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Embryonic stem cells (ESCs) are pluripotent stem cells with the ability to self-renew and to differentiate into any cell types of the 3 germ layers. Recent studies have demonstrated that there is a strong connection between mitochondrial function and pluripotency. Here, we report that methyltransferase like (Mettl) 17, identified from the clustered regularly interspaced short palindromic repeats knockout screen, is required for proper differentiation of mouse embryonic stem cells (mESCs). Mettl17 is located in mitochondria through its N-terminal targeting sequence and specifically interacts with 12S mitochondrial ribosomal RNA (mt-rRNA) as well as small subunits of mitochondrial ribosome (MSSUs). Loss of Mettl17 affects the stability of both 12S mt-rRNA and its associated proteins of MSSUs. We further showed that Mettl17 is an S-adenosyl methionine (SAM)-binding protein and regulates mitochondrial ribosome function in a SAM-binding-dependent manner. Loss of Mettl17 leads to around 70% reduction of m4C840 and 50% reduction of m5C842 of 12S mt-rRNA, revealing the first regulator of the m4C840 and indicating a crosstalk between the 2 nearby modifications. The defects of mitochondrial ribosome caused by deletion of Mettl17 lead to the impaired translation of mitochondrial protein-coding genes, resulting in significant changes in mitochondrial oxidative phosphorylation and cellular metabolome, which are important for mESC pluripotency.-Shi, Z., Xu, S., Xing, S., Yao, K., Zhang, L., Xue, L., Zhou, P., Wang, M., Yan, G., Yang, P., Liu, J., Hu, Z., Lan, F. Mettl17, a regulator of mitochondrial ribosomal RNA modifications, is required for the translation of mitochondrial coding genes.

Molecular weights of seven poly(phenylene ethynylene)-based water-soluble conjugated polyelectrolytes (CPEs) obtained through Sonogashira coupling are determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A standard sample preparation protocol is developed to characterize the seven CPEs using 2,5-dihydroxybenzoic acid as the matrix (M) and AgTFA as the cationization reagent (CR). High-quality MALDI mass spectra are obtained at volume mixing ratios (CPE/M/CR) of 5/5/1 for anionic polymers (P1–P4) and 5/50/1 for cationic polymers (P5–P7). Molecular weight, molecular weight distribution, and end-group information are analyzed. The effects of molecular weight of CPEs on optical and quenching properties are also studied. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017

RATIONALE Extractive electrospray ionization mass spectrometry (EESI-MS) was invented as a typical ambient mass spectrometry method (AMS) and has been used for analyzing complex liquid samples. Here, we designed a Venturi effect-based self-aspiration sampling device and applied it to the EESI-MS for high-throughput analysis of liquid sample. METHODS A special concentric nebulizer was designed and employed to produce a suction force for the direct aspiration of liquid samples, followed by ionization and detection. This sample aspiration process was explained and optimized using computational fluid dynamics (CFD) analysis. Experiment data were recorded to exhibit the sensitivity, memory effect, inter-day reproducibility, throughput, and applicability of the self-aspiration sampling EESI-MS. RESULTS The limit of detection (LOD) of this method was determined as 4.5 × 10(-10) g/mL (S/N = 3) for caffeine, and the sample throughput and relative standard deviation (RSD) for full scan mode can reach 0.67 samples/s and 4.76%, respectively. Even for MS/MS mode, the frequency can still be kept at 0.4 samples/s (RSD = 4.71%). Inter-day RSD examined in 1 week was below 10% for the signal of characteristic fragment ions of reserpine. Moreover, based on this method, the amount of caffeine in instant coffee was determined as 4.7%. This device was also proven to be suitable for the protein/peptide analysis. CONCLUSIONS These experiment results, especially the amazing results on sample throughput and inter-day RSD, suggest that we provide a valuable device which can be used for the direct high-throughput qualitative/quantitative mass spectrometry analysis of real liquid samples in ambient. Copyright © 2016 John Wiley & Sons, Ltd.

Rationale Because the ratio of tripeptide glutathione (GSH) to glutathione disulphide (GSSG) is tightly associated with the oxidative stress and antioxidant level of an organism, it is often considered to be an indicator of the redox states of cells. Therefore, developing an integrated protocol for rapid, efficient, and low-cost detection of GSH to measure antioxidant ability is of significant importance for the diagnosis of oxidative stress-associated diseases. Methods TiO2-Au/graphene nanocomposites were synthesized to integrate the characteristics of graphene as matrix for MALDI-MS, gold NPs as selective probes for GSH and the photocatalytic property of TiO2. Under UV–visible (UV–Vis) light irradiation, OH can be produced by the TiO2-Au/G composites, which can oxidize GSH to form GSSG. When various antioxidants are introduced into the aforementioned system, OH can be scavenged, thereby leaving part of GSH in its reductive format. Based on the ratio of GSH/GSSG, the antioxidative capability of various beverages and fruits can be determined. Results TiO2-Au/G composites were employed to enrich GSH, where 0.01 mg/mL GSH can be efficiently extracted by the nanocomposites from aqueous solution and detected by MS with high signal-to-noise ratio. The proposed strategy was applied to evaluate three often-used antioxidants: Vitamin C, Vitamin E, and β-carotene; and demonstrated that the antioxidative ability of VC is the strongest. To further evaluate the feasibility of the proposed strategy for antioxidative ability evaluation of complex sample, commercial juices and fresh fruit were also studied. Conclusions A novel strategy for sensitive and fast characterization of the antioxidative ability of various beverages and fruits was developed based on TiO2-Au/G nanocomposites coupled with MALDI-MS. Copyright © 2016 John Wiley & Sons, Ltd.

