Characterization of efficient proteolysis by trypsin loaded macroporous silica

Tryptic digestion of proteins in trypsin loaded porous silica has been shown to be highly efficient. Enzymatic silica-reactors were prepared by immobilizing trypsin into macroporous ordered siliceous foam (MOSF) and into mesoporous SBA-15 silica which has a smaller pore size. The tryptic products from the silica reactors were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and a higher proteolysis efficiency was obtained with MOSF. These results can be well interpreted by a sequential digestion model taking into account the confinement and concentration enrichment of both the substrates and enzymes within the silica pores. Proteins at low concentrations and proteins in urea and surfactant solutions were also successfully digested with the MOSF-based reactor and identified by MS. Considering that the immobilized trypsin could retain its enzymatic activity for weeks, this MOSF reactor provides many advantages compared to free enzyme proteolysis. As a proof-of-concept, the digest of a real complex sample extracted from the cytoplasm of mouse liver tissue using trypsin loaded MOSF yielded better results than the typical in-solution protocol.     

Characterisation of cisplatin coordination sites in cellular <Emphasis Type="Italic">Escherichia coli</Emphasis> DNA-binding proteins by combined biphasic liquid chromatography and ESI tandem mass spectrometry

Combined multidimensional liquid chromatography and electrospray ionisation tandem mass spectrometry was employed to analyse platinated tryptic peptides from Escherichia coli cells treated with the anticancer drug cis-[PtCl₂(NH₃)₂] at pH 7.0. Prerequisites for the LC/LC/MS/MS analysis of protein targets that are fulfilled by cisplatin are (a) that the original protein binding sites have a high kinetic stability over the range 2.3 < pH < 8.5, and (b) that the metal fragment remains coordinated to a significant number of b⁺ and y⁺ peptide ions under MS/MS fragmentation conditions. Matching the MS/MS spectra of the platinated tryptic peptides to sequences of proteins in the E. coli database enabled the identification of 31 protein targets for cisplatin. Whereas six of these are high-abundance enzymes and ribosomal proteins in E. coli cells, five low-abundance DNA-binding proteins were also identified as specific targets. These include the DNA mismatch repair protein mutS, the DNA helicase II (uvrD) and topoisomerase I (top1). Two efflux proteins (acrD, mdtA), the redox regulator thioredoxin 1 (thiO) and the external filament-like type-1 fimbrial protein A chain (fimA1) were also characterised as specific cisplatin-binding proteins. Kinetically favoured carboxylate (D, E) and hydroxy (S, T, Y) O atoms were identified as the Pt coordination sites in 18 proteins and methionyl S atoms in 9 proteins.     

Functional dual hydrophilic dendrimer‐modified metal‐organic framework for the selective enrichment of N‐glycopeptides

Analysis of protein glycosylation remains a significant challenge due to the low abundance of glycoproteins or N‐glycopeptides. Here we have synthesized an amino‐functionalized metal‐organic framework (MOF) MIL‐101(Cr)‐NH₂ whose surface is grafted with a hydrophilic dendrimer poly(amidoamine) (PAMAM) for N‐glycopeptide enrichment based on the hydrophilic interactions. The selected substrate MOF MIL‐101(Cr) owns high surface area which provides nice support for peptide adsorption. In addition, the MOF displayed a good hydrophilic property after being modified with amino groups. Most importantly, the grafted hydrophilic dendrimer PAMAM was firstly applied in the postsynthetic modification of MOFs. And this functionalization route using macromolecular dendrimer opens a new perspective in MOFs design. Owing to its long dendritic chains and abundant amino groups, our material displayed dual hydrophilic property. In the enrichment of standard glycoprotein HRP digestion, the functional MOF material was shown to have low detection limit (1 fmol/μL) and good selectivity when the concentration of nonglycopeptides was 100 fold higher than the target N‐glycopeptides. All the results proved that MIL‐101(Cr)‐NH₂@PAMAM has great potential in the glycoproteome analysis.     

