Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution ...conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.
•The reported LC-MS/MS method resolved 82 erectile dysfunction (ED) drugs in 10min.•ED drug extraction from different matrices gave recoveries from 92 to111%.•The screening method was successfully ...applied to more than 32 samples.•The method is faster and more sensitive than currently used and reported methods.•Identification of additional species, in an untargeted approach, is possible.
A rapid LC-MS/MS method has been developed to simultaneously separate 71 erectile dysfunction (ED) drugs and 11 natural ingredients that are sometimes found alongside ED drugs, present in suspected adulterated or counterfeit samples. The separation was achieved in 10min using 2.6μm fused-core C18 particles in a 100×2.1mm column coupled to an LTQ Orbitrap XL mass spectrometer operated in positive electrospray mode. Using a straightforward methanolic extraction procedure, recovery from real samples (tablets, capsules, oral liquids and herbal products) was 92–111% and the lower and upper limits of detection and quantification were in the sub ng/mL and the sub μg/mL ranges, respectively. The intra- and inter-assay precision were ≤3.2% and 10.4% respectively across three concentrations of standards (50, 250 and 1000ng/mL) measured for 4 representative drugs spiked into a tablet-based matrix. This behavior was consistently observed for all the other compounds. The mass accuracy was less than 3ppm. Moreover, an advantage of this method is that the full scan event in the acquisition method associated with the high resolution of the Orbitrap XL allows post-analysis identification, in an untargeted approach, of additional species in the complex matrices. Our LC-MS/MS method for ED drugs was successfully applied to 32 samples and the drug identifications were in 100% agreement with those obtained by the conventional methods HPLC-UV and GC-MS. Following the complete validation of the ED method, it has been introduced in the current counterfeit identification procedures at Health Canada.
Immobilized proteolytic enzymes present several advantages over their soluble form, not the least of which is suppression of autoproteolysis peaks even at high enzyme‐to‐substrate ratios. We have ...made immobilized chymotrypsin by directly crosslinking it with glutaraldehyde to produce polymeric particles. Digestion of two model substrates using the particles was followed by CE peptide mapping with detection by UV absorbance or LIF. Results showed that autoproteolysis was highly suppressed and that different storage conditions of the particles in the short term (24 h) did not affect digestion of denatured BSA. As well, the chymotrypsin particles were indifferent to the presence of fluorescein groups on a casein substrate. Glutaraldehyde crosslinking of chymotrypsin inside a fused silica capillary column to make an immobilized enzyme reactor (IMER) was achieved in a series of reagent addition and washing steps, entirely automated using a commercial CE instrument. Digestion of myoglobin in the IMER for 30 min at 37°C followed by peptide mapping by CE‐MS of the collected digest allowed identification of 17 chymotryptic peptides of myoglobin, or 83% primary sequence coverage.
Hydrogelation of small molecules in aqueous solutions results from a balance between solubilization and precipitation (or crystallization). The hydrophobic moieties of amphiphiles tend to aggregate ...and the hydrophilic units may stabilize the aggregates in aqueous solutions. Morphologies vary according to the chemical structure of the amphiphiles. The formation of nanofibers or worm-like micelles is a prerequisite for hydrogels. Molecular hydrogels often show better degradability and functional diversity than polymeric hydrogels and may be useful in biomedical applications. Bile acids have attracted increasing attention for designing various biomaterials, including molecular hydrogels. They are naturally occurring amphiphilic compounds that exist in our body and help with the dissolution and digestion of fat by the formation of micelles. This review highlights the recent progress in the field of molecular hydrogelators based on bile acids, including bile salts, anionic, cationic and neutral bile acid derivatives, two-component hydrogelation systems, and polymeric supramolecular hydrogels, along with their potential applications.
Bile acid derivatives can form molecular hydrogels that may be useful for drug delivery, tissue engineering and nanotemplating.
A mixture of a cholic acid dimer with a secondary amine group and formic acid at a molar ratio of 1/1 is regarded as an organic salt, and it self-assembles in aqueous solutions to form monodisperse ...nanofibers. The nanofibers are separated at low concentrations of the mixture but entangle with each other at high concentrations to form well-dispersed and randomly arranged 3D fibrous networks. Above the minimum gelation concentration of the dimer, the fibrous network is strong enough to gelate the aqueous solutions to form a hydrogel. Hydrogels obtained from the dimer salt at a lower concentration are isotropic and show extinction between crossed polarizers in the polarizing microscope, whereas they become anisotropic (i.e., nematic hydrogels) upon increasing the dimer salt concentration or under physical stirring. The parallel arrangement of nanofibers from randomly directed fibrous networks may be responsible for the formation of such nematic hydrogels.
