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Intrachain modifications of membrane glycerophospholipids (GPLs) due to formation of the carbon−carbon double bond (CC), cyclopropane ring, and methyl branching are crucial for bacterial membrane homeostasis. Conventional collision-induced dissociation (CID) of even-electron ions of GPL favors chargedirected fragmentation channels, and thus little structurally informative fragments can be detected for locating intrachain modifications. In this study, we report a radical-directed dissociation (RDD) approach for characterization of the intrachain modifications within phosphoethanolamines (PEs), a major lipid component in bacterial membrane. In this method, a radical precursor that can produce benzyl or pyridine methyl radical upon low-energy CID at high efficiency is conjugated onto the amine group of PEs. The carbon-centered radical ions subsequently initiate RDD along the fatty acyl chain, producing fragment patterns key to the assignment and localization of intrachain modifications including CC, cyclopropane rings, and methyl branching. Besides intrachain fragmentation, RDD on the glycerol backbone produces fatty acyl loss as radicals, allowing one to identify the fatty acyl chain composition of PE. Moreover, RDD of lyso-PEs produces radical losses for distinguishing the sn-isomers. The above RDD approach has been incorporated onto a liquid chromatography−mass spectrometry workflow and applied for the analysis of lipid extracts from Escherichia coli and Bacillus subtilis.

Oxidation reactions are fundamental transformations in organic synthesis and chemical industry. With oxygen or air as terminal oxidant, aerobic oxidation catalysis provides the most sustainable and economic oxidation processes. Most aerobic oxidation catalysis employs redox metal as its active center. While nature provides non-redox metal strategy as in pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenases (MDH), such an effective chemical version is unknown. Inspired by the recently discovered rare earth metal-dependent enzyme Ln-MDH, here we show that an open-shell semi-quinone anionic radical species in complexing with lanthanum could serve as a very efficient aerobic oxidation catalyst under ambient conditions. In this catalyst, the lanthanum(III) ion serves only as a Lewis acid promoter and the redox process occurs exclusively on the semiquinone ligand. The catalysis is initiated by 1e--reduction of lanthanum-activated ortho-quinone to a semiquinone-lanthanum complex La(SQ-.)2, which undergoes a coupled O-H/C-H (PCHT: proton coupled hydride transfer) dehydrogenation for aerobic oxidation of alcohols with up to 330 h−1 TOF.

The membrane proteins of microbes are at the forefront of host and parasite interactions. Having a general view of the functions of microbial membrane proteins is vital for many biomedical studies on microbiota. Nevertheless, due to the strong hydrophobicity and low concentration of membrane proteins, it is hard to efficiently enrich and digest the proteins for mass spectrometry analysis. Herein, we design an enzymatic nanoreactor for the digestion of membrane proteins using methylated wellordered hexagonal mesoporous silica (Met-SBA-15). The material can efficiently extract hydrophobic membrane proteins and host the proteolysis in nanopores. The performance of the enzymatic nanoreactor is first demonstrated using standard hydrophobic proteins and then validated using membrane proteins extracted from Escherichia coli (E. coli) or a mixed bacterial sample of eight strains. Using the nanoreactor, 431 membrane proteins are identified from E. coli, accounting for 38.5% of all membrane proteins of the species, which is much more than that by the widely used in-solution digestion protocol. From the mixed bacterial sample of eight strains, 1395 membrane proteins are identified using the nanoreactor. On the contrary, the traditional in-solution proteolysis workflow only leads to the identification of 477 membrane proteins, demonstrating that the Met-SBA-15 can be offered as an excellent tool for microbial membrane proteome research and is expected to be used in human microbiota studies, e.g. host−microbe interactions.

Chain modifications on fatty acyls, such as methyl branching, are important to modulate the biochemical and biophysical properties of lipids. The current lipid analysis workflows which mainly rely on collisional-induced...

