No abstract

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.