Summary Aims In our previous study, eEF1A1 was identified to be a new target for protecting brain ischemia injury, but the mechanism remains largely unknown. In this study, we screened the downstream cellular protein molecules interacted with eEF1A1 and found mechanism of eEF1A1 in brain ischemia protection. Methods and results Through co-immunoprecipitation and mass spectrometry for searching the interaction of proteins with eEF1A1 in bEnd3 cells, HSC70 was identified to be a binding protein of eEF1A1, which was further validated by Western blot and immunofluorescence. eEF1A1 or HSC70 knockdown, respectively, increased OGD-induced apoptosis of brain vascular endothelial cells, which was detected by Annexin V-FITC/PI staining. HSC70 or eEF1A1 knockdown enhances phosphorylated JNK, phosphorylation of c-JUN (Ser63, Ser73), cleaved caspase-9, and cleaved caspase-3 expression, which could be rescued by JNK inhibitor. Conclusion In summary, our data suggest that the presence of chaperone forms of interaction between eEF1A1 and HSC70 in brain vascular endothelial cells, eEF1A1 and HSC70 can play a protective role in the process of ischemic stroke by inhibiting the JNK signaling pathway activation.

In this work, polydopamine-coated magnetic graphene (MG@PDA) nanocomposites were synthesized by a facile method. Trypsin was then directly immobilized on the surface of the nanocomposites through simple PDA chemistry with no need for introducing any other coupling groups. The as-made MG@PDA nanocomposites inherit not only the large surface area of graphene which makes them capable of immobilizing high amount of trypsin (up to 0.175 mg/mg), but also the good hydrophilicity of PDA which greatly improves their biocompatibility. Moreover, the strong magnetic responsibility makes them easy to be separated from the digested peptide solution when applying a magnetic field. The feasibility of the trypsin-immobilized MG@PDA (MG@PDA-trypsin) nanocomposites for protein digestion was investigated and the results indicated their high digestion efficiency in a short digestion time (10 min). In addition, the reusability and stability of the MG@PDA-trypsin nanocomposites were also tested in our work. To further confirm the efficiency of MG@PDA-trypsin nanocomposites for proteome analysis, they were applied to digest proteins extracted from skimmed milk, followed by nano RPLC-ESI-MS/MS analysis, and a total of 321 proteins were identified, much more than those obtained by 16-h in-solution digestion (264 proteins), indicating the great potential of MG@PDA-trypsin nanocomposites as the supports for high-throughput proteome study.

In this study, heteroatomic ferrisilicalite-1 zeolite monolith (FS-1 ZM) was developed for affinitive recognition of intracellular phosphorylated protein in Tetrahymena thermophila after exposure to organoarsenic feed additives (roxarsone, ROX; p-arsanilic acid, PASA) and their photodegradates. ROX or PASA can be photodegradated into toxic inorganic arsenicals, which has become a public concern. After T. thermophila being exposed to ROX or PASA and their photodegradates, it is of significance to address intracellular protein phosphorylation alteration. However, it is challenging for effective capture of phosphorylated protein under the interference of arsenicals uptake. Followed by ferrisilicalite-1 zeolite monolith based immobilization process, sodium dodecyl sulfate–polyacrylamide electrophoresis, in-gel enzymatic digestion, and subsequent LC–MS/MS methodology, the integrated protocol was firstly attempted for identification of arsenic-induced phosphorylated protein alteration. It was found there was little impact of ROX or PASA on intracellular phosphorylated protein distribution. Alternatively, phosphorylated 14-3-3ϵ was down-regulated after the cells were exposed to photodegradates of ROX or PASA, which were similar to those following exposure to inorganic arsenicals. Overall, inorganic arsenicals present during the degradative pathways of ROX or PASA affect the signal transduction system of T. thermophila cells. The down-regulation of phosphorylated 14-3-3ϵ would be further studied as potential biomarker for organoarsenicals released into the environment.

Lectin array is becoming important in profiling targeted glycan/glycoprotein, but weak interaction between lectin and glycan causes low sensitivity of the approach. This study aims to develop a bead-based lectin array for improving the sensitivity of glycosylation profiling. Lectins are chemically coupled to fluorescent dye coated microbeads, and glycan-lectin recognition is carried out three dimensionally. The performance of this platform was evaluated, and the LOD of lectin Ricinus communis agglutinin 120 (RCA120) was 50 pg/mL (1 pM) of asialofetuin, providing the bead-based lectin microarray with the highest sensitivity among the reported lectin microarrays. Furthermore, multiplexed assay was performed, which allowed the simultaneous detection of multiple carbohydrate epitopes in a single reaction vessel. The glycosylation patterns of hepatocellular carcinoma associated immunoglobulin G were analyzed, and increased (α-1,6) core fucosylation and (α-2,6) sialylation patterns were observed, which may provide significant clinical evidence for disease diagnosis.

In this work, core-shell magnetic metal organic framework (MOF) microspheres were successfully synthesized by coating magnetite particles with mercaptoacetic acid and subsequent reactions with ethanol solutions of Cu(OAc)2 and benzene-1,3,5-tricarboxylic acid (designated as H3 btc) alternately. The resulting Fe3 O4 @[Cu3 (btc)2 ] possess strong magnetic responsiveness. We applied the novel nanocomposites in the enrichment of low-concentration standard peptides, peptides in MYO and BSA tryptic digests and in human urine in combination with MALDI-TOF MS analysis for the first time. In addition, the Cu3 (btc)2 MOF shells exhibit strong affinity to peptides, thus providing a rapid and convenient approach to the concentration of low-abundance peptides. Notably, peptides at an extremely low concentration of 10 pM could be detected by MALDI-TOF MS after enrichment with the magnetic MOF composites. In brief, the facile synthesis and efficient enrichment process of the Fe3 O4 @[Cu3 (btc)2 ] microspheres make them promising candidates for the isolation of peptides in even complex biological environments.

