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In the intrinsic pathway of apoptosis, stresses of mitochondrial reactive oxygen species (mitoROS) might be sensed as more effective signals than those in cytosol, as mitochondria are the major sources of reactive oxygen species (ROS) and pivotal components during cell apoptosis. Mitochondrial superoxide dismutase (SOD2) takes the leading role in eliminating mitoROS, and inhibition of SOD2 might induce severe disturbances overwhelming the mitochondrial oxidative equilibrium, which would elevate the intracellular oxidative stresses and drive cells to death. Herein, we report a general strategy to kill cancer cells by targeted inhibition of SOD2 using 2-methoxyestradiol (2-ME, an inhibitor for the SOD family) via a robust mitochondria-targeted mesoporous silica nanocarrier (mtMSN), with the expected elevation of mitoROS and activation of apoptosis in HeLa cells. Fe3O4@MSN was employed in the mitochondria-targeted drug delivery and selective inhibition of mitochondrial enzymes, and was shown to be stable with good biocompatibility and high loading capacity. Due to the selective inhibition of SOD2 by 2-ME/mtMSN, enhanced elevation of mitoROS (132% of that with free 2-ME) was obtained, coupled with higher efficiency in initiating cell apoptosis (395% of that with free 2-ME in 4 h). Finally, the 2-ME/mtMSN exhibited powerful efficacy in targeted killing of HeLa cells by taking advantage of both biological recognition and magnetic guiding, causing 97.0% cell death with only 2 μg/mL 2-ME/mtMSN, hinting at its great potential in cancer therapy through manipulation of the delicate mitochondrial oxidative balance.

Facile preparation of core-shell magnetic metal-organic framework nanospheres by a layer-by-layer approach is presented. The nanospheres have high surface area (285.89 cm(2) g(-1)), large pore volume (0.18 cm(3) g(-1)), two kinds of mesopores (2.50 and 4.72 nm), excellent magnetic responsivity (55.65 emu g(-1)), structural stability, and good dispersibility. The combination of porosity, hydrophobicity, and uniform magnetism was exploited for effective enrichment of peptides with simultaneous exclusion of high molecular weight proteins. The nanospheres were successfully applied in the selective enrichment of endogenous peptides in human serum.

A polyhedral oligomeric silsesquioxane (POSS) hybrid monolith was simply prepared by using octaglycidyldimethylsilyl POSS (POSS-epoxy) and cystamine dihydrochloride as monomers via ring-opening polymerization. The effects of composition of prepolymerization solution and polycondensation temperature on the morphology and permeability of monolithic column were investigated in detail. The obtained POSS hybrid monolithic column showed 3D skeleton morphology and exhibited high column efficiency of ∼71,000 plates per meter in reversed-phase mechanism. Owing to this POSS hybrid monolith essentially possessing a great number of disulfide bonds, the monolith surface would expose thiol groups after reduction with dithiothreitol (DTT), which supplied active sites to functionalize with various alkene monomers via thiol-ene click reaction. The results indicated that the reduction with DTT could not destroy the 3D skeleton of hybrid monolith. Both stearyl methylacrylate (SMA) and benzyl methacrylate (BMA) were selected to functionalize the hybrid monolithic columns for reversed-phase liquid chromatography (RPLC), while [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide (MSA) was used to modify the hybrid monolithic column in hydrophilic interaction chromatography (HILIC). These modified hybrid monolithic columns could be successfully applied for separation of small molecules with high efficiency. It is demonstrated that thiol-ene click reaction supplies a facile way to introduce various functional groups to the hybrid monolith possessing thiol groups. Furthermore, due to good permeability of the resulting hybrid monoliths, we also prepared long hybrid monolithic columns in narrow-bore capillaries. The highest column efficiency reached to ∼70,000 plates using a 1-m-long column of 75μm i.d. with a peak capacity of 147 for isocratic chromatography, indicating potential application in separation and analysis of complex biosamples.

Covalent organic polymers are an emerging class of materials with potential applications in areas including molecular separation, gas sorption, and catalysis. A novel fully conjugated organic polymer bearing bis(imino)pyridine (COP-BIP) and its catalytic function are reported here. Unlike previous COP materials, the imine bonds of COP-BIP act as both linkages and complexation sites for the binding of metal ions. A clear structure is presented based on ultraviolet-visible, Fourier transform infrared, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry characterization. The COP-BIP materials are thermally stable up to 440 °C and are insoluble in conventional solvents. In addition, COP-BIP complexes Pd ions on bis(imino)pyridine sites and forms a heterogeneous catalyst, which exhibits excellent catalytic activity in the Suzuki-Miyaura C–C coupling reaction.

Conventional N-glycoproteome analysis usually applies C18 reversed-phase (RP) adsorbent for sample purification, which will lead to unavoidable sample loss due to the high hydrophilicity of N-glycopeptides. In this study, a porous graphitized carbon (PGC) absorbent was combined with a C18 adsorbent for N-glycopeptide purification in comprehensive N-glycoproteome analysis based on the hydrophobic and polar interactions between carbon and N-glycans. It was observed that the small hydrophilic N-glycopeptides that cannot retain onto C18 adsorbent can be captured by the graphitized carbon, while the large hydrophobic N-glycopeptides that cannot retain onto the graphitized carbon can be feasibly captured by the C18 adsorbent. Comparing with sample purification by using C18 adsorbent only, 28.5 % more N-glycopeptides were identified by combining both C18 and PGC adsorbents. The C18-PGC strategy was further applied for both sample purification and pre-fractionation of a complex protein sample from HeLa cell. After hydrophilic interaction chromatography enrichment, 1,484 unique N-glycopeptides with 1,759 unique N-glycosylation sites were finally identified.

An enzymatic approach to label peptide N-termini with isotope-coded affinity tags is presented. This method exploits the high activity of trypsin for peptide synthesis in organic solvents. A cosubstrate containing a stable isotope-coded Arg residue and a biotin tag was synthesized. When the cosubstrate was incubated with tryptic peptides and trypsin in ethanol solution, the stable isotope-coded affinity tag was specifically coupled onto the N-termini of peptides via the formation of new peptide bonds. The labeled peptides were specifically enriched by avidin affinity chromatography and then were submitted to liquid chromatography-tandem mass spectrometry (LC/MS/MS) for quantification. This enrichment step effectively reduced the interference by unlabeled peptides. The excellent performance of this approach was demonstrated by labeling standard peptides as well as a mouse liver digest. In addition to one amino acid residue, a few dipeptide tags were also introduced to the N-termini of peptides successfully by this enzymatic approach. It was found that the identifications for samples labeled with these tags were highly complementary. Coupling a short sequence tag onto peptides could be an effective approach to improve the coverage for proteome analysis.

