Reproducible method to enrich membrane proteins with high purity and high yield for an LC‐MS/MS approach in quantitative membrane proteomics

The proportionately low abundance of membrane proteins hampers their proteomic analysis, especially for a quantitative LC‐MS/MS approach. To overcome this limitation, a method was developed that consists of one cell disruption step in a hypotonic reagent using liquid nitrogen, one isolation step using a low speed centrifugation, and three wash steps using high speed centrifugation. Pellets contained plasma, nuclear, and mitochondrial membranes, including their integral, peripheral, and anchored membrane proteins. The reproducibility of this method was verified by protein assay of four separate experiments with a CV of 7.7%, and by comparative LC‐MS/MS label‐free quantification of individual proteins between two experiments with 99% of the quantified proteins having a CV ≤30%. Western blot and LC‐MS/MS results of markers for cytoplasm, nucleus, mitochondria, and their membranes indicated that the enriched membrane fraction was highly pure by the absence of, or presence of trace amounts of, nonmembrane marker proteins. The average yield of membrane proteins was 237 μg/10 million HT29‐MTX cells. LC‐MS/MS analysis of the membrane‐enriched sample resulted in the identification of 2597 protein groups. In summary, the developed method is reproducible, produces a highly pure membrane fraction, and generates a high yield of membrane proteins.     

Electric migration of α‐hemolysin in supported n‐bilayers: A model for transmembrane protein microelectrophoresis

Proteome analysis involves separating proteins as a preliminary step toward their characterization. This paper reports on the translational migration of a model transmembrane protein (α‐hemolysin) in supported n‐bilayers (n, the number of bilayers, varies from 1 to around 500 bilayers) when an electric field parallel to the membrane plane is applied. The migration changes in direction as the charge on the protein changes its sign. Its electrophoretic mobility is shown to depend on size and charge. The electrophoretic mobility varies as 1/R², with R the equivalent geometric radius of the embedded part of the protein. Measuring mobilities at differing pH in our system enables us to determine the pI and the charge of the protein. Establishing all these variations points to the feasibility of electrophoretic transport of a charged object in this medium and is a first step toward electrophoretic separation of membrane proteins in n‐bilayer systems.     

基于微流控技术的蛋白质结晶及其筛选方法的研究进展简

微流控技术以其高通量、低消耗和集成化等优点成为蛋白质结晶微型化研究的重要手段. 本文综述了基于微流控技术的蛋白质结晶技术和方法,主要包括微泵微阀、液滴(Droplet)、滑动芯片(SlipChip)以及液滴实验室(DropLab)等技术. 此外,还针对当前膜蛋白在结构生物学研究中的重要地位,综述了应用于膜蛋白结晶的微流控技术的研究进展.     

Improvement of a sample preparation method assisted by sodium deoxycholate for mass‐spectrometry‐based shotgun membrane proteomics†

In current shotgun‐proteomics‐based biological discovery, the identification of membrane proteins is a challenge. This is especially true for integral membrane proteins due to their highly hydrophobic nature and low abundance. Thus, much effort has been directed at sample preparation strategies such as use of detergents, chaotropes, and organic solvents. We previously described a sample preparation method for shotgun membrane proteomics, the sodium deoxycholate assisted method, which cleverly circumvents many of the challenges associated with traditional sample preparation methods. However, the method is associated with significant sample loss due to the slightly weaker extraction/solubilization ability of sodium deoxycholate when it is used at relatively low concentrations such as 1%. Hence, we present an enhanced sodium deoxycholate sample preparation strategy that first uses a high concentration of sodium deoxycholate (5%) to lyse membranes and extract/solubilize hydrophobic membrane proteins, and then dilutes the detergent to 1% for a more efficient digestion. We then applied the improved method to shotgun analysis of proteins from rat liver membrane enriched fraction. Compared with other representative sample preparation strategies including our previous sodium deoxycholate assisted method, the enhanced sodium deoxycholate method exhibited superior sensitivity, coverage, and reliability for the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.     

Cathode catalysts degradation mechanism from liquid electrolyte to membrane electrode assembly

Single-cell and half-cell degradation test procedures were evaluated for carbon-supported Pt/C, PtCo/C and PtNi/C catalysts. Half-cell analyses were employed to understand the effect of the number of cycles and of the scan rate over the cathode catalysts degradation under potential cycling from 0.6 to 1.2 V. The data suggested a time-dependent degradation for all three catalytic systems. Single-cell measurements were used to evaluate the impact of catalyst degradation on fuel cell performance. The measurements in both setups showed similar ECSA and ORR mass activity losses. Specific degradation mechanisms related to Pt dissolution, Pt agglomeration, and transitional metal leaching were quantified and correlated with performance losses.     

