A Phosphorus Analogue of Carbonyl Nitrene.

Carbonyl nitrenes are versatile intermediates that have been extensively characterized; however, their phosphorus analogues remain largely unknown. Herein, we report the observation of a rare example of carbonyl phosphinidene NH2C(O)P, which was generated through the photolytic (193 nm) dehydrogenation of phosphinecarboxamide (NH2C(O)PH2) in a solid N2-matrix at 12 K. The characterization of NH2C(O)P in the triplet ground state with matrix-isolation IR and ultraviolet-visible (UV-vis) spectroscopy is supported by comprehensive isotope labeling experiments (D and 15N) and quantum chemical calculations. Upon visible-light irradiation at 680 nm, NH2C(O)P inserts into dihydrogen by the reformation of NH2C(O)PH2 with concomitant isomerization to the more stable aminophosphaketene (NH2PCO). Additionally, the photoisomerization of NH2C(O)PH2 to NH2C(OH) = PH along with decomposition by yielding hydrogen-bonded complexes HNCO···PH3 and HPCO···NH3 has been observed in the matrix.

Protein Editing using a Concerted Transposition Reaction.

Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processes in vitro. In general, this strategy necessitates a protein folding step following ligation of the unstructured fragments, a requirement that constrains the types of systems amenable to the approach. Here, we report an in vitro strategy that allows internal regions of target proteins to be replaced in a single operation. Conceptually, our system is analogous to a DNA transposition reaction, but employs orthogonal pairs of split inteins to swap out a designated region of a host protein with an exogenous molecular cassette. We show using isotopic labeling experiments that this 'protein transposition' reaction is concerted when the kinetics for the embedded intein pairs are suitably matched. Critically, this feature allows for efficient manipulation of protein primary structure in the context of a native fold. The utility of this method is illustrated using several protein systems including the multisubunit chromatin remodeling complex, ACF, where we also show protein transposition can occur in situ within the cell nucleus. By carrying out a molecular 'cut and paste' on a protein or protein complex under native folding conditions, our approach dramatically expands the scope of protein semisynthesis.

Water Microdroplets in Air: A Hitherto Unnoticed Natural Source of Nitrogen Oxides.

Water microdroplets are widespread in the atmosphere. We report a striking observation that micron-sized water droplets obtained from zero-volt spray sources (sonic spray, humidifier, spray bottle, steamer, etc.) spontaneously generate nitrogen oxides. The mechanistic investigation through the development of custom-designed sampling sources combined with mass spectrometry and isotope labeling experiments confirmed that air nitrogen reacts with the water at the air-water interface, fixing molecular nitrogen to its oxides (NO, NO2, and N2O) and acids (HNO2 and HNO3) at trace levels without any catalyst. These reactions are attributed to the consequence of an experimentally detected feeble corona discharge (breakdown of air) at the air-water interface, likely driven by the high intrinsic electric field at the surface of water microdroplets. The extent of this corona discharge effect varies depending on the pH, salinity/impurity, size, speed, and lifetime of microdroplets in the air. Thus, this study discloses that the air-water interface of microdroplets breaks the strong chemical bond of nitrogen (N2), producing nitrogen oxides in the environment, while lightning strikes and microbial processes in soil are considered their dominant natural sources. As nitrogen oxides are toxic air pollutants, their spontaneous formation at the air-water interface should have important implications in atmospheric reactions, requiring further investigations.

Revealing the structural evolution of CuAg composites during electrochemical carbon monoxide reduction

Comprehending the catalyst structural evolution during the electrocatalytic process is crucial for establishing robust structure/performance correlations for future catalysts design. Herein, we interrogate the structural evolution of a promising Cu-Ag oxide catalyst precursor during electrochemical carbon monoxide reduction. By using extensive in situ and ex situ characterization techniques, we reveal that the homogenous oxide precursors undergo a transformation to a bimetallic composite consisting of small Ag nanoparticles enveloped by thin layers of amorphous Cu. We believe that the amorphous Cu layer with undercoordinated nature is responsible for the enhanced catalytic performance of the current catalyst composite. By tuning the Cu/Ag ratio in the oxide precursor, we find that increasing the Ag concentration greatly promotes liquid products formation while suppressing the byproduct hydrogen. CO2/CO co-feeding electrolysis and isotopic labelling experiments suggest that high CO concentrations in the feed favor the formation of multi-carbon products. Overall, we anticipate the insights obtained for Cu-Ag bimetallic systems for CO electroreduction in this study may guide future catalyst design with improved performance.