A novel hierarchically ordered macro/mesoporous silica was functionalized with polyboronic acid (HOMMS@PolyVPBA). This composite possesses the attractive features of high surface area and large accessible porosity, and was applied to enrich glycopeptides. Here the HOMMS material was introduced into glycoproteome for the first time. The HOMMS@PolyVPBA gave remarkable selectivity for glycopeptides even at a low molar ratio of glycopeptides/nonglycopeptides (1:50) with a very rapid enrichment speed (only 10 min) and low detection (1 ng μL-1 ). Therefore the synthetic HOMMS@PolyVPBA might be a promising selective enrichment material for glycoproteome analysis.

A sensitive microchip-based electrochemical immunosensor has been developed for quantification of low level S100B, which is an important biomarker of neurological diseases. Poly(ethyleneimine) (PEI), containing NH2 groups, was firstly modified on the surface of the poly(methyl methacrylate) (PMMA) microchannel to capture monoclonal anti-S100B. Afterward, S100B and second polyclonal anti-S100 were sequentially coupled by antigen-antibody specific interaction, and the alkaline phosphatase conjugates (ALP-conjugates) were bound to the second antibodies in a PMMA microchannnel. Using differential pulse voltammetry (DPV), the immunosensor achieved the detection limit of 0.1 pg mL−1 with the linear concentration range of 0.1 pg mL−1–100 pg mL−1. The results indicate that the proposed strategy not only has high specificity and sensitivity, but also resists nonspecific protein adsorption. The disposable microchip immunosensor shows good promise for clinical screening of important biomarkers and convenient point-of-care diagnostics.

Transplantation of cardiomyocytes derived from stem cells is a promising option for cardiac repair. However, how to obtain efficient cardiomyocytes from stem cells is still a great challenge. Understanding of the mechanism that regulates the cardiac differentiation of stem cells is necessary for the effective induction of cardiomyocytes. A clonal derivative named P19CL6 cells can easily differentiate into cardiomyocytes with 1% dimethyl sulfoxide (DMSO) treatment, which offers a valuable model to study cardiomyocytes differentiation in vitro. In this study, the isobaric tags for relative and absolute quantitation (iTRAQ) proteomics were performed to identify proteins associated with cardiomyocytes differentiation of P19CL6 cells induced by DMSO. Out of 543 non-redundant proteins identified, 207 proteins showed significant changes during differentiation with � 1.2-fold or � 0.83-fold changes cut-offs. Nine proteins were confirmed by the quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis respectively. Notably, broad consistency was well showed between mRNA and protein expression for down-regulation of nucleosome assembly protein 1-like 1 (Nap1l1). Further study revealed that knockdown of Nap1l1 by stable transfection of shRNA vector significantly accelerated DMSO-induced cardiomyocytes differentiation of P19CL6 cells characterized by increases in expression of cardiac specific transcription factors, genes, and proteins (GATA4, MEF-2C, ANP, BNP, cTNT, and b-MHC). Therefore, Nap1l1 is a novel protein that regulates cardiomyocytes differentiation of P19CL6 cells induced by DMSO. J. Cell. Biochem. 113: 3788–3796, 2012. 2012 Wiley Periodicals, Inc.

Lysine acetylation is a dynamic and reversible modification, which has been proved to be a key posttranslational modification in cellular regulation. However, the low amounts of the acetylated proteins could hardly be detected before enrichment. In this study, for the first time, antibody-immobilized magnetic carbonaceous microspheres were developed for selective enrichment of acetylated proteins and peptides. At first, standard proteins composed of acetylated bovine serum albumin, myoglobin, α-casein and ovalbumin were used as model proteins to verify the enrichment efficiency. Then, the synthesized peptide was employed to confirm the selectivity of the method. Besides, the antibody-immobilized magnetic particles were successfully applied to analyze mouse mitochondrial proteins. After database search, 29 acetylated sites in 26 proteins were identi?ed.

Mesoporous carbon nanospheres (MCNs) with small diameters of ≈90 nm are developed as an efficient transmembrane delivery vehicle of an anticancer drug, doxorubicin (DOX). MCNs exhibit a high loading capacity toward DOX due to hydrophobic interactions and the supramolecular π stacking between DOX and the carbonaceous structures, on which the pH-dependent drug release are successfully achieved. Specifically, DOX can be loaded onto MCNs in basic solution and in a physiological pH range, while release occurs in acidic solution in its ionized state. By effective passive and active targeting, MCNs can be readily internalized into HeLa cells, where the carried DOX can be efficiently released in the acidic microenvironment of the tumors for further therapy. The endocytosis and cytotoxicity of DOX@MCNs toward HeLa cells are investigated by confocal microscopy and MTT assay. This smart pH-dependent drug loading and release property of DOX@MCNs makes it possible to reduce the cytotoxicity to normal tissues during circulation in the body since the normal physiological pH is ≈7.4.