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.

Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) materials have been increasingly attractive in glycopeptide enrichment. However, the traditional ZIC-HILIC materials are modified with monolayer zwitterionic molecules on the surface, therefore, the hydrophilicity, detection sensitivity and loading capacity are limited. In this work, we synthesized novel silica nanoparticles with uniform poly(2-(methacryloyloxy)ethyl)dimethyl-(3-sul-fopropyl)ammonium hydroxide (PMSA) brushes grafted onto the surface via reversible addition-fragmentation chain transfer (RAFT) polymerization (denoted as SiO2-RAFT@PMSA). The resulting SiO2-RAFT@PMSA nanoparticles demonstrated low detection limit (10 fmol) and high recovery yield (over 88%) for glycopeptide enrichment from tryptic digest of human IgG. The SiO2-RAFT@PMSA nanoparticles were further applied for the analysis of mouse liver glycoproteome, a total number of 303 unique N-glycosylation sites corresponding to 185 glycoproteins was reliably profiled in three replicate nano-LC-MS/MS runs. Significantly, more glycopeptides were identified than those of nanoparticles, monolayer MSA molecules modified SiO2@single-MSA and nonuniform multi-layer PMSA brushes coated SiO2@PMSA, as well as commercial ZIC@HILIC beads and Click Maltose beads. The excellent performance of SiO2-RAFT@PMSA nanoparticles results from the non-fouling property, a large quantity of functional molecules and suitable link arms provided by uniform PMSA brushes, as well as efficient interaction between glycopeptides and uniform PMSA brushes. It is concluded that the synthesized SiO2-RAFT@PMSA nanoparticles exhibit great potential in glycoproteome analysis. Moreover, this strategy to modify nanopaticles with uniform polymer brushes via RAFT polymerization can also be explored to design other types of materials for bioseparation application.

A strategy for the absolute quantitation of metabolic enzymes in rat liver microsomes was developed based on shotgun-based proteomics approach. Rat liver microsomes were digested with trypsin, and drug metabolizing enzymes were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) using standard curves based on custom synthesized specific peptides for each enzyme. The assays for cytochrome P450 (CYP450) and uridine diphosphoglucuronosyl transferase (UGT) showed good linearity ( r > 0.995) with lower limits of quantitation of 10 nM. A synthetic isotope labeled specific peptide was then used as internal standard to determine UGT1A1 by stable isotope dilution method. The labeled peptide behaved similarly to the unlabeled peptide in LC-MS/MS and the assay was linear in matrix solution. The UGT1A1 concentration was detected by the labeled peptide method to be 18.2 pmol mg −1 protein compared with 17.3 pmol mg −1 protein obtained by the standard curve method. Although the consistent results were achieved by the two methods, the stable isotope dilution method was more convenient and more suitable for high throughput determinations in complex systems.

Protein phosphorylation is one of the most common post-translational modifications. It plays key roles in regulating diverse biological processes of liver tissues. To better understand the role of protein phosphorylation in liver functions, it is essential to perform in-depth phosphoproteome analysis of human liver. Here, an enzyme assisted reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach with both RPLC separations operated with optimized acidic mobile phase was developed. High orthogonal separation was achieved by trypsin digestion of the Glu-C generated peptides in the fractions collected from the first RPLC separation. The phosphoproteome coverage was further improved by using two types of instruments, i.e. TripleTOF 5600 and LTQ Orbitrap Velos. A total of 22,446 phosphorylation sites, corresponding to 6526 nonredundant phosphoproteins were finally identified from normal human liver tissues. Of these sites, 15,229 sites were confidently localized with Ascore ≥ 13. This dataset was the largest phosphoproteome dataset of human liver. It can be a public resource for the liver research community and holds promise for further biology studies. Biological significance The enzyme assisted approach enabled the two RPLC separations operated both with optimized acidic mobile phases. The identifications from TripleTOF 5600 and Orbitrap Velos are highly complementary. The largest phosphoproteome dataset of human liver was generated.

Highly selective and efficient enrichment of trace phosphorylated proteins or peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. In this study, a novel immobilized metal affinity chromatography (IMAC) material has been synthesized to improve the enrichment specificity and sensitivity for phosphopeptides by introducing a titanium phosphate moiety on a multilayer polysaccharide (hyaluronate (HA) and chitosan (CS)) coated Fe3O4@SiO2 nanoparticle (denoted as Fe3O4@SiO2@(HA/CS)10–Ti4+ IMAC). The thicker multilayer polysaccharide endows excellent hydrophilic properties and a higher binding capacity of the titanium ion to the IMAC material. Due to the combination of uniform magnetic properties, highly hydrophilic properties and enhanced binding capacity of the titanium ion, the Fe3O4@SiO2@(HA/CS)10–Ti4+ nanoparticle possesses many merits, such as high selectivity for phosphopeptides (phosphopeptides/non-phosphopeptides at a molar ratio of 1 : 2000), extreme detection sensitivity (0.5 fmol), large binding capacity (100 mg g−1), high enrichment recovery (85.45%) and rapid magnetic separation (within 10 s). Moreover, the as-prepared IMAC nanoparticle provides effective enrichment of phosphopeptides from real samples (human serum and nonfat milk), showing great potential as a tool for the detection and identification of low-abundance phosphopeptides in biological samples.

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.

The origin of atmospheric dissolved organic nitrogen (DON) deposition is not very clear at present. Across China, the DON deposition was substantially larger than that of world and Europe, and we found significant positive correlation between contribution of DON and the deposition flux with pristine site data lying in outlier, possibly reflecting the acute air quality problems in China. For a case study in Northeast China, we revealed the deposited DON was mainly derived from intensive agricultural activities rather than the natural sources by analyzing the compiled dataset across China and correlating DON flux with NH 4 + –N and NO 3 − –N. Crop pollens and combustion of fossil fuels for heating probably contributed to summer and autumn DON flux respectively. Overall, in Northeast China, DON deposition could exert important roles in agro-ecosystem nutrient management and carbon sequestration of natural ecosystems; nationally, it was suggested to found rational network for monitoring DON deposition.