Esterification of Cyclic N6-Threonylcarbamoyladenosine During RNA Sample Preparation.

The continuous deciphering of crucial biological roles of RNA modifications and their involvement in various pathological conditions, together with their key roles in the use of RNA-based therapeutics, has reignited interest in studying the occurrence and identity of non-canonical ribonucleoside structures during the past years. Discovery and structural elucidation of new modified structures is usually achieved by combination of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) at the nucleoside level and stable isotope labeling experiments. This approach, however, has its pitfalls as demonstrated in the course of the present study: we structurally elucidated a new nucleoside structure that showed significant similarities to the family of (c)t6A modifications and was initially considered a genuine modification, but subsequently turned out to be an in vitro formed glycerol ester of t6A. This artifact is generated from ct6A during RNA hydrolysis upon addition of enzymes stored in glycerol containing buffers in a mildly alkaline milieu, and was moreover shown to undergo an intramolecular transesterification reaction. Our results demand for extra caution, not only in the discovery of new RNA modifications, but also with regard to the quantification of known modified structures, in particular chemically labile modifications, such as ct6A, that might suffer from exposure to putatively harmless reagents during the diverse steps of sample preparation.

Efficient Segmental Isotope Labeling of Integral Membrane Proteins for High-Resolution NMR Studies.

High-resolution structural NMR analyses of membrane proteins are challenging due to their large size, resulting in broad resonances and strong signal overlap. Among the isotope labeling methods that can remedy this situation, segmental isotope labeling is a suitable strategy to simplify NMR spectra and retain high-resolution structural information. However, protein ligation within integral membrane proteins is complicated since the hydrophobic protein fragments are insoluble, and the removal of ligation side-products is elaborate. Here, we show that a stabilized split-intein system can be used for rapid and high-yield protein trans-splicing of integral membrane proteins under denaturing conditions. This setup enables segmental isotope labeling experiments within folded protein domains for NMR studies. We show that high-quality NMR spectra of markedly reduced complexity can be obtained in detergent micelles and lipid nanodiscs. Of note, the nanodisc insertion step specifically selects for the ligated and correctly folded membrane protein and simultaneously removes ligation byproducts. Using this tailored workflow, we show that high-resolution NMR structure determination is strongly facilitated with just two segmentally isotope-labeled membrane protein samples. The presented method will be broadly applicable to structural and dynamical investigations of (membrane-) proteins and their complexes by solution and solid-state NMR but also other structural methods where segmental labeling is beneficial.

Photosynthetic cultivation of Chlamydomonas reinhardtii with formate as a novel carbon source to the protein production

Formate, which can be produced from CO2 with green energy, has received increasing attentions as a carbon source for bioconversion. Microalgae have the native advantages of use captured light energy to facilitate the formate assimilation. However, the low efficient natural conversion and inhibition to the electron transfer chain obstacle its applications’ development. Here, using green microalga C. reinhardtii 21gr as the model, an approach to improve formate-based biomass and protein production was raised and verified through isotope labeling experiments, and the pathway of formate which presented in photosynthetic organisms was related to amino acids. By the enhancement of chloroplast glyceraldehyde-3-phosphate dehydrogenase (cGAPDH) expression, together with the adaptive laboratory evolution (ALE) over regular lethal dose of formate, the yields of the biomass and proteins were promoted with 34 % and 55 % to the wild type, respectively. Indicated by the photosynthetic activity variations, it was proposed that the balance of cellular energy and reduction power is necessary for microalgae to achieve high productivity with formate. Further investigated by proteomics, 6-phosphogluconate dehydrogenase (6-PGD) was identified as the key enzyme to formate and nitrogen’s co-assimilation.