Proteolysis affects every protein at some point in its life cycle. Many biomarkers of disease or cancer are stable proteolytic fragments in biological fluids. There is great interest and a challenge in proteolytically modified protein study to identify physiologic protease-substrate relationships and find potential biomarkers. In this study, two human hepatocellular carcinoma (HCC) cell lines with different metastasis potential, MHCC97L, and HCCLM6, were researched with a high-throughput and sensitive PROTOMAP platform. In total 391 proteins were found to be proteolytically processed and many of them were cleaved into persistent fragments instead of completely degraded. Fragments related to 161 proteins had different expressions in these two cell lines. Through analyzing these significantly changed fragments with bio-informatic tools, several bio-functions such as tumor cell migration and anti-apoptosis were enriched. A proteolysis network was also built up, of which the CAPN2 centered subnetwork, including SPTBN1, ATP5B, and VIM, was more active in highly metastatic HCC cell line. Interestingly, proteolytic modifications of CD44 and FN1 were found to affect their secretion. This work suggests that proteolysis plays an important role in human HCC metastasis.

In this paper, magnetic mesoporous silica microspheres with C8-modified interior pore-walls were prepared through a facile one-pot sol-gel coating strategy, and were successfully applied for selective enrichment of endogenous peptides in mouse brain for peptidome analysis. Through the one-pot sol-gel approach with surfactant (CTAB) as a template, tetraethyl orthosilicate (TEOS) and n-ctyltriethoxysilane (C8TEOS) as the precursors, C8-modified magnetic mesoporous microspheres (C8-Fe(3)O(4)@mSiO(2)) consisting magnetic core and mesoporous silica shell with C8-groups exposed in the mesopore channels were synthesized. The obtained microspheres possess highly open mesopores of 3.4 nm, high surface area (162.5 m(2)/g), large pore volume (0.17 cm(3)/g), excellent magnetic responsivity (56.3 emu/g) and good dispersibility in aqueous solution. Based on the abundant surface silanol groups, functional C8 groups and the strong magnetic responsivity of the core-shell C8-Fe(3) O(4) @mSiO(2) microspheres, efficient and fast enrichment of peptides was achieved. Additionally, the C8-Fe(3)O(4)@mSiO(2) microspheres exhibit excellent performance in selective enrichment of endogenous peptides from complex samples that are consist of peptides, large proteins and other compounds, including human serum and mouse brain followed by automated nano-LC-ESI-MS/MS analysis. These results indicate C8-Fe(3)O(4)@mSiO(2) microspheres would be a potential candidate for endogenous peptides enrichment and biomarkers discovery in peptidome analysis.

Graphene nanosheets offer intriguing electronic, thermal, and mechanical properties and are expected to find a variety of applications in high-performance nanocomposite materials. Dispersal of graphene nanosheets in polymer hosts and precise interface control are challenging due to their strong interlayer cohesive energy and surface inertia. Here, an efficient strategy is presented for growing polymers directly from the surface of reduced graphene oxide (GO). This method involves the covalent attachment of Br-containing initiating groups onto the surface of hydrazine hydrate reduced GO via a diazonium addition and the succeeding linking of poly(tert-butyl methacrylate) (PtBMA) chains (71.7 wt % grafting efficiency) via surface-initiated single-electron-transfer living radical polymerization (SET-LRP) to graphene nanosheets. The resulting materials were characterized by using a range of testing techniques and it was proved that polymer chains were successfully introduced to the surface of exfoliated graphene sheets. After grafting with PtBMA, the modified graphene sheets still maintained the separated single layers, and the dispersibility was improved significantly. The method is believed to offer possibilities for optimizing the processing properties and interface structure of graphene–polymer nanocomposites. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011.

This study developed a simple and rapid purification method for plasma membrane with high yields from adherent cells. The plasma membrane (PM) sheets could be absorbed specifically by the cationic silica-magnetite nanocomposites (CSMN) under acidic conditions, and recovered directly in cell-lysis-buffer with no need for precipitation. The binding between CSMN and PM sheets was confirmed by electron microscopy. Western blot analysis demonstrated a >10-fold relative enrichment factor. Up to 422 integral membrane proteins were identified from 10(7) Huh7 cells. Notably, we found 29 Ras family proteins by classification according to their biological functions. The whole enrichment procedure took

In the work, aminophenylboronic acid (APB)-functionalized magnetic mesoporous silica, which holds the attractive features of high magnetic responsivity and large surface area, was developed to enrich glycopeptides. At first, magnetic mesoporous silica nanocomposites were prepared. And then, the nanocomposites were functioned with glycidoxypropyltrimethoxysilane (GLYMO) for boronic acid immobilization. Due to that the boronic acid group on the surface of magnetic mesoporous silica nanocomposites can form tight yet reversible covalent bond with glycopeptides containing cis-1,2-diols groups, the magnetic mesoporous silica nanocomposites were successfully applied to selective enrichment of glycopeptides. APB functionalized magnetic mesoporous silica was also demonstrated to have high selectivity for the glycopeptides in the presence of a 10-fold excess bovine serum albumin (BSA) over horseradish peroxidase (HRP) in the tryptic digest. We also find that magnetic mesoporous silica has better sensitivity in HRP digest compared with that of commercial aminophenylboronic acid-functionalized magnetic nanoparticles beads. The limit of detection for glycopeptides from glycoprotein HRP is about 0.01 ng/µL.

The post translational modifications of histone variants are playing an important role in the structure of chro- matin, the regulation of gene activities and the diagnosis of diseases, and conducting in-depth researches and discovering new sites depend on new and rational analytical methods to some extent. In this work, the combinatorial method of high resolution LTQ-Orbitrap mass spectrometry and multiple enzymes was employed to identify the post translational modifications (PTMs) of histone H4 of human liver cells. The novel methylation site, argnine 67 (R 67), was observed besides some sites reported previously such as lysine 31 (K 31), lysine 44 (K 44), argnine 55 (R 55) and lysine 59 (K 59) in the global domain. Meanwhile, various combinations of acetylation of lysine 5 (K 5), lysine 8 (K 8), lysine 12 (K 12), lysine 16 (K 16) and methylation of lysine 20 (K 20) in the NH2-terminal tails were also identified after the LC-MS/MS analysis of trypsin, Arg-C, Glu-C and chymotrypsin digests.