Post-translational modifications (PTMs) are covalent processing events of proteins. It is the reason that the complex biological process can be regulated under the limited gene number. As many as 300 PTMs of proteins play important roles in many living activities such as regulation of cell cycle, signal transduction and immune response. Most of the protein PTMs have the characteristics such as low abundance, high dynamic range and complex structures. Therefore, there are enormous challenges for the large scale analysis of protein PTMs. The PTMs proteomics approach which is based on the mass spectrometry emerges as an ideal strategy for the global analysis of protein PTMs. Now, the large scale analysis of protein PTMs has been the key research area of proteomics. In this review, we focus on the development of new method and technology of PTMs proteomics, including the enrichment strategies, separation method, mass spectrometry techniques and quantitative methods of the protein PTMs.

A new organic-inorganic hybrid monolith was prepared by the ring-opening polymerization of octaglycidyldimethylsilyl polyhedral oligomeric silsesquioxane (poss) with 1,4-butanediamine (bda) using 1-propanol, 1,4-butanediol, and peg 10 000 as a porogenic system. benefiting from the moderate phase separation process, the resulting poly(poss-co-bda) hybrid monolith possessed a uniform microstructure and exhibited excellent performance in chromatographic applications. neutral, acidic, and basic compounds were successfully separated on the hybrid monolith in capillary lc (clc), and high column efficiencies were achieved in all of the separations. in addition, as the amino groups could generate a strong eof, the hybrid monolith was also applied in cec for the separation of neutral and polar compounds, and a satisfactory performance was obtained. these results demonstrate that the poly(poss-co-bda) hybrid monolith is a good separation media in chromatographic separations of various types of compounds by both clc and cec.

Terminal sialylation is very important in cancer biology and has been extensively investigated for the discovery of potential clinical biomarkers of cancers. In this study, we presented a novel approach, by using of Ti(IV)-IMAC, to enrich sialic-acid-containing N-glycopeptides for the analysis of terminal sialylation. Compared with conventional method using TiO2 , this approach obtained 2.5 times more glycopeptides and glycosylation sites. Then, a simple integrated system combining filter-aided sample preparation, ACN-improved digestion, and Ti(IV)-IMAC enrichment was established for efficient analysis. In this system, protein digestion, glycopeptide enrichment, and deglycosylation were integrated and were performed sequentially in a single filter unit without any need for desalting, lyophilization, or sample transfer procedures. As a result, the number of identifications was improved by 1.5-fold and the total processing time was drastically reduced to only 7-8 h. By using this system, fast and efficient analysis of human serum sialylated N-glycoproteome was achieved. From only 1 μL of human serum, 217 unique glycopeptides and 194 glycosylation sites were successfully identified.

Despite the importance of protein N-glycosylation in a series of biological processes, in-depth characterization of protein glycosylation is still a challenge due to the high complexity of biological samples and the lacking of highly sensitive detection technologies. We developed a monolithic capillary column based glycoproteomic reactor enabling high-sensitive mapping of N-glycosylation sites from minute amounts of sample. Unlike the conventional proteomic reactors with only strong-cation exchange or hydrophilic-interaction chromatography columns, this novel glycoproteomic reactor was composed of an 8 cm long C12 hydrophobic monolithic capillary column for protein digestion and a 6 cm long organic-silica hybrid hydrophilic monolithic capillary column for glycopeptides enrichment and deglycosylation, which could complete whole-sample preparation including protein purification/desalting, tryptic digestion, enrichment, and deglycosylation of glycopeptides within about 3 h. The developed reactor exhibited high detection sensitivity in mapping of N-glycosylation sites by detection limit of horseradish peroxidase as low as 2.5 fmol. This reactor also demonstrated the ability in complex sample analysis, and in total, 486 unique N-glycosylation sites were reliably mapped in three replicate analyses of a protein sample extracted from ∼10(4) HeLa cells.

A simple approach to fabricate hybrid monolithic column within the confines of fused-silica capillaries (75 mu m i.d.) was introduced. A polyhedral oligomeric silsesquioxanes (POSS) reagent containing a methacrylate group was selected as functional monomer, and copolymerized with bisphenol A dimethacrylate (BPADMA) or ethylene dimethacrylate (EDMA) in the presence of porogenic solvents via thermally initiated free radical polymerization. After optimization of the preparation conditions, two POSS-containing hybrid monoliths were successfully prepared and exhibited good permeability and stability. By comparison of the separation efficiencies of the resulting poly(POSS-co-BPADMA) and poly(POSS-co-EDMA) monoliths in capillary electrochromatography (CEC) and capillary liquid chromatography (cLC), it was indicated the former has better column efficiencies for alkylbenzenes, phenols, anilines and PAHs in CEC and cLC than the latter. Particularly, the hybrid poly(POSS-co-BPADMA) monolith is more suitable for separation of PAHs due to pi-pi interaction between the analytes and aromatic rings in the surface of monolithic stationary phase. (C) 2012 Elsevier B.V. All rights reserved.

As human serum is an important source for early diagnosis of many serious diseases, analysis of serum proteome and peptidome has been extensively performed. However, the serum phosphopeptidome was less explored probably because the effective method for database searching is lacking. Conventional database searching strategy always uses the whole proteome database, which is very time-consuming for phosphopeptidome search due to the huge searching space resulted from the high redundancy of the database and the setting of dynamic modifications during searching. In this work, a focused database searching strategy using an in-house collected human serum pro-peptidome target/decoy database (HuSPep) was established. It was found that the searching time was significantly decreased without compromising the identification sensitivity. By combining size-selective Ti (IV)-MCM-41 enrichment, RP-RP off-line separation, and complementary CID and ETD fragmentation with the new searching strategy, 143 unique endogenous phosphopeptides and 133 phosphorylation sites (109 novel sites) were identified from human serum with high reliability.

