Pressure-assisted digestion of proteins, also known as pressure cycling technology (PCT), using a Barocycler NEP 2320 was compared with the conventional method using atmospheric pressure. Our objective was to demonstrate that PCT provides more controlled enzymatic digestion of proteins than prolonged digestion at atmospheric pressure ranging from 18 to 24 h. More controlled digestion would be beneficial for studies of highly posttranslationally modified protein such as histones. For the comparison of these two techniques, recombinant and native histone H4 were used as model proteins. PCT was optimized for pressure and time, and it was found to be most effective at 15 kpsi for 120 min of incubation. In conclusion, the PCT method was found to be much faster than using atmospheric pressure. PCT was also found to allow for unambiguous control of digestion parameters and to provide a high yield of sequence coverage compared with atmospheric pressure.
Histones; Proteomics; High-performance liquid chromatography; Barocycler NEP 2320; Sample preparation
Glutathione (GSH), an intracellular tripeptide that combats oxidative stress, must be continually replaced due to loss through conjugation and destruction. Previous methods, estimating the synthesis of GSH in vivo, used constant infusions of labeled amino acid precursors. We developed a new method based on incorporation of 2H from orally supplied 2H2O into stable C-H bonds on the tripeptide. The incorporation of 2H2O into GSH was studied in rabbits over a two week period. The method estimated N, the maximum number of C-H bonds in GSH that equilibrate with 2H2O as amino acids. GSH was analyzed by liquid chromatography mass spectrometry after derivatization to yield GSH-N-ethylmaleimide (GSNEM). A model, which simulated the expected abundance at each mass isotopomer for the GSNEM ion at various values for N, was used to find the best fit to the data. The plateau labeling fit best a model with N= 6 of a possible 10 C-H bonds. Thus, the amino acids precursors do not completely equilibrate with 2H2O prior to GSH synthesis. Advantages of this new method include replacing costly amino acid infusions with the oral administration of 2H2O and a statistical basis for estimating N.
glutathione; deuterated water; models; isotopomers; mass spectrometry; lc/ms
Defining the biologic roles of human milk oligosaccharides (HMOS) requires an efficient, simple, reliable, and robust analytical method for simultaneous quantification of oligosaccharide profiles from multiple samples. The HMOS fraction of milk is a complex mixture of polar, highly branched, isomeric structures that contain no intrinsic facile chromophore, making their resolution and quantification challenging. A liquid chromatography-mass spectrometry (LC-MS) method was devised to resolve and quantify 11 major neutral oligosaccharides of human milk simultaneously. Crude HMOS fractions are reduced, resolved by porous graphitic carbon HPLC with a water/acetonitrile gradient, detected by mass spectrometric specific ion monitoring, and quantified. The HPLC separates isomers of identical molecular weights allowing 11 peaks to be fully resolved and quantified by monitoring mass to charge (m/z) ratios of the deprotonated negative ions. The standard curves for each of the 11 oligosaccharides is linear from 0.078 or 0.156 to 20 μg/mL (R2 > 0.998). Precision (CV) ranges from 1% to 9%. Accuracy is from 86% to 104%. This analytical technique provides sensitive, precise, accurate quantification for each of the 11 milk oligosaccharides and allows measurement of differences in milk oligosaccharide patterns between individuals and at different stages of lactation.
Oligosaccharides; Human Milk; LC-MS
Sedimentation velocity analytical ultracentrifugation is a classical biophysical technique that is commonly used to analyze the size, shape and interactions of biological macromolecules in solution. Fluorescence detection provides enhanced sensitivity and selectivity relative to the standard absorption and refractrometric detectors, but data acquisition is more complex and can be subject to interference from several photophysical effects. Here, we describe methods to configure sedimentation velocity measurements using fluorescence detection and evaluate the performance of the fluorescence optical system. The fluorescence detector output is linear over a concentration range of at least 1- 500 nM of fluorescein and Alexa Fluor 488. At high concentration, deviations from linearity can be attributed to the inner filter effect. A duplex DNA labeled with Alexa Fluor 488 was used as a standard to compare sedimentation coefficients obtained using fluorescence and absorbance detectors. Within error, the sedimentation coefficients agree. Thus, the fluorescence detector is capable of providing precise and accurate sedimentation velocity results that are consistent with measurements performed using conventional absorption optics, provided the data are collected at appropriate sample concentrations and the optics are configured correctly.
analytical ultracentrifugation; sedimentation velocity; fluorescence
Cannabinoids receptors (CB) are being targeted therapeutically for the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. More recently, cannabinoid agonists have displayed anti-proliferative activity against breast cancer and prostate cancer in animal models.