Analysis of feeding mechanism in a pitcher ofNepenthes hybrida

The process of digestion of captured feeds in a pitcher, an insect-trapping organ, ofNepenthes was studied. Changes in bacterial population, pH and NH₄ ⁺ concentrations in pitcher juice were examined. Strong activities of both acid- and alkaline phosphatase, phosphoamidase, esterase C4 and esterase C8 were found in the pitcher juice. Optimum pH of proteases in the juice and those secreted from bacteria showed pH 3.0 and pH 8.0–9.0, respectively. Twenty six strains of bacteria were isolated from 4 pitchers: 10 strains were gram positive, 16 strains were gram negative (10 strains had casein hydrolase activity). A proton excretion was induced by NH₄ ⁺ released from the added solutions, and accordingly, the pH of the solutions fell. As a simulation model of the digestion process of feeds in pitcher juice and polypeptone solution was added into the washed pitcher. A good correlation was found among the NH₄ ⁺ concentration, pH and bacterial cell titer.     

Gel‐free mass spectrometry analysis of Drosophila melanogaster heads

Membrane proteins play key roles in several fundamental biological processes such as cell signalling, energy metabolism and transport. Despite the significance, these still remain an under‐represented group in proteomics datasets. Herein, a bottom‐up approach to analyse an enriched membrane fraction from Drosophila melanogaster heads using multidimensional liquid chromatography (LC) coupled with tandem‐mass spectrometry (MS/MS) that relies on complete solubilisation and digestion of proteins, is reported. An enriched membrane fraction was prepared using equilibrium density centrifugation on a discontinuous sucrose gradient, followed by solubilisation using the filter‐aided sample preparation (FASP), tryptic and sequential chymotryptic digestion of proteins. Peptides were separated by reversed‐phase (RP) LC at high pH in the first dimension and acidic RP‐LC in the second dimension coupled directly to an Orbitrap Velos Pro mass spectrometer. A total number of 4812 proteins from 114 865 redundant and 38 179 distinct peptides corresponding to 4559 genes were identified in the enriched membrane fraction from fly heads. These included brain receptors, transporters and channels that are most important elements as drug targets or are linked to disease. Data are available via ProteomeXchange with identifier PXD001712 ( http://proteomecentral.proteomexchange.org/dataset/PXD001712 ).     

Synthesis of folic acid functionalized terbium‐doped dendritic fibrous nano‐silica and Interaction with HEK 293 normal,MDA breast cancer and HT 29 colon cancer cells

A novel folic acid functionalized terbium‐doped dendritic fibrous nanoparticle (Tb@KCC‐1‐NH₂‐FA) with high surface area was synthesized using a novel hydrothermal protocol. In the present work, we report the fluorescent Tb‐doted nanomaterial with emission wavelength at 497 nm which confirms the formation of Tb@KCC‐1‐NH₂‐FA. Synthesized nanoparticles were investigated through transmission electron microscope, field emission scanning electron Microscopy, Fourier transform infrared spectra, Brunauer‐Emmett‐Teller, energy dispersive X‐ray, Zeta potential and particle size distribution values and AFM (Atomic force microscopy) techniques. Specially, our desired nanomaterial which has FA moieties on the surface of Tb@KCC‐1‐NH2‐FA where interact with folate receptor (FR) which there is on the surface of the various cancer cells. For this purpose, fluorescence microscopy images were used to prove the uptake of FA based nanomaterial with FR‐positive MDA breast cancer and HT 29 colon cancer cells. Also HEK 293 normal cells as FR‐negative cells verified the specificity of our desired nanomaterial toward the FR‐positive cells. The cytotoxicity survey of Tb@KCC‐1‐NH₂‐FA was examined by MTT assays against MDA breast cancer, HT 29 colon cancer and HEK 293 Normal cell lines which confirmed their biocompatible nature with any significant cytotoxic effects even for concentration higher than 900 μg/mL which could be used as a non‐toxic catalyst or carrier in biological ambient. Hence, Tb@KCC‐1‐NH₂‐FA were synthesized using green and hydrothermal method; the process was simple with good productivity and desired nanocomposite was non‐toxic.     