We developed a reversed-phase monolithic column based enzyme reactor for protein analysis. The effects of buffer solution, porous structure of the column, digestion time and hydrophobicity of solid phase on the digestion performance of enzyme reactor were systematically investigated in terms of the number of identified unique peptides, sequence coverage and the level of missed cleavage as the evaluation indicators. The results demonstrated that the reversed-phase monolithic column based enzyme reactor had high digestion efficiency and might be further extended to the proteome analysis of complex samples.

A butyl-silica hybrid monolithic column for bioseparation by capillary liquid chromatography (cLC) was prepared with butyl methacrylate and alkoxysilanes through a "one-pot" process. The effects of polycondensation temperature, volume percentage of N,N'-dimethylformamide, and content of cetyltrimethylammonium bromide and butyl methacrylate on the morphologies of the hybrid monolithic columns prepared were investigated in detail. Baseline separations of proteins and small peptides on the hybrid monolithic column were achieved by cLC with gradient elution. In addition, the resulting hybrid column was also applied for analysis of tryptic digests of bovine serum albumin by cLC coupled with tandem mass spectrometry. The results demonstrate its potential application in separation of complex biological samples.

Hybrid organic-silica monolithic columns, regarded as a second generation of silica-based monoliths, have received much interest due to their unique properties over the pure silica-based monoliths. This review mainly focuses on development in the fields of preparation of hybrid monolithic columns in a capillary and their application for CEC and capillary liquid chromatography separation, as well as for sample pretreatment of solid-phase microextraction and immobilized enzyme reactor since July 2010. The preparation approaches are comprehensively summarized with three routes: (i) general solgel process using trialkoxysilanes and tetraalkoxysilanes as coprecursors; (ii) one-pot process of alkoxysilanes and organic monomers concomitantly proceeding solgel chemistry and free radical polymerization; and (iii) other polymerization approaches of organic monomers containing silanes. The modification of hybrid monoliths containing reactive groups to acquire the desired surface functionality is also described.

Protein methylation is receiving more and more attention for its important regulating role in diverse biological processes including epigenetic regulation of gene transcription, RNA processing, DNA damage repair, and signal transduction. Global analysis of protein methylation at the proteome level requires the enrichment of methylated peptides with various forms; unfortunately, the immunoaffinity purification method can only enrich a subset of them due to lacking of pan specific antibody. Because methylation does not significantly alter the physicochemical properties of arginine or lysine residues, chemical approach for global methylome analysis is still at infancy. In this study, by exploiting the fact that the methylation on Arg and Lys prohibiting the cleavage by proteases for these sites, we developed an antibody-free method to enrich methylated peptides, which enabled the identification of 887 methylation forms on 768 sites from HepG2 cells. This technique allows the simultaneous analysis of both Lys and Arg methylation while it has better performance for the identification of Arg methylation. It should find broad applications in studying methylation regulated biological processes.

Suppressing the background interferences and enhancing the analytes signals are long-term goals in high performance electrospray ionization mass spectrometry (ESI-MS) analyses. We observed that performing electrospray in the presence of a concentrated acetonitrile atmosphere suppresses background interferences and enhances peptide signals. An enclosed nanoESI source was utilized to provide a stable atmosphere of concentrated acetonitrile vapor for high performance ESI-MS analyses. The median MS signal intensity increased by 5 times for a set of 23 BSA tryptic peptides in direct ESI-MS analysis. Further, the number of reproducibly and precisely quantified peptides could be improved 67% in six replicate label-free quantitative proteome analyses by this strategy.

Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti(4+)-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO(2) enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti(4+)-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 μg of a HeLa cell lysate digest. In comparison, ∼ 2000 unique phosphopeptides could be identified by Ti(4+)-IMAC with HFP and close to 3000 by TiO(2). We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti(4+)-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 μg of triple dimethyl labeled MCF-7 digest. This analysis led to the identification of over 9,000 unique phosphorylation sites. The use of three peptide activation methods confirmed that ETD is best capable of sequencing multiply charged peptides. Collectively, our data show that the combination of SCX and Ti(4+)-IMAC is particularly advantageous for phosphopeptides with multiple basic residues.

Nano-bio interface has been paid much attention recently, though with the lack of methodology to predict the potential responses in biological systems such as cells induced by nanomaterials. In this study, we described a proteomic approach to investigate the proteome change in K562 cells exposed to oxidized single-walled carbon nanotubes (o-SWCNTs). 605 proteins were identified by semi-quantitative proteomic analysis (SQPA), including 29 significantly changed proteins with spectra count (SpC) ratios lager than 2 or less than 0.5. Three of them including HBA, CFL1 and LMAN2 were further validated by western blotting. The differential proteins were further classified by Ingenuity Pathways Analysis (IPA) to integrate them into a signaling network. Based on the information by this network, we predict that o-SWCNT treatment activated cell aggregation, decreased cell migration, but had no effect on cell death. And these cellular responses were further experimentally demonstrated. The protein signaling network established in this study would greatly benefit the studies on the bio-applications of o-SWCNTs and their toxicity studies. Our study demonstrated that proteomics could be used as a predicting tool to study nano-bio interface at cellular level.

Although thousands of metal-organic frameworks (MOFs) have been fabricated and widely applied in gas storage/separations, adsorption, catalysis, and so on, few kinds of MOFs have been used as adsorption materials while simultaneously serving as matrixes to analyze small molecules for laser desorption/ionization mass spectrometry (LDI-MS). Herein, a new concept is introduced to design and synthesize MOFs as both adsorption materials and matrixes according to the structure of ligands and common matrixes. The proof of concept design was demonstrated by selection of 2,5-pyridinedicarboxylic acid (PDC) and 2,5-dihydroxyterephthalic acid (DHT) as ligands for synthesis of MOFs. Two Zr(IV)-based MOFs of UiO-66-PDC and UiO-66-(OH)2 were synthesized and applied for the first time as new matrixes for analysis of small molecules by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Both of them showed low matrix interferences, high ionization efficiency, and good reproducibility when used as matrixes. A variety of small molecules, including saccharides, amino acids, nucleosides, peptides, alkaline drugs, and natural products, were analyzed. In addition, UiO-66-(OH)2 exhibited potential for application in the quantitative determination of glucose and pyridoxal 5'-phosphate. Furthermore, thanks to its intrinsically large surface area and highly ordered pores, UiO-66-(OH)2 also showed sensitive and specific enrichment of phosphopeptides prior to MS analysis. These results demonstrated that this strategy can be used to efficiently screen tailor-made MOFs as matrixes to analyze small molecules by MALDI-TOF-MS.