Visible‐Light‐Induced Oxidation of Silanes with SF6 for Efficient Synthesis of Siloxanes

A visible‐light‐induced strategy utilizing sulfur hexafluoride (SF6) as distinctive oxidant and water as the oxygen source to transform silanes into siloxanes has been developed under mild conditions, realizing the efficient utilization and degradation of the strong greenhouse gas, sulfur hexafluoride, simultaneously. Mechanistic experiments demonstrate that the reaction proceeds through a radical pathway, and it has been confirmed that the oxygen atoms in the resulting siloxanes indeed originate from water by isotopic labeling experiments with 18O.

High-Throughput screening of metal nitrides for electrochemical nitrogen reduction

Metal nitrides have garnered considerable attention in the context of nitrogen reduction reactions (NRR) due to their rich lattice nitrogen and abundant nitrogen vacancies to activate N2. However, the identification of suitable metal nitrides with superior stability, activity, and selectivity (vs. hydrogen evolution reaction, HER) for electrochemical NRR under rigorous isotope labeling experiments remains a perplexing challenge in experiments. In this study, we implemented a systematic screening protocol to assess the electrochemical stability and efficiency of various metal nitrides for NRR by high-throughput density functional theory calculations, aiming to search out promising metal nitride catalysts. Through a set of predetermined criteria, a subset of 6 candidates emerges as the most promising metal nitrides for NRR from a pool of 668 metal nitrides sourced from the Materials Project Database, all of which catalyze NRR following the Mars-van Krevelen pathway. Importantly, based on the analysis of these candidates, the unique structural property (i.e., four(or three)-coordinate lattice nitrogen) and energy descriptor (i.e., the formation energy of the lattice nitrogen vacancy is around 2.1 eV) of potential metal nitrides for electrochemical NRR were extracted, which could be used as the direction to rationally design metal nitride catalyst. This research not only serves as a valuable guide for experimental investigations into potential metal nitrides for NRR, but also provides an efficient method for high-throughput screening of potential catalysts for other electrochemical reactions.

Electrochemical nitrogen reduction reaction catalyzed by a cucurbit[6]uril-phosphomolybdic-copper cation hybrid

The electrocatalytic nitrogen reduction reaction (NRR) provides an energy-saving and environmentally friendly method to synthesize ammonia. Herein, a molybdenum-containing hybrid material (Q[6]-PMA-Cu) was prepared form the assembly of Keggin-type phosphomolybdic acid (PMA) with cucurbit[6]uril (Q[6]) and Cu2+ cations. Q[6]-PMA-Cu with multiple active sites and stable properties not only enables a high selectivity for NRR, but also shows a distinct advantage to suppress HER process, affording a high ammonia yield of 135.04±4.25 µg h−1 mgcat−1 with a Faradaic efficiency (FE) of 85.48±3.33%. 15N2 isotope labeling and control experiments were conducted to verified the nitrogen resource of ammonia formation. The results of experiments and DFT calculations demonstrates the reaction mechanism along the alternate association pathway, of which the metal active sites (Mo & Cu) play crucial role in electron transfer and the presence of hydrophobic Q[6] is responsible to inhibit side reaction of HER.

Reassignment of the Structure of a Tryptophan-Containing Cyclic Tripeptide Produced by the Biarylitide Crosslinking Cytochrome P450blt.

The structure of the sidechain crosslinked Tyr-Leu-Trp peptide produced by the biarylitide crosslinking cytochrome P450Blt from Micromonospora sp. MW-13 has been reanalysed by a series of NMR, computational and isotope labelling experiments and shown to contain a C-N rather than a C-O bond. Additional in vivo experiments using such a modified peptide show there is a general tolerance of biarylitide crosslinking P450 enzymes for histidine to tryptophan mutations within their minimal peptide substrate sequences despite the lack of such residues noted in natural biarylitide gene clusters. This work further highlights the impressive ability of P450s from biarylitide biosynthesis pathways to act as biocatalysts for the formation of a range of sidechain crosslinked tripeptides.