Ammonium uptake and cellular alkalisation in roots of Arabidopsis thaliana: The involvement of cytoplasmic calcium1

Using roots from Arabidopsis thaliana expressing the recombinant calcium indicator aequorin, we show that NH₃ uptake and alkalisation of plant cells act as a stimulus which induces transient elevations of the cytoplasmic free calcium concentration ([Ca²⁺]_c). The magnitudes of these [Ca²⁺]_c elevations are dependent on the concentration of the membrane permeable form, NH₃, and hence, particularly dependent on the pH in the external medium. EGTA and La³⁺ are able to significantly suppress the [Ca²⁺]_c transients showing that Ca²⁺ influx through the plasma membrane is likely to be involved. Verapamil and nifedipine had no inhibitory effects, which suggests that Ca²⁺ release from internal stores might not contribute significantly to the NH₃‐triggered [Ca²⁺]_c response. Pre‐incubation in l ‐methionine‐dl ‐sulphoximine – an inhibitor of the glutamine synthetase – did not alter the NH₃‐induced [Ca²⁺]_c responses at all. These results are consistent with previous studies where NH₃‐induced changes of cytoplasmic and vacuolar pH were investigated in maize roots. Furthermore, the similarity between the kinetics of NH₃‐driven cellular pH changes demonstrated in previous studies and the [Ca²⁺]_c transients shown here suggests a direct relationship between [Ca²⁺]_c and cellular alkalisation (cytoplasmic pH and/or vacuolar pH). However, the mechanism behind this possible causal relation remains to be elucidated.     

Verification of protein disulfide bond arrangement by in‐gel tryptic digestion under entirely neutral pH conditions

To develop a concise proteomic procedure to verify the protein disulfide bond arrangement, non‐reductive trypsin digestion of neuregulin 1‐β1 (176–246), a model disulfide‐containing protein, was assessed by a proteolytic ¹⁸O‐labeling analysis. As a result, the commonly used in‐gel tryptic digestion method has been improved for use entirely under neutral pH conditions. With this procedure, the disulfide arrangement of proteins could represent a clinical index candidate in pathological proteomic studies.     

Efficient enrichment of phosphopeptides by magnetic TiO₂-coated carbon-encapsulated iron nanoparticles

Titanium dioxide (TiO₂) has been widely used for phosphopeptide enrichment. Several approaches have been reported to produce magnetic TiO₂ affinity probes. In this report, we present a facile approach to immobilize TiO₂ onto poly(acrylic acid)‐functionalized magnetic carbon‐encapsulated iron nanoparticles as affinity probes for efficient enrichment of phosphopeptides. By using the new magnetic TiO₂ affinity probes, denoted as TiO₂‐coated Fe@CNPs, rapid and effective MALDI‐TOF MS profiling of phosphopeptides was demonstrated in different model systems such as tryptic digests of β‐casein, and complex β‐casein/BSA mixture. The TiO₂‐coated Fe@CNPs out‐performed the commercial TiO₂‐coated magnetic beads for detection of phosphopeptides from tryptic digests of β‐casein/BSA mixture with a molar ratio of 1:100. The new TiO₂‐coated magnetic probes were also proven to be applicable for real life samples. The magnetic TiO₂‐coated Fe@CNPs were employed to selectively isolate phosphopeptides from tryptic digests of HeLa cell lysates and out‐performed the commercial magnetic TiO₂ beads in the number of identified phosphopeptides and phosphorylation sites. In a 200‐μg equivalent of HeLa cell lysates, we identified 1415 unique phosphopeptides and 1093 phosphorylation sites, indicating the good performance of the new approach.     