Co3O4/UiO-66@α-Al2O3陶瓷膜对VOCs废气的分离催化性能

以α-Al₂O₃为支撑层, UiO-66为分离层, Co₃O₄为催化层, 构建了Co₃O₄/UiO-66@α-Al₂O₃陶瓷膜.对其形貌结构进行了表征, 并研究了其对挥发性有机物(VOCs)的分离催化性能. 结果表明, 该陶瓷膜对喷涂行业废气中苯与吡啶两种主要成分体现出良好的分离性, 透过侧的气体中吡啶与苯的摩尔浓度比值可由给料侧的1提高至17; 体系中引入臭氧后, 给料侧苯的浓度明显降低, 其去除率可达到89%. 透过侧的吡啶去除率仅为27%, 得到了较大程度的保留, 剩余的吡啶经收集后可进行单独的深度处理. Co₃O₄/UiO-66@α-Al₂O₃陶瓷膜在对喷涂行业废气中苯成分高效降解的同时, 能够有效解决喷涂行业废气处理过程中氮氧化物的排放问题, 有望成为喷涂行业废气预处理工艺的理想选择.     

Pore Engineering for Covalent Organic Framework Membranes

Membrane technology is of particular significance for the sustainable development of society owing to its potential capacity to tackle the energy shortage and environmental pollution. Membrane materials are the core part of membrane technology. Researchers have always been pursuing predictable structures of advanced membrane materials, which provides a possibility to fully unlock the potential of membranes. Covalent organic frameworks(COFs), with the advantage of controllable pore microenvironment, are considered to be promising candidates to achieve this design concept. The customizable function of COF membranes through pore engineering does well in the enhancement of selective permeability performance, which offers COF membranes with great application potentials in separation and transportation fields. In this context, COF-based membranes have been developed rapidly in recent years. Herein, we present a brief overview on the strategies developed for pore engineering of COF membranes in recent years, including skeleton engineering, pore surface engineering, host-guest chemistry and membrane fabrication. Moreover, the features of transmission or separation of molecules/ions based on COF membranes and corresponding applications are also introduced. In the last part, the challenges and prospects of the development of COF membranes are discussed.     

MXene: A two-dimensional material in selective water separation via pervaporation

MXene, well-identified as Ti₃C₂T_X, belongs to the family of two-dimensional (2D) materials, which have been currently explored in various applications. Very recently, such materials have been pointed out as potential nanomaterials for advanced solute separations when introduced in membranes, such as ion separation, gas separation, nanofiltration, chiral molecular separation, and solvent separation. This latter separation, generally named Pervaporation (PV), is identified as a highly selective technology for water separations. To date, few pieces of research have been released but providing interesting insights into several solvent (including water) separations. Hence, this brief review aims to analyze and discuss the latest advances for utilizing MXenes for PV membranes. Particular emphasis has been devoted to the relevant outcomes in the field, along with the strategies followed by researchers to tailor membranes. Based on the current findings, the perspectives in the field are also stated.     

Improved Stabilities of Labeling Probes for the Selective Modification of Endogenous Proteins in Living Cells and In Vivo

To date, various affinity-based protein labeling probes have been developed and applied in biological research to modify endogenous proteins in cell lysates and on the cell surface. However, the reactive groups on the labeling probes are also the cause of probe instability and nonselective labeling in a more complex environment, e. g., intracellular and in vivo. Here, we show that labeling probes composed of a sterically stabilized difluorophenyl pivalate can achieve efficient and selective labeling of endogenous proteins on the cell surface, inside living cells and in vivo. As compared with the existing protein labeling probes, probes with the difluorophenyl pivalate exhibit several advantages, including long-term stability in stock solutions, resistance to enzymatic hydrolysis and can be customized easily with diverse fluorophores and protein ligands. With this probe design, endogenous hypoxia biomarker in living cells and nude mice were successfully labeled and validated by in vivo, ex vivo, and immunohistochemistry imaging.     

Trehalose-protected lipid membranes for determining membrane protein structure and insertion

Trehalose preserves lipid bilayers during dehydration and rehydration by replacing water to form hydrogen bonds between its own OH groups and lipid headgroups. We compare the lipid conformation and dynamics between trehalose-protected lyophilized membranes and hydrated membranes, to assess the suitability of the trehalose-containing membrane as a matrix for membrane protein structure determination. (31)P spectra indicate that the lipid headgroup of trehalose-protected dry POPC membrane (TRE-POPC) have an effective phase transition temperature that is approximately 50K higher than that of the hydrated POPC membrane. In contrast, the acyl chains have similar transition temperatures in the two membranes. Intramolecular lipid (13)C'-(31)P distances are the same in TRE-POPC and crystalline POPC, indicating that the lipid headgroup and glycerol backbone conformation is unaffected by trehalose incorporation. Intermolecular (13)C-(31)P distances between a membrane peptide and the lipid headgroups are 10% longer in the hydrated membrane at 226 K than in the trehalose-protected dry membrane at 253 K. This is attributed to residual motions in the hydrated membrane, manifested by the reduced (31)P chemical shift anisotropy, even at the low temperature of 226 K. Thus, trehalose lyoprotection facilitates the study of membrane protein structure by allowing experiments to be conducted at higher temperatures than possible with the hydrated membranes.