Lysophosphatidylcholine acyltransferase-1 (LPCAT1) plays a critical role in the remodeling of phosphatidylcholines (PCs) in cellular lipidome. However, evidence is scarce regarding its sn-selectivity, viz. the preference of assembling acyl-Coenzyme A (CoA) at the C1 or C2-hydroxyl on a glycerol backbone because of difficulty to quantify the thus-formed PC snisomers. We have established a multiplexed assay to measure both sn-and acyl-chain selectivity of LPCAT1 toward a mixture of acyl-CoAs by integrating isomerresolving tandem mass spectrometry. Our findings reveal that LPCAT1 shows exclusive sn-1 specificity regardless of the identity of acyl-CoAs. We further confirm that elevated PC 18 : 1/16:0 relative to its snisomer results from an increased expression of LPCAT1 in human hepatocellular carcinoma (HCC) tissue as compared to normal liver tissue. MS imaging via desorption electrospray ionization of PC 18 : 1/16:0 thus enables visualization of HCC margins in human liver tissue at a molecular level.Glycerophospholipids (GPLs) are essential components of all biological membranes. [1] This important category of lipids shares three common building blocks, a glycerol backbone, two fatty acyls linked to the C-1 and C-2 hydroxyls of the glycerol, and a phosphate-containing head group esterified at the C-3 hydroxyl. For individual GPL molecules, saturated and monounsaturated fatty acids tend to be esterified at the C-1 position or so-called sn-1 position of the glycerol, whereas polyunsaturated fatty acids, such as arachidonic acid, are commonly located at the C-2 position or sn-2 position. [2] Such an asymmetric distribution of acyl chains is regulated by the remodeling pathway (Land's cycle), [3] where phospholipase A 1 or A 2 (PLA 1 /PLA 2 ) converts GPLs to 2-acyl lysophospholipids or 1-acyl lysophospholipids (LPLs), while lysophospholipid acyltransferases (LPLATs) re-acylate LPLs back to GPLs. For instance, the expression patterns of lysophosphocholine acyltransferases (LPCATs) and their activities toward different acyl-CoAs in a specific tissue type contributes to the molecular diversity of phosphatidylcholines (PCs) in the tissue. [4] LPCAT1 is highly expressed in lung tissue and it catalyzes the production of dipalmitoyl-phosphatidylcholine (PC 16:0/ 16:0), a major lipid component of pulmonary surfactant. [5] A previous study suggests that LPCAT1 has a preference toward saturated fatty acyl-CoAs, e.g., C16:0 and C14:0. [5b] Recently, several groups reported that the compositions of PC sn-isomers were significantly different in cancer tissue relative to normal control, showing a great potential in disease phenotyping. [6] Knowing the sn-selectivity of LPCAT1 will thus provide valuable insights into the metabolic origins of the altered lipid compositions.Mass spectrometry (MS) has become the method of choice for developing new enzymatic assays, owing to its high sensitivity, structural specificity, and high-throughput analysis capability. [7] Assays based on liquid chromatography-mass spectrometry (LC-MS) have b...

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Hepatic ischemia‐reperfusion injury (HIRI) is a critical complication after liver surgery that negatively affects surgical outcomes of patients with the end‐stage liver‐related disease. Reactive oxygen species (ROS) are responsible for the development of ischemia‐reperfusion injury and eventually lead to hepatic dysfunction. Selenium‐doped carbon quantum dots (Se‐CQDs) with an excellent redox‐responsive property can effectively scavenge ROS and protect cells from oxidation. However, the accumulation of Se‐CQDs in the liver is extremely low. To address this concern, the fabrication of Se‐CQDs‐lecithin nanoparticles (Se‐LEC NPs) is developed through self‐assembly mainly driven by the noncovalent interactions. Lecithin acting as the self‐assembly building block also makes a pivotal contribution to the therapeutic performance of Se‐LEC NPs due to its capability to react with ROS. The fabricated Se‐LEC NPs largely accumulate in the liver, effectively scavenge ROS and inhibit the release of inflammatory cytokines, thus exerting beneficial therapeutic efficacy on HIRI. This work may open a new avenue for the design of self‐assembled Se‐CQDs NPs for the treatment of HIRI and other ROS‐related diseases.