During cell division, accurate chromosome segregation is tightly regulated by Polo-like kinase 1 (PLK1) and opposing activities of Aurora B kinase and protein phosphatase 1 (PP1). However, the regulatory mechanisms underlying the aforementioned hierarchical signaling cascade during mitotic chromosome segregation have remained elusive. Sds22 is a conserved regulator of PP1 activity, but how it regulates PP1 activity in space and time during mitosis remains elusive. Here we show that Sds22 is a novel and cognate substrate of PLK1 in mitosis, and the phosphorylation of Sds22 by PLK1 elicited an inhibition of PP1-mediated dephosphorylation of Aurora B at threonine 232 (Thr232) in a dose-dependent manner. Overexpression of a phosphomimetic mutant of Sds22 causes a dramatic increase in mitotic delay, whereas overexpression of a non-phosphorylatable mutant of Sds22 results in mitotic arrest. Mechanistically, the phosphorylation of Sds22 by PLK1 strengthens the binding of Sds22 to PP1 and inhibits the dephosphorylation of Thr232 of Aurora B to ensure a robust, error-free metaphase-anaphase transition. These findings delineate a conserved signaling hierarchy that orchestrates dynamic protein phosphorylation and dephosphorylation of critical mitotic regulators during chromosome segregation to guard chromosome stability.

Although many affinity adsorbents have been developed for phosphopeptides enrichment, high-specifically capturing the multi-phosphopeptides is still a big challenge. Here, we investigated the mechanism of phosphate ion coordination and substitution on affinity adsorbents surfaces and modulated the selectivity of affinity adsorbents to multi-phosphopeptides based on the different capability of mono- and multi-phosphopeptides in competitively substituting the pre-coordinated phosphate ions at strong acidic condition. We demonstrated both the species of pre-coordinated phosphate ions and the substituting conditions played crucial roles in modulating the enrichment selectivity to multi-phosphopeptides, and the pre-coordinated affinity materials with relative more surfaces positive charges exhibited better enrichment efficiency due to the cooperative effect of electrostatic interaction and competitive substitution. Finally, an enrichment selectivity of 85% to multi-phosphopeptides was feasibly achieved with 66% improvement in identification numbers for complex protein sample extracted from HepG2 cells. Data are available via ProteomeXchange with identifier PXD004252.

In this study, we developed a Ti(IV) monolithic spin tip for phosphoproteome analysis of a minute amount of biological sample for the first time. The surface of polypropylene pipet tip was activated by the photoinitiator benzophenone under UV light radiation followed by polymerization of ethylene glycol methacrylate phosphate and bis-acrylamide in the tip to form a porous monolith with reactive phosphate groups. The as-prepared tips grafted with monolithic adsorbent were then chelated with titanium(IV) ion for phosphopeptide enrichment. It was found that the tips enabled fast and efficient capture of phosphopeptides from microscale complex samples. The monolithic tip was demonstrated to have a detection limit as low as 5 fmol β-casein tryptic digest, along with an exceptionally high specificity to capture phosphopeptides from complex tryptic digest mixed with an unphosphorylated protein and a phosphorylated protein at a molar ratio up to 1000:1. When the tip was applied to enrich phosphopeptides from 5 μg of tryptic digest of complex HeLa cell proteins, 1185 high confidence of phosphorylated sites were successfully identified with the specificity as high as 92.5%. So far, this is the most sensitive phosphoproteomics analysis using a standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for proteome-wide phosphorylation analysis in mammalian cells.

In cancer cells, the mammalian target of rapamycin complex 1 (mTORC1) that requires hormonal and nutrient signals for its activation, is constitutively activated. We found that overexpression of pyruvate kinase M2 (PKM2) activates mTORC1 signaling through phosphorylating mTORC1 inhibitor AKT1 substrate 1 (AKT1S1). An unbiased quantitative phosphoproteomic survey identified 974 PKM2 substrates, including serine202 and serine203 (S202/203) of AKT1S1, in the proteome of renal cell carcinoma (RCC). Phosphorylation of S202/203 of AKT1S1 by PKM2 released AKT1S1 from raptor and facilitated its binding to 14-3-3, resulted in hormonal- and nutrient-signals independent activation of mTORC1 signaling and led accelerated oncogenic growth and autophagy inhibition in cancer cells. Decreasing S202/203 phosphorylation by TEPP-46 treatment reversed these effects. In RCCs and breast cancers, PKM2 overexpression was correlated with elevated S202/203 phosphorylation, activated mTORC1 and inhibited autophagy. Our results provided the first phosphorylome of PKM2 and revealed a constitutive mTORC1 activating mechanism in cancer cells.

This study investigated the relationship between peptide profiles and the bioactivity character of yogurt in simulated gastrointestinal trials. A total of 250, 434 and 466 peptides were identified by LC-MS/MS analyses of yogurt, gastric digest and pancreatic digest. Forty peptides of yogurt survived in gastrointestinal digestion. κ-CN and β-CN contributed the diversity of peptides during the fermentation process and gastrointestinal digestion, respectively. The favorite of κ-CN by lactic acid bacteria complemented gut digestion by hydrolyzing κ-CN, the low abundance milk proteins. The potential bioactivities were evaluated by in vitro ACE and DPP-IV inhibition assays. The ACE inhibition rate of the pancreatic digests was ~ 4 - and ~ 2 - fold greater than that of yogurt and the gastric digests. The ACE inhibitory peptides generated during gastrointestinal digestion improved the ACE inhibitory activity of the gastric and pancreatic digests. The DPP-IV inhibition rate of the pancreatic digest was ~ 6 - and ~ 3 - fold greater than that of yogurt and the gastric digest. The numbers of potential DPP-IV inhibitory peptides were positively correlated to the DPP-IV inhibitory activity of the gastric and pancreatic digests. Biological significance The present study describes the characters and bioactivities of peptides from yogurt in a simulated gastrointestinal digestion. The number of peptides identified from yogurt and gastrointestinal digests by LC-MS/MS increased in the simulated gastrointestinal trials. The in vitro ACE and DPP-IV inhibition bioactivities revealed that the bioactivity of yogurt was enhanced during gastrointestinal digestion. The correlation between peptides and bioactivity in vitro indicated that not only the peptides amount but also the proportion of peptides with high bioactivities contributed to increased bioactivity during gastrointestinal digestion. The study of peptides identified from yogurt and digests revealed that the number of released peptides was not determined by the abundance of the parent proteins but by whether the enzymes favored the protein. In summary, peptide profiling and bioactivities of yogurt in simulated gastrointestinal digestion helped to elucidate the health benefits of yogurt peptides. The results further revealed that pre-digestion of milk by lactic acid bacteria are complementary to generate bioactive peptides and to provide particular yogurt functions.