Specific interaction of central nervous system myelin basic protein with lipids. Specific regions of the protein sequence protected from the proteolytic action of trypsin

The tryptic hydrolysis of the basic protein of central nervous system myelin (A₁ basic protein) and of A₁ basic-lipid complexes was studied. The tryptic digestion was monitored by “finger printing”, column chromatography and amino acid analysis of the resulting pure peptides.Specific regions of the protein sequence were found to be protected from the hydrolytic action of the trypsin only after the protein was recombined with specific lipids. The degree of protection was in the order: cerebroside sulphate > acidic lipid fraction of myelin > phosphatidylsrine = total lipid extract of myelin. The protected Lys-X, Arg-X bonds were all situated in the region amino acid 20 to amino acid 113 of the intact protein. This region contains the (proline)₃ bend in the protein which is stabilized by interaction with lipids and also the encephalitogenic site for monkey and rabbit.From the results reported in this publication we would like to suggest a specific interaction between a region of the A₁ basic protein molecule and cerebroside sulphate. Differences in A₁ basic protein-lipid interaction in different animals arising from differences in lipid composition and fatty acid composition of the different lipid species combined with minor changes in the protein sequence could explain the species variability of the encephalitogenic sites of the A₁ basic protein.     

The P2 protein of bovine root myelin: partial chemical characterization.

The P2 protein of bovine root myelin has chemical characteristics which differentiate it from other myelin basic proteins. The tryptic peptide map of the bovine P2 protein is distinctly different from the map of either the rabbit PI protein or the bovine CNS myelin basic protein. The tryptic peptides of the P2 protein show no overlap in either map positions or amino acid content with the peptides of the CNS myelin basic protein. Analysis of the individual peptides in the P2 map accounted for all of the amino acids present in the analysis of the whole protein. The P2 protein has a blocked NH₂-terminus, lysine at its COOH-terminus and no hexosamine. CD studies revealed that the P2 protein has a very stable β-structure in aqueous solution at neutral or basic pH and retains much of this structure in acid and in 8 M urea. It is suggested that these structural properties are relevant to the dual role of the P2 protein as a membrane constituent and as an antigen.     

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two‐dimensional electrophoresis

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH₄HCO₃ + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.     

Altered cell wall properties are responsible for ammonium‐reduced aluminium accumulation in rice roots

The phytotoxicity of aluminium (Al) ions can be alleviated by ammonium (NH₄⁺) in rice and this effect has been attributed to the decreased Al accumulation in the roots. Here, the effects of different nitrogen forms on cell wall properties were compared in two rice cultivars differing in Al tolerance. An in vitro Al‐binding assay revealed that neither NH₄⁺ nor NO₃^? altered the Al‐binding capacity of cell walls, which were extracted from plants not previously exposed to N sources. However, cell walls extracted from NH₄⁺‐supplied roots displayed lower Al‐binding capacity than those from NO₃^?‐supplied roots when grown in non‐buffered solutions. Fourier‐transform infrared microspectroscopy analysis revealed that, compared with NO₃^?‐supplied roots, NH₄⁺‐supplied roots possessed fewer Al‐binding groups (‐OH and COO‐) and lower contents of pectin and hemicellulose. However, when grown in pH‐buffered solutions, these differences in the cell wall properties were not observed. Further analysis showed that the Al‐binding capacity and properties of cell walls were also altered by pHs alone. Taken together, our results indicate that the NH₄⁺‐reduced Al accumulation was attributed to the altered cell wall properties triggered by pH decrease due to NH₄⁺ uptake rather than direct competition for the cell wall binding sites between Al³⁺ and NH₄⁺.     