Novel tools are necessary to explore proteins related to human immunodeficiency virus (HIV) infection. In this work, proteomic and glycoproteomic technology were employed to examine plasma samples from HIV-positive patients. Through comparative proteome analysis of normal and HIV-positive plasma samples, 19 differentially expressed protein spots related to 12 non-redundant proteins were identified by ESI-ion trap MS. Among these, the 130-kDa isoform of α-1-antitrypsin was found to be decreased in HIV-positive patients while another variant with a molecular weight of 40 kDa was increased. SWISS-2-D-PAGE reference gel and protein sequence comparisons of the 40-kDa protein showed homology with α-1-antitrypsin minus the N-terminus, and its identity was further confirmed by 1-D Western blotting and glycoproteomic analysis. In all, our results showed that proteomics and glycoproteomics are powerful tools for discovering proteins related to HIV infection. Furthermore, this 40-kDa variant of α-1-antitrypsin found in the plasma of HIV-positive individuals may prove to be a potentially useful biomarker for anti-HIV research according to bioinformatics analysis.

A novel approach of sequence pattern correlation has been applied to predict an expected amino acid sequence from CID ESI-MS spectra. The proposed approach deduces sequence patterns with no help from known protein database such that it is useful to identify an unknown peptide or new protein. The algorithm applies a cross-correlation to match an experimental CID spectrum with predicted sequence pattern generated from fragmentation information. The fragmentation knowledge of both y-series and other non y-series are utilized to generate the predicted sequence patterns. In contrast to the normal de novo approach, the proposed approach is insensitive to mass tolerance and non-susceptive to spectral integrality with no need for selection of a starting point.

In this work, we report the development of a novel enrichment protocol for peptides by using the microspheres composed of Fe(3)O(4)@nSiO(2) Core and perpendicularly aligned mesoporous SiO(2) shell (designated Fe(3)O(4)@nSiO(2)@mSiO(2)). The Fe(3)O(4)@nSiO(2)@mSiO(2) microspheres possess useful magnetic responsivity which makes the process of enrichment fast and convenient. The highly ordered nanoscale pores (2 nm) and high-surface areas of the microspheres were demonstrated to have good size-exclusion effect for the adsorption of peptides. An increase of S/N ratio over 100 times could be achieved by using the microspheres to enrich a standard peptide, and the application of the microspheres to enrich universal peptides was performed by using myoglobin tryptic digest solution. The enrichment efficiency of re-used Fe(3)O(4)@nSiO(2)@mSiO(2) microspheres was also studied. Large-scale enrichment of endogenous peptides in rat brain extract was achieved by the microspheres. Automated nano-LC-ESI-MS/MS was applied to analyze the sample after enrichment, and 60 unique peptides were identified in total. The facile and low-cost synthesis as well as the convenient and efficient enrichment process of the novel Fe(3)O(4)@nSiO(2)@mSiO(2) microspheres makes it a promising candidate for selectively isolation and enrichment of endogenous peptides from complex biological samples.

We have developed a smart nanodevice for the highly efficient and selective detection of glycoproteins. This polyfunctional device is fabricated through the rational functionalization of macroporous silica foam (MOSF) materials with a boron species (B-MOSF) and amino groups (NH(2)-MOSF), and then the integration of MOSF, B-MOSF and NH(2)-MOSF materials. In such a device, a macroporous structure with very large-pore sizes (diameters approximately 100 nm) and high-pore volumes (0.65 cm(3) g(-1)) is advantageous to efficiently fasten the enzymatic reaction. The targeted specific glycopeptides of the products can be selectively isolated and enriched in B-MOSF through the chemo-affinity between boronic acid and glycol groups, while the non-specific peptides are released to the solutions, or further purified by MOSF and NH(2)-MOSF, which have opposite charges. As a result, the protein digestion and glycol-peptide isolation can be simultaneously achieved in the functionalized macroporous materials in one step, which is a great advantage compared to conventional multi-procedure and time-consuming techniques.

In mammalian cells, when tandem affinity purification approach is employed, the existence of untagged endogenous target protein and repetitive washing steps together result in overall low yield of purified/stable complexes and the loss of weakly and transiently interacting partners of biological significance. To avoid the trade-offs involving in methodological sensitivity, precision, and throughput, here we introduce an integrated method, biotin tagging coupled with amino acid-coded mass tagging, for highly sensitive and accurate screening of mammalian protein-protein interactions. Without the need of establishing a stable cell line, using a short peptide tag which could be specifically biotinylated in vivo, the biotin-tagged target/bait protein was then isolated along with its associates efficiently by streptavidin magnetic microbeads in a single step. In a pulled-down complex amino acid-coded mass tagging serves as "in-spectra" quantitative markers to distinguish those bait-specific interactors from non-specific background proteins under stringent criteria. Applying this biotin tagging coupled with amino acid-coded mass tagging approach, we first biotin-tagged in vivo a multi-functional protein family member, 14-3-3epsilon, which was expressed at close to endogenous level. Starting with approximately 20 millions of 293T cells which were significantly less than what needed for a tandem affinity purification run, 266 specific interactors of 14-3-3epsilon were identified in high confidence.