Nucleic acid associated proteins (NAaP) play the essential roles in gene regulation and protein expression. The global analysis of cellular NAaP would give a broad insight to understand the interaction between nucleic acids and the associated proteins, such as the important proteinous regulation factors on nucleic acids. Proteomic analysis presents a novel strategy to investigate a group of proteins. However, the large scale analysis of NAaP is yet impossible due to the lack of approaches to harvest target protein groups with a high efficiency. Herein, a simple and efficient method was developed to collect cellular NAaP using magnetic oxidized carbon nanotubes based on the strong interaction between carbon nanotubes and nucleic acids along with corresponding associated proteins. We found that the magnetic oxidized carbon nanotubes demonstrated a nearly 100% extraction efficiency for intracellular nucleic acids from cells in vitro. Importantly, the proteins associated on nucleic acids could be highly efficiently harvested using magnetic oxidized carbon nanotubes due to the binding of NAaP on nucleic acids. 1594 groups of nuclear NAaP and 2595 groups of cellular NAaP were extracted and identified from about 1,000,000 cells, and 803 groups of NAaP were analyzed with only about 10,000 cells, showing a promising performance for the proteomic analysis of NAaP from minute cellular samples. This highly efficient extraction strategy for NAaP is a simple approach to identify cellular nucleic acid associated proteome, and we believed this strategy could be further applied in systems biology to understand the gene expression and regulation.

As the altered glycosylation expressions of cell surface proteins are associated with many diseases, glycoproteomics approach has been widely applied to characterization of surface glycosylation alteration. In general, the abundances of proteolytic glycopeptides derived from corresponding glycoproteins can be measured to determine the abundances of glycoproteins. However, this quantification strategy cannot distinguish whether the changes are results from changes of protein abundance or changes in glycosite occupancy. For the accurate and specific quantification of the cell surface glycosylation profile, we proposed a modified cell surface-capturing strategy where the glycopeptides were submitted to LC-MS/MS analysis directly for identification of glycoproteins and the non-glycopeptides were isotopically labelled for quantification of glycoproteins. This strategy was applied to comparatively analyze cell surface glycoproteins of two human cell lines, i.e. Chang Liver and HepG2 cells. Totally 341 glycoproteins were identified with 82.4% specificity for cell membrane proteins and 33 glycoproteins were quantified with significant expression change between the two cell lines. The differential expressions of two selected proteins (EMMPRIN and BCAM) were validated by Western blotting. This method enables specific and accurate analysis of the cell surface glycoproteins and may have broad application in the field of biomarker and drug target discovery.

Various enzyme reactors and online enzyme digestion strategies have been developed in recent years. These reactors greatly enhanced the detection sensitivity and proteome coverage in qualitative proteomics. However, these devices have higher rates of miscleavage in protein digestion. Therefore, we investigated the effect of online enzyme digestion on the quantification accuracy of quantitative proteomics using chemical or metabolic isotope labeling approaches. The incomplete digestion would introduce some unexpected variations in comparative quantification when the samples are digested and then chemically isotope labeled in different aliquots. Even when identical protein aliquots are processed on these devices using post-digestion chemical isotope labeling and the CVs of the ratios controlled to less than 50% in replicate analyses, about 10% of the quantified proteins have a ratio greater than two-fold, whereas in theory the ratio is 1:1. Interestingly, the incomplete digestion with enzyme reactor is not a problem when metabolic isotope labeling samples were processed because the proteins are isotopically labeled in vivo prior to their simultaneous digestion within the reactor. Our results also demonstrated that both high quantification accuracy and high proteome coverage can be achieved in comparative proteome quantification using online enzyme digestion even when a limited amount of metabolic isotope labeling samples is used (1683 proteins comparatively quantified from 10(5) Hela cells).

The Ser/Thr protein kinases fall into three major subgroups, pro-directed, basophilic, and acidophilic, on the basis of the types of substrate sequences that they preferred. Despite many phosphoproteomics efforts that have been taken for global profiling of phosphopeptides, methodologies focusing on analyzing a particular type of kinase substrates have seldom been reported. Selective enrichment of phosphopeptides from basophilic kinase substrates is difficult because basophilic motifs are cleaved by trypsin during digestion. In this study, we develop a negative enrichment strategy to enhance the identification of basophilic kinase substrates. This method is based on an observation that high pH strong anion exchange (SAX) chromatography can separate tryptic phosphopeptides according to the number of acidic amino acidic residues that they have. Thus, SAX was applied to deplete acidic phosphopeptides from the phosphopeptide mixture, which improved the coverage for the detection of basophilic kinase substrates. The SAX depletion approach was further combined with online SCX-RP separation for large-scale analysis of mouse liver phosphoproteome, which resulted in the identification of 6944 phosphorylated sites. It was found that motifs associated with basophilic kinases prevail for these identified phosphorylated sites.

The human serum proteome is closely associated with the state of the body. Endogenous peptides derived from proteolytic enzymes cleaving on serum proteins are widely studied due to their potential application in disease-specific marker discovery. However, the reproducibility of peptidome analysis of endogenous peptides is significantly influenced by the proteolytic enzymes within body fluids, thereby limiting the clinical use of the endogenous peptides. We comprehensively investigated the N and C terminus of endogenous peptides using peptidomics. The cleavage site patterns of the N and C terminus and adjacent sites from all the identified endogenous peptides were highly conserved under different sample preparation conditions, including long-term incubation at 37°C and pretreatment with repeated freeze-thaw cycles. Furthermore, a distinguishable cleavage site pattern was obtained when a different disease serum was analyzed. The conserved cleavage site pattern derived from proteolytic enzymes holds potential in highly specific disease diagnosis.