Regenerable amine-based solvents used for post-combustion CO
2
capture, primarily monoethanolamine and piperazine, are known to undergo degradation and secondary reactions over time forming, amongst ...other species, N-nitrosamines. These carcinogenic species can eventually make their way from treated wastewater into environmental waters. The United States Environmental Protection Agency (US EPA) recommends that the concentration of N-nitrosamines in surface water not exceed 1.24 μg/L. We have developed a straightforward method to quantify N-nitrosopiperazine in treated wastewater by hydrophilic interaction liquid chromatography – mass spectrometry (HILIC–MS) after sample preparation by supported liquid extraction (SLE). To achieve the best extraction recovery and method limits of quantification (MLOQ), standards were prepared in a high-salt synthetic matrix to mimic the treated wastewater effluent. To further improve the MLOQ, the drying steps after extraction were optimized. HILIC separation of the highly polar analytes was achieved using an ethylene-bridged hybrid amide stationary phase. Detection was achieved using a triple quadrupole mass spectrometer operated in positive electrospray ionisation and multiple reaction monitoring mode, providing a final MLOQ of 0.25 μg/L for N-nitrosopiperazine. Validation of the method was carried out to ensure good confidence in the data obtained for a treated wastewater sample from a post-combustion CO
2
capture facility. In addition, N-nitrosopiperazine was quantified with the developed SLE-HILIC–MS method in eight degraded carbon capture samples that had not yet undergone wastewater treatment.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Bile acids are natural compounds that can be made into dimers by covalently linking two of them through diethylenetriamine. A cholic acid dimer of this kind is synthesized and is found to form ...thermally reversible hydrogels with selected carboxylic acids through combined hydrogen bonding and ionic interactions. The gelation and viscoelastic properties of the hydrogels may be varied by judicious choice of the carboxylic mono- and diacids. The total organic content (the dimer and carboxylic acid) represents about 2% or less by weight in the ternary mixture. The molecular arrangement between the dimer and carboxylic acid is proposed to illustrate the formation mechanism of the hydrogels. The marginal solubility of the dimer–acid mixtures seems to be the deciding factor in obtaining the hydrogels.
Immobilized enzyme microreactors based on proteases for proteomics studies are usually made from enzyme bound to a solid-phase support such as particles packed into a cartridge. Our group has ...developed a supportless immobilization strategy that uses glutaraldehyde-mediated crosslinking to render proteolytic enzymes insoluble for facile protein digestion. Although this works well in batch format, in-situ crosslinking within a microcolumn-based enzyme microreactor is less straight-forward. A microreactor was fabricated by immobilizing chymotrypsin, a proteolytic enzyme, inside a 250-µm i.d. fused silica capillary that was first functionalized with amino groups before adding glutaraldehyde and enzyme. The extent and location of enzyme immobilization within the capillary tubing was probed by reacting fluorescein isothiocyanate with residual amino groups in the microreactor and imaging the capillary by confocal laser fluorescence microscopy. The images imply that chymotrypsin immobilization occurred mostly near the wall and did not extend into the center of the microreactor as a crosslinked porous network. On the other hand, this structure facilitates the passage of substrate through the reactor. Digestion of denatured bovine serum albumin by flow through the 43-cm long crosslinked chymotrypsin microreactor for a total of 3.2 min produced twenty-nine peptides, corresponding to 34% primary sequence coverage.
The immobilization conditions and kinetic behaviour of trypsin, covalently immobilized via the 1,4-diisothiocyanatobenzene (DITC) linker onto aminopropylated controlled pore glass (CPG) particles, ...have been evaluated to establish a rapid and efficient protocol for fabrication of an immobilized enzyme microreactor (IMER) for protein hydrolysis and subsequent peptide mapping. Addition of calcium ions to either the immobilization reaction solution or hydrolysis assay was studied for a synthetic substrate. Activity was slightly higher when immobilization was carried out in the presence of Ca
2+ whereas more enzyme could be immobilized in its absence. A protocol requiring less than 3
h was devised to obtain maximal enzymatic activity with the lowest ratio of soluble trypsin to DITC–CPG particles. The resulting immobilized enzyme was found to retain an acceptable percentage (
ca. 35%) of its activity after immobilization. The particles were dry-packed into a capillary to make a microscale IMER. Repeatability, reusability and digestion efficiency of the μIMER were investigated for the substrate β-casein using capillary electrophoretic-based peptide mapping. In initial tests, a single device showed reproducible peptide maps for 21 digestions lasting 2
h each, carried out over a period of 2 months. Complete digestion of β-casein could be achieved in a few minutes (86
s residence time in the μIMER followed by a wash step).
The characterization of protein glycosylation can be a complex and time-consuming procedure, especially for prokaryote O-linked glycoproteins, which often comprise unusual oligosaccharide structures ...with no known glycosylation motif. In this report, we describe a “top-down” approach that provides information on the extent of glycosylation, the molecular masses, and the structure of oligosaccharide residues on bacterial flagella, important structural proteins involved in the motility of pathogenic bacteria. Flagella from four bacterial pathogens, namely, Campylobacter jejuni, Helicobacter pylori, Aeromonas caviae, and Listeria monocytogenes, were analyzed by this top-down mass spectrometry approach. The approach needs minimal sample preparation and can be performed within a few minutes compared to the tedious and often time-consuming “bottom-up” approach involving proteolytic digestion and LC−MS−MS analyses of the suspected glycopeptides. Multiply protonated protein precursor ions subjected to low-energy collisional activation in a quadrupole time-of-flight instrument showed extensive and specific gas-phase deglycosylation resulting in the formation of abundant oxonium ions with very few fragment ions from peptidic bond cleavages. Structural information on individual carbohydrate residues is obtained using a second-generation product ion scan of oxonium ions formed by collisional activation of the intact protein ions in the source region. The four bacterial flagella examined differed not only by the extent of glycosylation but also by the nature of carbohydrate substituents. For example, the flagellin from the Gram-positive bacterium, L. monocytogenes showed O-linked GlcNAc residues at up to 6 sites/protein monomer. In contrast, the three Gram-negative bacterial pathogens C. jejuni, H. pylori and A. caviae displayed up to 19 Ser/Thr O-linked sites modified with residues structurally related to N-acetylpseudaminic acid (Pse5Ac7Ac) and in the case of Campylobacter include a novel N-acetylglutamine substituent on Pse5Am7Ac.
PDF