Given the importance of secreted proteins as a source for early detection and diagnosis of disease, secreted proteins have been arousing considerable attention. However, the analysis of secreted proteins represents a challenge for current proteomic techniques. One of the difficulties in secretomic study is to concentrate proteins from large volume of growth media, particularly, the low abundant and low molecular weight proteins (molecular weight

In the work, 4-mercaptophenylboronic acid (4-MPBA)-functionalized gold nanoparticles were synthesized via a facile approach. At first, gold nanoparticles (about 50 nm) were prepared by a simple and convenient hydrothermal method based on a polyol process. Then, gold nanoparticles were modified with 4-MPBA by the well-known reaction of Au with the thiol groups. The MPBA-functionalized gold nanoparticles were characterized by Fourier transform infrared spectra and UV/Vis adsorption spectra. Due to the fact that the boronic acid group on the surface of 4-MPBA-modified gold particles can form tight yet reversible covalent bonds with glycopeptides containing cis-1,2-diols groups, the MPBA-modified gold nanoparticles were successfully applied to selective enrichment of glycopeptides. Isolation and enrichment of glycopeptides in a standard protein (asialofetuin and horseradish peroxidase) digestion and a complex sample were performed using MPBA-modified gold nanoparticles, followed by matrix-assisted laser desorption/ionization quadruple ion trap time-of-flight (MALDI-QIT-TOF) mass spectrometric analysis. The experimental results demonstrated that MPBA-modified gold nanoparticles synthesized by the facile approach have the powerful potential for selective enrichment of glycopeptides, and can be an alternative tool in glycoproteomics.

A core-satellite-structured composite material has been successfully synthesized for capturing glycosylated peptides or proteins. This novel hybrid material is composed of a silica-coated ferrite "core" and numerous "satellites" of gold nanoparticles with lots of "anchors". The anchor, 3-aminophenylboronic acid, designed for capturing target molecules, is highly specific toward glycosylated species. The long organic chains bridging the gold surface and the anchors could reduce the steric hindrance among the bound molecules and suppress nonspecific bindings. Due to the excellent structure of the current material, the trap-and-release enrichment of glycosylated samples is quite simple, specific, and effective. Indeed, the composite nanoparticles could be used for enriching glycosylated peptides and proteins with very low concentrations, and the enriched samples can be easily separated from bulk solution by a magnet. By using this strategy, the recovery of glycopeptides and glycoproteins after enrichment were found to be 85.9 and 71.6% separately, whereas the adsorption capacity of the composite nanoparticles was proven to be more than 79 mg of glycoproteins per gram of the material. Moreover, the new composite nanoparticles were applied to enrich glycosylated proteins from human colorectal cancer tissues for identification of N-glycosylation sites. In all, 194 unique glycosylation sites mapped to 155 different glycoproteins have been identified, of which 165 sites (85.1%) were newly identified.

A novel strategy based on carboxy group derivatization is presented for specific characterization of phosphopeptides. By tagging the carboxy group with 1-(2-pyrimidyl) piperazine (PP), the ion charge states of phosphopeptides can be largely enhanced, showing great advantages for sequencing phosphorylated peptides with electron-transfer dissociation MS. Besides, after PP-derivatization, most non-specific bindings can be avoided by eliminating the interaction between the carboxy group and TiO 2 , greatly improving the specificity of TiO 2 -based phosphopeptide enrichment strategy. Moreover, being tagged with a hydrophobic group, the retention time of phosphopeptides in RPLC can be prolonged, overcoming the difficulty of separating phosphopeptides in RPLC-based approach. Together with several other advantages, such as ease of handling, rapid reaction time, broad applicability and good reproducibility, this PP-derivatization method is promising for high-throughput phospho-proteome research.

In this report, alternating current-assisted on-plate proteolysis has been developed for rapid peptide mapping. Protein solutions containing trypsin were allowed to digest directly on the spots of a stainless steel MALDI plate with the assistance of low-voltage alternating current electricity. Alternating current (AC) was allowed to pass through the protein solutions via the MALDI plate and a platinum disc electrode. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of BSA and cytochrome c (Cyt-c). It was demonstrated that AC substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min. The digests were identified by MALDI-TOF MS with sequence coverages of 42% (BSA) and 77% (Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion. The present proteolysis strategy is simple and efficient, offering great promise for MALDI-TOF MS peptide mapping.

Sinusoidal alternating voltages (typically 5 V) were employed to enhance the efficiency of proteolysis for peptide mapping in this work. Protein solutions containing trypsin were allowed to digest with the assistance of alternating electric fields (AEFs) between a pair of platinum wire electrodes in Eppendorf tubes. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of several standard proteins. It was demonstrated that AEFs significantly accelerated in-solution proteolysis and the digestion time was substantially reduced to 5 min. The digests were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with sequence coverages that were comparable to those obtained by using conventional 12-h in-solution proteolysis. The suitability of AEF-assisted proteolysis to real protein samples was demonstrated by digesting and identifying human serum albumin in gel separated from human serum by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). The present proteolysis strategy is simple and efficient and will find a wide range of applications in protein identification.

In this report, infrared (IR) radiation was employed to enhance the efficiency of tryptic proteolysis for peptide mapping. Protein solutions containing trypsin in sealed transparent Eppendorf tubes were allowed to digest under an IR lamp at 37 degrees C. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of BSA and myoglobin (MYO) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF MS with the sequence coverages of 69% (BSA) and 90% (MYO) that were much better than those obtained by conventional in-solution tryptic digestion. The present IR-assisted proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.

In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH 3-5 LIEF fraction and the unfractionated sample were separated by pH 3-6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3-5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples.

