Zheyi Liu

Preparation of Monolithic Polymer Columns with Homogeneous Structure via Photoinitiated Thiol-yne Click Polymerization and Their Application in Separation of Small Molecules

Two monolithic polymer columns were directly prepared in the UV-transparent fused-silica capillaries via photoinitiated thiol-yne click polymerization of 1,7-octadiyne (ODY) with a dithiol (1,6-hexanedithiol, 2SH) or a tetrathiol (pentaerythriol tetrakis(3-mercaptopropionate), 4SH) within 15 min. The rapid polymerization provided a time-saving approach to optimize preparation conditions. Then, two porogenic systems of diethylene glycol diethyl ether (DEGDE)/tetrahydrofuran (THF) and DEGDE/poly(ethylene glycol) (PEG, Mn = 200) were found to effectively control the porous structure of two kinds of polymeric monoliths (O2SH and O4SH), respectively. The almost disappearance of thiol and alkynyl vibrations (2560 and 2115 cm(-1), respectively) in infrared spectra and Raman spectra indicated a high conversion of the thiol-yne polymerization reaction. The thiol-yne polymerization was further proved by analyzing the energy-dispersive X-ray spectrum (EDS), MALDI-TOF mass spectrum, and elemental data. Scanning electron microscopy (SEM) images showed the monolithic polymer columns with homogeneous porous structure and macropore size of 0.5-1.0 μm, which facilitated the minimum plate heights of 10.0-12.0 μm for alkylbenzenes in reversed-phase liquid chromatography (RPLC). The low values of the A and C terms (<1.0 μm and <15.5 ms, respectively) in the van Deemter equation were similar to those obtained by some monolithic silica columns. The BSA tryptic digest was also separated on the monolithic polymer column by cLC-MS/MS. The result with 85% protein coverage was better than those given by some hybrid monolithic columns. The monolithic polymer columns were further applied for separation of phenols, natural products, and standard proteins and demonstrated satisfactory separation ability.

Novel features of eukaryotic photosystem II revealed by its crystal structure analysis from a red alga

Photosystem II (PSII) catalyzes light-induced water splitting, leading to the evolution of molecular oxygen indispensible for life on the earth. The crystal structure of PSII from cyanobacteria has been solved at an atomic level, but the structure of eukaryotic PSII has not been analyzed. Because eukaryotic PSII possesses additional subunits not found in cyanobacterial PSII, it is important to solve the structure of eukaryotic PSII to elucidate their detailed functions, as well as evolutionary relationships. Here we report the structure of PSII from a red alga Cyanidium caldarium at 2.76 Å resolution, which revealed the structure and interaction sites of PsbQ′, a unique, fourth extrinsic protein required for stabilizing the oxygen-evolving complex in the lumenal surface of PSII. The PsbQ′ subunit was found to be located underneath CP43 in the vicinity of PsbV, and its structure is characterized by a bundle of four up-down helices arranged in a similar way to those of cyanobacterial and higher plant PsbQ, although helices I and II of PsbQ′ were kinked relative to its higher plant counterpart because of its interactions with CP43. Furthermore, two novel transmembrane helices were found in the red algal PSII that are not present in cyanobacterial PSII; one of these helices may correspond to PsbW found only in eukaryotic PSII. The present results represent the first crystal structure of PSII from eukaryotic oxygenic organisms, which were discussed in comparison with the structure of cyanobacterial PSII.

Separation of intact proteins by using polyhedral oligomeric silsesquioxane based hybrid monolithic capillary columns.

High-efficient separation of intact proteins is still a huge challenge in proteome analysis of complex biological samples by using capillary columns. In this study, four POSS-based hybrid monolithic capillary columns were prepared and applied in nano-flow liquid chromatography (Nano-LC) separation of intact proteins. It was observed that the POSS-based hybrid monolithic columns exhibit high permeability, good LC separation reproducibility and column efficiency for intact protein separation. The effects of different LC separation conditions such as flow rate, gradient steepness, column length and mobile phase additives on the LC separation efficiency of the POSS-based hybrid monolithic column were systematically examined. Finally, fast LC separation of 7 proteins mixture was realized in 2.5 min by using the optimized conditions on the 100 μm i.d. POSS-based hybrid monolithic capillary column.

Fabrication of a novel magnetic yolk–shell Fe3O4@mTiO2@mSiO2 nanocomposite for selective enrichment of endogenous phosphopeptides from a complex sample

A yolk–shell nanocomposite composed of a magnetic mesoporous anatase TiO2 (Fe3O4@mTiO2) core, a medium cavity and an outermost mesoporous silica (mSiO2) shell was successfully fabricated. Due to a combination of the strong magnetic response, improved diffusion of peptides, numerous affinity sites towards phosphopeptides and the size-exclusion effect, the nanocomposite demonstrated high enrichment efficacy and selectivity towards endogenous phosphopeptides from human serum.

Intrinsic factor controlling the deformation and ductile-to-brittle transition of metallic glasses

The energetic driving force and resistance for shearing and cracking in metallic glasses (MGs) are quantitatively evaluated. A universal thermodynamic criterion is proposed for better understanding the intrinsic correlations between fracture toughness and Poisson’s ratio, the competitions between various deformation modes and the ductile-to-brittle transition in MGs and other materials. A new cooperation parameter δ is also introduced to depict quantitatively the relative propensity of shearing versus cracking. This work could provide insights into the long-standing issues of deformation mechanisms of glassy materials, and be helpful in searching for ductile and tough MGs.