Sample handling procedures including protein digestion, glycopeptide enrichment, and deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.

Hydrophobic organicinorganic hybrid monolithic columns were synthesized via thermally initiated free radical polymerization with the confines of 75 mu m id capillary using a polyhedral oligomeric silsesquioxane (poss) reagent containing eight or more methacrylate groups as the crosslinker. three organic functional monomers, butyl methacrylate (buma), lauryl methacrylate (lma) and methacrylic acid (maa), were selected and copolymerized with the poss in the presence of 1-propanol and 1,4-butanediol to prepare the poly(poss-co-buma), poly(poss-co-lma), and poly(poss-co-maa) monoliths, respectively. the 2-acrylamido-2-methyl-1-propanesulfonic acid (amps) was copolymerized as ionizable monomer into the poly(poss-co-buma) and poly(poss-co-lma) for the generation of eof in capillary electrochromatography (cec). a hybrid poly(poss-co-lma-co-maa) monolith was also similarly prepared by copolymerizing ternary monomers of poss, lma, and maa, and compared with poly(poss-co-buma), poly(poss-co-lma), and poly(poss-co-maa) monoliths. the resulting four kinds of poss-contained hybrid monoliths exhibited good permeability and mechanical stability. their column efficiencies were evaluated by the separation of alkylbenzene homologues and polar compounds in cec. the results indicated that the highest efficiencies of 194?100 and 102?100 theoretical plates per meter for thiourea and benzene were obtained on the poly(poss-co-lma-co-maa) monolith. additionally, the poly(poss-co-lma-co-maa) monolith exhibited better selectivity for separation of polar compounds than those of other hybrid monoliths.

Complete coverage of all phosphorylation sites in a proteome is the ultimate goal for large-scale phosphoproteome analysis. However, only making use of one protease trypsin for protein digestion cannot cover all phosphorylation sites, because not all tryptic phosphopeptides are detectable in MS. To further increase the phosphoproteomics coverage of HeLa cells, we proposed a tandem digestion approach by using two different proteases. By combining the data set of the first Glu-C digestion and the second trypsin digestion, the tandem digestion approach resulted in the identification of 8062 unique phosphopeptides and 8507 phosphorylation sites in HeLa cells. The conventional trypsin digestion approach resulted in the identification of 3891 unique phosphopeptides and 4647 phosphorylation sites. It was found that the phosphorylation sites identified from the above two approaches were highly complementary. By combining above two data sets, in total we identified 10899 unique phosphopeptides and 11262 phosphorylation sites, corresponding to 3437 unique phosphoproteins with FDR < 1% at peptide level. We also compared the kinase motifs extracted from trypsin, Glu-C, or a second trypsin digestion data sets. It was observed that basophilic motifs were more frequently found in the trypsin and the second trypsin digestion data sets, and the acidic motifs were more frequently found in the Glu-C digestion data set. These results demonstrated that our tandem digestion approach is a good complement to the conventional trypsin digestion approach for improving the phosphoproteomics analysis coverage of HeLa cells.

A simple single-step thermal-treatment "one-pot" approach for the preparation of organic-silica hybrid capillary monolithic columns is described. In this improved method, the cross-linker vinyltrimethoxysilane (VTMS) was replaced by 3-methacryloxypropyltrimethoxysilane (γ-MAPS), which is more active in polymerization reactions, and only one thermal treatment step was required in the preparation of hybrid monoliths. Two zwitterionic organic-silica monolithic columns were successfully synthesized by using [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (MSA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) as the organic monomers. The effects of the tetramethoxysilane (TMOS)/γ-MAPS molar ratio, content of monomer, composition of porogenic solvent, and reaction temperature on the morphologies of the hybrid monoliths were investigated. The MSA-silica and MPC-silica hybrid monolithic columns exhibited good permeability and good mechanical stability. The monolithic columns were used for the separation of polar compounds by capillary hydrophilic-interaction chromatography (cHILIC). A typical HILIC retention mechanism was observed at higher organic solvent contents (>50% ACN). The MSA monoliths were further investigated in the separation of various neutral, basic, and acidic analytes, as well as small peptides, by capillary liquid chromatography (cLC), and high efficiency and satisfactory reproducibility were achieved. In addition, the analysis of a tryptic digest of bovine serum albumin (BSA) by cLC tandem mass spectrometry (cLC-MS/MS) with an MSA monolith further demonstrated its potential in the separation of biological samples.

A phenyl-silica hybrid monolithic column for capillary liquid chromatography (cLC) was prepared through “one-pot” process by con-currently using benzyl methacrylate and alkoxysilanes. The effects of the molar ratio of tetramethoxysilane/vinyltrimethoxysilane (TMOS/VTMS), polycondensation temperature, content of supramolecule template (cetyltrimethylammonium bromide, CTAB), ratio of N,N′-dimethylformamide/methanol (v/v), the volume of benzyl methacrylate on the morphologies of the prepared phenyl-silica hybrid monolithic columns were investigated in detail. The permeability of the hybrid monolithic column was calculated as 3.23 × 10 −13 m 2 , and the minimum plate height was determined as 8.38 μm which corresponding to 119,000 theoretical plates per meter. Separation of various neutral, polar and basic analytes as well as small peptides on the hybrid monolithic column was achieved by cLC and showed high efficiency and satisfactory reproducibility. Moreover, the prepared hybrid monolithic column was also applied for the analysis of tryptic digests of bovine serum albumin (BSA), ovalbumin, α-casein, cytochrome C and myoglobin by cLC tandem mass spectrometry (cLC–MS/MS), and the results showed that the separation performance was close to that of the octadecylsilane (C18) packed capillary column which demonstrating its potential in proteome analysis. Moreover, since the prepolymerization system was mainly consisted of organic solvents (methanol and N,N′-dimethylformamide), various hydrophobic monomers could be potentially used to prepare organic-silica hybrid monolithic columns through “one-pot” approach.