In this work, we present, to our knowledge, the first demonstration of the utility of iron oxide magnetic microspheres coated with gallium oxide for the highly selective enrichment of phosphopeptide prior to mass spectrometric analysis. These microspheres that we prepared not only have a shell of gallium oxide, giving them a high-trapping capacity for the phosphopeptides, but also their magnetic property enables easy isolation by positioning an external magnetic field. Tryptic digest products of phosphoproteins including beta-casein, ovalbumin, casein, as well as five protein mixtures were used as the samples to exemplify the feasibility of this approach. In very short time (only 0.5 min), phosphopeptides sufficient for characterization by MALDI-TOF-MS were selectively enriched by the Ga(2)O(3)-coated Fe(3)O(4) microspheres. The performance of the Ga(2)O(3)-coated Fe(3)O(4) microspheres were further compared with Fe(3+)-immobilized magnetic silica microspheres, commercial Fe(3+)-IMAC resin, and TiO2 beads for enrichment of peptides originating from tryptic digestion of beta-casein and BSA with a molar ratio of 1:50, and the results proved a stronger selective ability of Ga(2)O(3)-coated Fe(3)O(4) microspheres over the other materials. Finally, the Ga(2)O(3)-coated Fe(3)O(4) microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. All results show that Ga(2)O(3)-coated Fe(3)O(4) microsphere is an effective material for selective isolation and concentration of phosphopeptides.

alpha1,6-Fucose residues within the N-glycan core structures were commonly observed in many glycoproteins. Our previous studies showed that aberrantly alpha1,6-fucosylated glycoproteins might be associated with metastasis of hepatocellular carcinoma (HCC). Little is known about human normal liver tissues (HNLTs) in the literatures. In this study, a target glycoproteomic approach which consists of lectin-affinity chromatography, 2-DE, protein immunoprecipitation and lectin blot, and MALDI-MS/MS, was utilized to screen physiologically alpha1,6-fucosylated glycoproteins. Lens culinaris agglutinin (LCA)-affinity glycoprotein profiles of HNLT were established and analyzed, which allowed identification of 53 proteins by MS analysis, including haptoglobin precursor, alpha-enolase, etc. Gene ontology (GO) annotation proved that these proteins distribute predominately in organelle and play crucial roles in binding and catalytic reactions. The present methodology enabled the identification of all the specific subsets of glycoprotein, and the corresponding data could contribute to the finding of more aberrantly alpha1,6-fucosylated glycoproteins related to liver diseases.

A nanoreactor based on mesoporous silicates is described for efficient tryptic digestion of proteins within the mesochannels. Cyano-functionalized mesoporous silicate (CNS), with an average pore diameter of 18 nm, is a good support for trypsin, with rapid in situ digestion of the model proteins, cytochrome c and myoglobin. The generated peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Proteolysis by trypsin-CNS is much more efficient than in-solution digestion, which can be attributed to nanoscopic confinement and concentration enrichment of the substrate within the mesopores. Proteins at concentrations of 2 ng muL(-1) were successfully identified after digestion for 20 min. A biological complex sample extracted from the cytoplasm of human liver tissue was digested by using the CNS-based reactor. Coupled with reverse-phase HPLC and MALDI-TOF MS/MS, 165 proteins were identified after standard protein data searching. This nanoreactor combines the advantages of short digestion time with retention of enzymatic activity, providing a promising way to advance the development of proteomics.

SUMMARY 1 Our previous study showed that Nogo-B was highly expressed in endothelial cells and downregulated in endothelial cells following induction by lysophatidycholine, which contirubted to atherosclerotic lesions. However, the role of Nogo-B in the development of aortic aneurysms remains unclear. 2 In the present study, segments of thoracic aortic aneurysms (TAA) and adjacent normal thoracic aortic tissues (NTA) without aneurysmal changes were obtained from 31 patients undergoing graft surgery. The mRNA and protein expression levels of Nogo-B were measured with semiquantitative reverse transcription–polymerase chain reaction, western blotting and immunohistochemistry. 3 The results demonstrate that Nogo-B mRNA expression levels in TAA lesions decreased to 45% compared with levels in NTA lesions and that protein levels in TAA decreased to 35%. Tissue Nogo immunohistochemical staining in aortic specimens suggested the involvement of Nogo in neovascularization and smooth muscle cell proliferation. The weaker brown staining of endothelial cells in TAA lesions suggested the lower expression of Nogo-B in TAA lesions. 4 These results demonstrate that Nogo-B mRNA and protein expression are downregulated in TAA lesions. It is concluded that the reduction of Nogo-B protein expression in TAA lesions is closely correlated to the formation of aneurysm and that Nogo-B may play a protective role in the pathological process of aneurysms.

We present here an effective technique for the large-scale separation and identification of N-linked glycoproteins from Chang liver cells, the human normal liver cells. To enrich N-linked glycoproteins from the whole cells, a procedure containing the lysis of human liver cells, the solubilization of total proteins, lectin affinity chromatography including Concanavalin A and wheat germ agglutinin was established. Furthermore, captured N-linked glycoproteins were separated by 2-DE, and identified by MS and database searching. Finally, we found 63 N-glycoproteins in Chang liver cells. In addition, using the above method, we identified 7 remarkably up-regulated glycoproteins from MHCC97-H cells, highly metastatic liver cancer cells, compared to Chang liver cells. These up-regulated glycoproteins were associated with liver cancer and might be used as biomarkers for tumor diagnosis. Results showed that we established a high-throughput proteomic analysis for separating N-linked glycoproteins from human liver cells. This strategy greatly improved the glycoprotein analysis method associated with proteome-wide glycosylation changes related to liver cancer. Our work was part of the HUPO Human Liver Proteome Project (HLPP) studies and was supported by CHINA HUPO.