High-Sensitivity N‑Glycoproteomic Analysis of Mouse Brain Tissue by Protein Extraction with a Mild Detergent of N‑Dodecyl β‑D-Maltoside

N-dodecyl β-D-maltoside (DDM), a mild detergent with the ability to maintain the enzyme activity and solubilize hydrophobic proteins without changing their structures, was applied for N-glycoproteomic analysis of minute protein sample from mouse brain tissue. After combining with the capillary-based glycoproteomic reactor, 281 N-glycosylation sites were successfully characterized from 50 μg of mouse brain tissue, which was 110% higher at least than those obtained by conventional strategies.

Detecting Proteins Glycosylation by a Homogeneous Reaction System with Zwitterionic Gold Nanoclusters

Homogeneous gold nanoclusters (Au NCs) have been widely utilized in drug delivery, chemical sensing, bioassays, and biolabeling due to their unique physical and chemical properties. However, little attention has been paid to their application in detecting protein post-translational modifications. Herein, we describe the development of a homogeneous reaction system with water-soluble zwitterionic Au NCs to capture glycopeptides from complex biological samples. The unique characteristics of Au NCs, such as their molecular-like properties, the excellent homogeneity in aqueous solution, the organic solvent responsive precipitation, and the easy preparation in only 4.5 h, contribute to the high efficiency and high throughput for capturing the targeted glycopeptides. Compared with the conventional heterogeneous system with solid-state adsorbents, the number of characterized glycosylation sites was improved by 35%. Finally, an MS detection limit as low as 50 amol was achieved for the standard glycoprotein (IgG), and 1576 glycosylation sites from 713 glycoproteins were identified from only 60 μg of mouse liver protein. Data are available via ProteomeXchange with identifier PXD005635.

One-Pot Approach to Prepare Organo-silica Hybrid Capillary Monolithic Column with Intact Mesoporous Silica Nanoparticle as Building Block

A facile “one-pot” approach to prepare organo-silica hybrid capillary monolithic column with intact mesoporous silica nanoparticle (IMSN) as crosslinker and building block was described. An IMSN crosslinked octadecyl-silica hybrid capillary monolithic column (IMSN-C18 monolithic column) was successfully prepared, and the effects of fabrication conditions (e.g. concentration of intact mesoporous silica nanoparticle, polycondensation temperature, content of vinyltrimethoxysilane and stearyl methacrylate) on the structures of the IMSN-C18 monolithic column were studied in detail. The IMSN-C18 hybrid monolithic column possessed uniform morphology, good mechanical and pH stability (pH 1.1–11), which was applied to the separations of alkyl benzenes, polycyclic aromatic hydrocarbons (PAHs), as well as proteins. The minimum plate height of 10.5 μm (corresponding to 95000 N m−1) for butylbenzene and high reproducibility were achieved. The analysis of tryptic digest of bovine serum albumin (BSA) was carried out on the IMSN-C18 monolithic column by cLC coupled mass spectrometry (cLC-MS/MS), with the protein sequence coverage of 87.5% for BSA, demonstrating its potential application in proteomics.

Preparation of organic-silica hybrid monolithic columns via crosslinking of functionalized mesoporous carbon nanoparticles for capillary liquid chromatography

An organic-silica hybrid monolithic capillary column was fabricated by crosslinking (3-aminopropyl)trimethoxysilane (APTMS) modified mesoporous carbon nanoparticles (AP-MCNs) with tetramethoxysilane (TMOS) and n-butyltrimethoxysilane (C4-TriMOS). Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy, mercury intrusion porosimetry and inverse size-exclusion chromatography characterization proved the successful immobilization of mesoporous carbon nanoparticles (MCNs). The crosslinking of AP-MCNs into the hybrid monolithic matrix has significantly increased the reversed-phase retention of alkylbenzenes and chromatographic performance for small molecules separations in comparison with the neat one without MCNs. The resulting column efficiency of the mesoporous carbon nanoparticle-based butyl-silica hybrid monolithic column (MCN-C4-monolith) was up to ca. 116,600N/m for the capillary liquid chromatography (cLC) separation of butylbenzene. Enhanced performance of proteins separation was achieved on the MCN-C4-monolith in comparison with the butyl-silica hybrid monolithic column without MCN (C4-monolith). The separation of peptides from bovine serum albumin (BSA) digest was carried out on the MCN-C4-monolith by capillary liquid chromatography-tandem mass spectrometry (cLC-MS/MS) with protein sequence coverage of 81.9%, suggesting its potential application in proteomics.

Probing the Lysine Proximal Microenvironments within Membrane Protein Complexes by Active Dimethyl Labeling and Mass Spectrometry

Positively charged lysines are crucial to maintaining the native structures of proteins and protein complexes by forming hydrogen bonds and electrostatic interactions with their proximal amino acid residues. However, it is still a challenge to develop an efficient method for probing the active proximal microenvironments of lysines without changing their biochemical/physical properties. Herein, we developed an active covalent labeling strategy combined with mass spectrometry to systematically probe the lysine proximal microenvironments within membrane protein complexes (∼700 kDa) with high throughput. Our labeling strategy has the advantages of high labeling efficiency and stability, preservation of the active charge states, as well as biological activity of the labeled proteins. In total, 121 lysines with different labeling levels were obtained for the photosystem II complexes from cyanobacteria, red algae, and spinach and provided important insights for understanding the conserved and nonconserved local structures of PSII complexes among evolutionarily divergent species that perform photosynthesis.