A Cytochrome P450 Enzyme Catalyses Oxetane Ring Formation in Paclitaxel Biosynthesis

Oxetane synthase (TmCYP1), a novel cytochrome P450 enzyme from Taxus × media cell cultures, has been functionally characterized to efficiently catalyse the formation of the oxetane ring in tetracyclic taxoids. Transient expression of TmCYP1 in Nicotiana benthamiana using 2α,5α,7β,9α,10β,13α‐hexaacetoxytaxa‐4(20),11(12)‐diene (1) as a substrate led to the production of a major oxetane derivative, 1β‐dehydroxybaccatin IV (1a), and a minor 4β,20‐epoxide derivative, baccatin I (1b). However, feeding the substrate decinnamoyltaxinine J (2), a 5‐deacetylated derivative of 1, yielded only 5α‐deacetylbaccatin I (2b), a 4β,20‐epoxide. A possible reaction mechanism was proposed on the basis of substrate‐feeding, 2H and 18O isotope labelling experiments, and density functional theory calculations. This reaction could be an intramolecular oxidation‐acetoxyl rearrangement and the construction of the oxetane ring may occur through a concerted process; however, the 4β,20‐epoxide might be a shunt product. In this process, the C5‐O‐acetyl group in substrate is crucial for the oxetane ring formation but not for the 4(20)‐epoxy ring formation by TmCYP1. These findings provide a better understanding of the enzymatic formation of the oxetane ring in paclitaxel biosynthesis.

A Cytochrome P450 Enzyme Catalyses Oxetane Ring Formation in Paclitaxel Biosynthesis.

Oxetane synthase (TmCYP1), a novel cytochrome P450 enzyme from Taxus×media cell cultures, has been functionally characterized to efficiently catalyse the formation of the oxetane ring in tetracyclic taxoids. Transient expression of TmCYP1 in Nicotiana benthamiana using 2α,5α,7β,9α,10β,13α-hexaacetoxytaxa-4(20),11(12)-diene (1) as a substrate led to the production of a major oxetane derivative, 1β-dehydroxybaccatin IV (1 a), and a minor 4β,20-epoxide derivative, baccatin I (1 b). However, feeding the substrate decinnamoyltaxinine J (2), a 5-deacetylated derivative of 1, yielded only 5α-deacetylbaccatin I (2 b), a 4β,20-epoxide. A possible reaction mechanism was proposed on the basis of substrate-feeding, 2H and 18O isotope labelling experiments, and density functional theory calculations. This reaction could be an intramolecular oxidation-acetoxyl rearrangement and the construction of the oxetane ring may occur through a concerted process; however, the 4β,20-epoxide might be a shunt product. In this process, the C5-O-acetyl group in substrate is crucial for the oxetane ring formation but not for the 4(20)-epoxy ring formation by TmCYP1. These findings provide a better understanding of the enzymatic formation of the oxetane ring in paclitaxel biosynthesis.

Low-crystallinity porous Ni2P2O7 nanowires with excellent CO2 capture ability for highly-efficient CO2 reduction

In this study, we prepared the porous Ni2P2O7 nanowires with low-crystallinity (LC-NPO) and high-crystallinity Ni2P2O7 nanosheets (HC-NPO), and employed them for the first time as catalyst in the CO2 photoreduction reaction. The photocatalytic performance of them was tested under the UV–Vis light irradiation and compared with the TiO2 commercial powder (P25). The CO2 photoreduction experiments shown that the LC-NPO had the highest ability than the HCNPO and the P25. Specifically, with triethanolamine (TEOA) as the sacrificial agent, LC-NPO achieved CO and CH4 yields of approximately 56.06 and 12.13 μmol·g−1 after 4 h UV–Vis light irradiation, which were 4.67 and 5.00 times of P25, respectively. The experimental results, and Density Functional Theory calculations proved that the unique porous nanowire structure enables LC-NPO to have a larger specific surface area, higher quantity of oxygen defects, and superior CO2 capture ability compared to HC-NPO. These features maybe the primary reasons for the excellent CO2 photoreduction ability. Besides, the photo-electrochemical characterizations revealed that the LC-NPO had the much superior photoelectrochemical properties than the HC-NPO, which was another reason for enhanced photocatalytic ability. In-situ FTIR and 13C isotope labeling experiments were used to monitor the CO2 reduction process, and finally a potential enhanced mechanism had been discussed.