An easily replaceable enzymatic microreactor has been fabricated based on the glass microchip with trypsin-immobilized magnetic silica microspheres (MS microspheres). Magnetic microspheres with small size (approximately 300 nm in diameter) and high magnetic responsivity to magnetic field (68.2 emu/g) were synthesized and modified with tetraethyl orthosilicate (TEOS). Aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were then introduced to functionalize the MS microspheres for enzyme immobilization. Trypsin was stably immobilized onto the MS microspheres through the reaction of primary amines of the proteins with aldehyde groups on the MS microspheres. The trypsin-immobilized MS microspheres were then locally packed into the microchannel by the application of a strong field magnet to form an on-chip enzymatic microreactor. The digestion efficiency and reproducibility of the microreactor were demonstrated by using cytochrome c (Cyt-C) as a model protein. When compared with an incubation time of 12 h by free trypsin in the conventional digestion approach, proteins can be digested by the on-chip microreactor in several minutes. This microreactor was also successfully applied to the analysis of an RPLC fraction of the rat liver extract. This opens a route for its further application in top-down proteomic analysis.

The tegument proteins of schistosome have attracted the most attention in studies of host-parasite interplay, while the host proteins acting at the host-parasite interface remained largely elusive. Here, we undertook a high-throughput proteomic approach to characterize the schistosome-adsorbed host proteins. Fifty five distinct host proteins were confidently identified in S. japonicum samples, including cercaria, schistosomula, adults, eggs, and miracidia, together with tegument and eggshell preparations, of which 23 and 38 host proteins were identified in adult worms and eggs, respectively. Among the schistosome-adsorbed host proteins, host neutrophil elastases were found in the granuloma initiated by schistosome egg deposition, implying that the host innate immune molecules could participate in the granuloma formation for fighting against schistosome invasion, except for the adaptive immune system. In addition, some host proteins, such as proteinase inhibitor and superoxide dismutase, might be utilized by schistosome to counteract or attenuate the host attacks. These parasite-adsorbed host proteins will provide new insights into the host immune responses against schistosome infection, the evasive behavior of the adult worms, and the granuloma formation, which could render an in-depth understanding for the host-parasite interplay.

The current "shotgun" proteomic analysis, strong cation exchange-RPLC-MS/MS system, is a widely used method for proteome research. Currently, it is not suitable for complicated protein sample analysis, like mammal tissues or cells. To increase the protein identification confidence and number, an additional separation dimension for sample fractionation is necessary to be coupled prior to current multi-dimensional protein identification technology (MudPIT). In this work, SEC was elaborately selected and applied for sample prefractionation in consideration of its non-bias against sample and variety of choice of mobile phases. The analysis of the global lysate of normal human liver tissue sample provided by the China Human Liver Proteome Project, were performed to compare the proteome coverage, sequence coverage (peptide per protein identification) and protein identification efficiency in MudPIT, 3-D LC-MS/MS identification strategy with preproteolytic and postproteolytic fractionation. It was demonstrated that 3-D LC-MS/MS utilizing protein level fractionation was the most effective method. A MASCOT search using the MS/MS results acquired by QSTAR(XL) identified 1622 proteins from 3-D LC-MS/MS identification approaches. A primary analysis on molecular weight, pI and grand average hydrophobicity value distribution of the identified proteins in different approaches was made to further evaluate the 3-D LC-MS/MS analysis strategy.

Peptide mapping analysis, utilizing an easily replaceable and regenerable on-column enzymatic microreactor with metal-ion-chelated adsorption of enzyme on magnetic silica microspheres, combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was developed. Firstly, magnetic microspheres of small size and strong magnetism were prepared through solvothermal reaction. Thereafter, by introducing tetraethyl orthosilicate (TEOS), magnetic silica (MS) microspheres were formed. Trypsin could then be immobilized onto the MS microspheres based on the Lewis acid-base interaction through the divalent cation chelators such as iminodiacetic acid (IDA), which was chemically bound to the microspheres through the introduction of glycidoxypropyltrimethoxysilane (GLYMO). The trypsin-immobilized MS microspheres were then locally packed into the capillary by the application of a strong magnetic field using a magnet. The performance of the method was exemplified with digestion of bovine serum albumin for 5 min at 50°C and the result was comparable to the 12 h in-solution digestion. The ability of regeneration of the prepared on-column microreactor and good reproducibility of microreactor before and after regeneration were also demonstrated. Copyright © 2007 John Wiley & Sons, Ltd.

Liver stem/progenitor cells play a key role in liver development and maybe also in liver cancer development. In our previous study a population of c-Kit−(CD45/TER119)− liver stem/progenitor cells in mouse fetal liver, was successfully sorted with large amount (106–107) by using immuno-magnetic microbeads. In this study, the sorted liver stem/progenitor cells were used for proteomic study. Proteins of the sorted liver stem/progenitor cells and unsorted fetal liver cells were investigated using two-dimensional electrophoresis. A two-dimensional proteome map of liver stem/progenitor cells was obtained for the first time. Proteins that exhibited significantly upregulation in liver stem/progenitor cells were identified by peptide mass fingerprinting and peptide sequencing. Nineteen protein spots corresponding to 12 different proteins were identified as showing significant upregulation in liver stem/progenitor cells and seem to play important roles in such cells in cell metabolism, cell cycle regulation, and stress. An interesting finding is that most of the upregulated proteins were overexpressed in various cancers (11 of 12, including 6 in human hepatocellular carcinoma (HCC)) and involved in cancer development as reported in previous studies. Some of the identified proteins were validated by real-time PCR, Western blotting, and immunostaining. Taken together, the data presented provide a significant new protein-level insight into the biology of liver stem/progenitor cells, a key population of cells that might be also involved in liver cancer development. J. Cell. Biochem. 102: 936–946, 2007. © 2007 Wiley-Liss, Inc.