Purification and molecular characterization of human fibroblast interferon

Human fibroblast interferon was purified from serum-containing culture medium by a combination of concanavalin A or Blue Dextran Sepharose affinity chromatography with high-performance liquid chromatography to material exhibiting a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The interferon could be chromatographed and purified at acidic pH in volatile buffers on RP-8, RP-18, cyclohexyl, phenylalkyl, diphenyl, cyanopropyl, and diol supports. A specific activity averaging around 4 × 10⁸ units/mg was found for the pure material with a molecular weight of 20,000–21,000 after 20,000- to 50,000-fold purifications. In some preparations, low activity levels were also found at positions corresponding to 10,000, 17,000–18,000, 35,000, and 40,000 daltons. Amino acid and amino sugar analysis, partial NH₂- and COOH-terminal sequences, and tryptic peptide patterns determined at the picomole level are reported for the purified interferon.     

Morphological and transport characteristics of swollen chitosan‐based proton exchange membranes studied by molecular modeling

Chitosan biopolymer has been extensively applied in direct methanol fuel cells (DMFCs) as a potential replacement to conventional Nafion membrane for its considerably reduced methanol crossover. Here, we computationally explored the influences of methanol concentration, temperature, and pH parameters upon the nanostructure and dynamics, particularly the methanol crossover, in chitosan proton‐exchange membrane (PEM) through molecular dynamics simulations. Theoretical results demonstrated the increased swelling and radius of gyration of chitosan chains at higher concentrations. Structural examinations further revealed that an increase in methanol loading weakened the water interactions with chitosan functionalities (amine? NH₂, hydroxyl? OH, and methoxy? CH₂OH) whereas improved the methanol affinities toward chitosan, reflecting higher methanol sorption capability of chitosan at enhanced concentrations. Additionally, it was found that interactions between solvents and chitosan strengthened under acidic pH conditions on account of amine protonation. The water diffusivity inside the swollen chitosan diminished by increasing CH₃OH uptake, and in contrast diffusivity of methanol was noted to enhance. Furthermore, it was observed that an enhancement in temperature or a decrease in pH intensified solvent mobility. These insights imply that supplying methanol‐concentrated and/or acidic feed solutions into DMFCs based on chitosan PEMs could lower membrane performance due to the significant methanol transport dynamics.     

Calcium Dynamics During Physiological Acidification in Xenopus Oocyte

Interplays between intracellular pH (pHi) and calcium ([Ca²⁺]_i) variations remain unclear, though both proton and calcium homeostasis changes accompany physiological events such as Xenopus laevis oocyte maturation. In this report, we used NH₄Cl and changes of extracellular pH (pHe) to acidify the cytosol in a physiological range. In oocytes voltage-clamped at −80 mV, NH₄Cl triggered an inward current, the main component of which is a Ca²⁺-dependent chloride current. Calcium imaging confirmed that NH₄Cl provoked a [Ca²⁺]_i increase. The mobilized sources of calcium were discriminated using the triple-step protocol as a means to follow both the calcium-activated chloride currents (ICl-Ca) and the hyperpolarization- and acid-activated nonselective cation current (I_In). These currents were stimulated during external addition of NH₄Cl. This upregulation was abolished by BAPTA-AM, caffeine and heparin. By both buffering pHi changes with MOPS and by inhibiting calcium influx with lanthanum, intracellular acidification, initiated by NH₄Cl and extracellular acidic medium, was shown to trigger a [Ca²⁺]_i increase through both calcium release and calcium influx. The calcium pathways triggered by pHe changes are similar to those activated by NH₄Cl, thus suggesting that there is a robust signaling mechanism allowing the cell to adjust to variable environmental conditions.     

pH affects ammonium,nitrate and proton fluxes in the apical region of conifer and soybean roots