Nitrogen (N) is an important nutrient and signal for plant growth and development. However, to date, our knowledge of how plants sense and transduce the N signals is very limited. To better understand the molecular mechanisms of plant N responses, we took two-dimensional gel-based proteomic and phosphoproteomic approaches to profile the proteins with abundance and phosphorylation state changes during nitrate deprivation and recovery in the model plant Arabidopsis thaliana. After 7-day-old seedlings were N-deprived for up to 48 h followed by 24 h recovery, a total of 170 and 38 proteins were identified with significant changes in abundance and phosphorylation state, respectively. Bioinformatic analyses implicate these proteins in diverse cellular processes including N and protein metabolisms, photosynthesis, cytoskeleton, redox homeostasis, and signal transduction. Functional studies of the selected nitrate-responsive proteins indicate that the proteasome regulatory subunit RPT5a and the cytoskeleton protein Tubulin alpha-6 (TUA6) play important roles in plant nitrate responses by regulating plant N use efficiency (NUE) and low nitrate-induced anthocyanin biosynthesis, respectively. In conclusion, our study provides novel insights into plant responses to nitrate at the proteome level, which are expected to be highly useful for dissecting the N response pathways in higher plants and for improving plant NUE.

Direct mass spectrometric analysis of aberrant protein glycosylation is a challenge to the current analytical techniques. Except lectin affinity chromatography, no other glycosylation enrichment techniques are available for analysis of aberrant glycosylation. In this study, we developed a combined chemical and enzymatic strategy as an alternative for the mass spectrometric analysis of aberrant glycosylation. Sialylated glycopeptides were enriched with reverse glycoblotting, cleaved by endoglycosidase F3 and analyzed by mass spectrometry with both neutral loss triggered MS3 in collision induced dissociation (CID) and electron transfer dissociation (ETD). Interestingly, a great part of resulted glycopeptides were found with fucose attached to the N-acetylglucosamine (N-GlcNAc), which indicated that the aberrant glycosylation that is carrying both terminal sialylation and core fucosylation was identified. Totally, 69 aberrant N-glycosylation sites were identified in sera samples from hepatocellular carcinoma (HCC) patients. Following the identification, quantification of the level of this aberrant glycosylation was also carried out using stable isotope dimethyl labeling and pooled sera sample from liver cirrhosis and HCC was compared. Six glycosylation sites demonstrated elevated level of aberrancy, which demonstrated that our developed strategy was effective in both qualitative and quantitative studies of aberrant glycosylation.

It is well known that some discontinuous Bi particles and voids often appear at the interfaces for the SnBi/Cu couples, leading to the interfacial embrittlement during the thermo-aging procedure. In the current study, the experimental results confirmed that Bi segregation at the SnBi/Cu interface mainly originated from the Bi diffusion during reflowing and aging procedure. Small amounts of Ag, Al, Sn or Zn elements were deliberately added into the Cu substrate to restrain the interfacial Bi embrittlement of the SnBi/Cu couples. It does not only restrain the Bi segregation, but also eliminate the formation of voids at the interfaces. Besides, it is interesting to find that the addition of these elements above can significantly improve the mechanical properties of SnBi/Cu alloy joints even after long-term aging. Based on the experimental evidence, it is suggested that alloying Cu substrate is beneficial to the wide application of SnBi and other lead-free solders, and has the potential of being a common substrate in the electronic packaging field in the future.

A novel silica-based stationary phase with branched octadecyl groups was prepared by the sequential employment of the Michael addition reaction and photoinduced thiol-yne click chemistry with 3-aminopropyl-functionalized silica microspheres as the initial material. The resulting stationary phase denoted as SiO2 -N(C18)4 was characterized by elemental analysis, FTIR spectroscopy and Raman spectroscopy, demonstrating the existence of branched octadecyl groups in silica microspheres. The separations of benzene homologous compounds, acid compounds and amine analogues were conducted, demonstrating mixed-mode separation mechanism on SiO2 -N(C18)4 . Baseline separation of basic drugs mixture was acquired with the mobile phase of acetonitrile/H2 O (5%, v/v). SiO2 -N(C18)4 was further applied to separate Corydalis yanhusuo Wang water extracts, and more baseline separation peaks were obtained for SiO2 -N(C18)4 than those on Atlantis dC18 column. It can be expected that this new silica-based stationary phase will exhibit great potential in the analysis of basic compounds.

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 A 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.

A simple approach was developed for rapid preparation of polymeric monolithic columns in UV-transparent fused-silica capillaries via photoinitiated thiol-acrylate polymerization of pentaerythritol diacrylate monostearate (PEDAS) and trimethylolpropane tris(3-mercaptopropionate) (TPTM) within 10min, in which the acrylate homopolymerized and copolymerized with the thiol simultaneously. The morphology, permeability and chromatographic performance of the resulting poly(PEDAS-co-TPTM) monoliths were studied. It could be observed from SEM that the morphology of poly(PEDAS-co-TPTM) monolith was rather different from that of poly(PEDAS) monolith, which was fabricated via photo-induced free radical polymerization using PEDAS as the sole monomer. Compared with poly(PEDAS) monolith, poly(PEDAS-co-TPTM) monolith possessed better permeability when they were fabricated under the same preparation conditions. By adjusting the composition of porogenic solvents, poly(PEDAS-co-TPTM) monolith exhibited lower plate heights (15.7-17.7μm) than poly(PEDAS) monolith (19.1-37.9μm) in μLC. In addition, 66 unique peptides were positively identified on poly(PEDAS-co-TPTM) monolith when tryptic digest of four proteins was separated by μLC-MS/MS, demonstrating its potential in proteome analysis.

Deciphering the kinase-substrate relationship is vital for the study of phosphorylation network. The use of immobilized proteins on protein chip as the library for screening of potential kinase substrates is a tried-and-tested method. However, information on phosphorylation sites is lacking and the creation of the library with proteins of whole proteome by recombinant expression is costly and difficult. In this study, a new solid-phase approach by immobilization of proteins from cell lysate onto beads as a protein library for kinase substrate screening was developed. It was found that consensus phosphorylation sites motif for kinase substrates could be accurately determined and hundreds of in vitro kinase substrates and their phosphorylation sites could be identified by using this method.