The effect of pH on nitrate and ammonium uptake in the high‐affinity transport system and low‐affinity transport system ranges was compared in two conifers and one crop species. Many conifers grow on acidic soils, thus their preference for ammonium vs nitrate uptake can differ from that of crop plants, and the effect of pH on nitrogen (N) uptake may differ. Proton, ammonium and nitrate net fluxes were measured at seedling root tips and 5, 10, 20 and 30 mm from the tips using a non‐invasive microelectrode ion flux measurement system in solutions of 50 or 1500 µM NH₄NO₃ at pH 4 and 7. In Glycine max and Pinus contorta, efflux of protons was observed at pH 7 while pH 4 resulted in net proton uptake in some root regions. Pseudotsuga menziesii roots consistently showed proton efflux behind the root tip, and thus appear better adapted to maintain proton efflux in acid soils. P. menziesii's ability to maintain ammonium uptake at low pH may relate to its ability to maintain proton efflux. In all three species, net nitrate uptake was greatest at neutral pH. Net ammonium uptake in G. max and net nitrate uptake in P. menziesii were greatly reduced at pH 4, particularly at high N concentration, thus N concentration should be considered when determining optimum pH for N uptake. In P. menziesii and G. max, net N uptake was greater in 1500 than 50 µM NH₄NO₃ solution, but flux profiles of all ions varied among species.     

Carboxylation, the completion step in prothrombin biosynthesis

It has been found that [¹⁴C]CO₂ is incorporated into prothrombin $\text{in vivo}$ in two hours. The amount of incorporation is increased 3 to 4 fold by the administration of vitamin K₁ to the warfarin-treated vitamin K-deficient rat, over incorporation in the “normal” rat. The radioactivity is found in one acidic peptide following trypsin digestion and following pronase and aminopeptidase digestion is found in one acidic amino acid. The [¹⁴C] is lost on heating of this amino acid at pH 2, leaving unlabeled glutamic acid. It appears that the vitamin K-dependent step in the “completion” of prothrombin is carboxylation of a glutamyl residue of the preformed protein molecule.     

The Reproducible Isolation of Inactive Insulin Protein Complex

Physiologically inactive insulin protein complex (IPC) was isolated from human plasma by a new, readily-reproducible procedure. Pooled human plasma was passed through a Sephadex C-50 cation exchange column. Anionic and neutral materials were eluted from the column by a 0.005 M (NH₄)₂CO₃ solution, pH 7.8. A gradient of 0.005 to 1.0 M (NH₄)₂CO₃, pH 7.8, was used to elute the cationic fractions. Protein-containing elution peaks were adjusted to pH 7.3 ± 0.1 with 0.5 M acetic acid, and de-salting and concentrating the IPC was achieved with an Amicon Ultrafiltrator using a UM 10 membrane. The concentrated solutions from the individual elution peaks were freeze-dried and tested for insulin-like activity with the rat hemidiaphragm technique.     

Are Amazonian fish more sensitive to ammonia? Toxicity of ammonia to eleven native species

Little is known about the tolerance of Amazonian fish to ammonia. However, elevated ammonia of anthropogenic origin may now occur. As Amazonian fish evolved in waters which are generally acidic (i.e., low NH₃), we hypothesized that they would be more sensitive to ammonia than other freshwater fish. The acute (96-h) toxicity of NH₄Cl was tested in native ion-poor soft water (pH 7.0, ~28 °C) using semi-static tests with 11 species. Species sensitivity distributions (SSDs) for LC50_96 h and LC10_96 h and calculations of the hazardous concentrations to the most sensitive 5% (HC5 values) were tabulated. Values of LC50_96 h/LC10_96 h (in mM total ammonia) ranged from 2.24/0.78 for Paracheirodon axelrodi (most sensitive) to 19.53/16.07 for Corydoras schwartzi (most tolerant). These results confirm our hypothesis that Amazonian fish are more sensitive to ammonia than other freshwater species. High levels of ammonia may be associated with hypoxia, especially during dry periods. Simultaneous hypoxia (15–20% saturation) exacerbated ammonia toxicity in the most sensitive species (P. axelrodi), but not in Astronotus ocellatus or Corydoras schwartzi, a facultative air-breather where prevention of air access doubled ammonia toxicity. The present data are useful in generating regulatory guidelines in Amazonian waters and indicate that further studies incorporating hypoxia and air access/denial are needed.