In order to study cannabinoid receptor ligands, we have developed a novel plate-based assay that measures internalization of CB1/CB2 receptors by determining the change in the intracellular levels of the radiolabeled agonists: [3H]-Win 55-212-2 for CB1 and [3H]-CP 55-940 for CB2. The developed plate-based assay was validated by determining IC50 values for known antagonists: AM251, AM281, AM630 and AM6545. The data obtained was consistent with previously reported values, therefore confirming that the assay can be used to determine the functional binding activities (IC50) of antagonists for the CB1 and CB2 receptors.
In addition, we demonstrated that the plate-based assay may be used for screening against complex matrices. Specifically, we demonstrated that the plate-based assay was able to identify which extracts of several species of the genus Zanthoxylum had activity at the CB1/CB2 receptors.
plate-based assay; Zanthoxylum; isosteviol; CB1 antagonists; CB2 antagonist
The development of new antibacterial agents has become necessary to treat the large number of emerging bacterial strains resistant to current antibiotics. Despite the different methods of resistance developed by these new strains, the A-site of the bacterial ribosome remains an attractive target for new antibiotics. To develop new drugs that target the ribosomal A-site, a high-throughput screen is necessary to identify compounds that bind to the target with high affinity. To this end, we present an assay that uses a novel fluorescein-conjugated neomycin (F-neo) molecule as a binding probe to determine the relative binding affinity of a drug library. We show here that the binding of F-neo to a model Escherichia coli ribosomal A-site results in a large decrease in the fluorescence of the molecule. Furthermore, we have determined that the change in fluorescence is due to the relative change in the pKa of the probe resulting from the change in the electrostatic environment that occurs when the probe is taken from the solvent and localized into the negative potential of the A-site major groove. Finally, we demonstrate that F-neo can be used in a robust, highly reproducible assay, determined by a Z′-factor greater than 0.80 for 3 consecutive days. The assay is capable of rapidly determining the relative binding affinity of a compound library in a 96-well plate format using a single channel electronic pipette. The current assay format will be easily adaptable to a high-throughput format with the use of a liquid handling robot for large drug libraries currently available and under development.
Ribosome RNA; Aminoglycoside; Fluorescence; A-site; F-neo; Ribosome binding ligands; Screening
A simple off-column capillary electrophoretic (CE) assay for measuring acetyl coenzyme A carboxylase holoenzyme (holo-ACC) activity and inhibition was developed. The two reactions catalyzed by the holo-ACC components, biotin carboxylase (BC) and carboxyltransferase (CT), were simultaneous monitored in this assay. Acetyl coenzyme A (CoA), malonyl-CoA, adenosine triphosphate (ATP), and adenosine diphosphate (ADP) were separated by CE, and the depletion of ATP and acetyl-CoA as well as the production of ADP and malonyl-CoA were monitored. Inhibition of holo-ACC by the biotin carboxylase inhibitor, 2-amino-N,N-dibenzyloxazole-5-carboxamide, and the carboxyltransferase inhibitor, andrimid, was confirmed using this assay. A previously reported off-column CE assay for only the CT component of ACC was optimized, and an off-column CE assay for the BC component of ACC also was developed.
capillary electrophoresis; enzyme assay; acetyl CoA carboxylase; enzyme inhibition
We report systematic and large inaccuracies in the recorded elapsed time in data files from the analytical ultracentrifuge, leading to overestimates of the sedimentation coefficients of up to 10%. This far exceeds previously considered factors contributing to the uncertainty in this parameter, and has significant ramifications for derived parameters, such as hydrodynamic shape and molar mass estimates. The source of this error is at present unknown, but we found it to be quantitatively consistent across different instruments, increasing with rotor speed. Furthermore, its occurrence appears to correlate with the use of the latest data acquisition software from the manufacturer, in use in some of our laboratories for nearly two years. Many of the recently published sedimentation coefficients may need to be re-examined. The problem can be easily recognized by comparing the file time-stamps provided by the operating system with the elapsed scan times recorded within the data files. We therefore implemented a routine in SEDFIT that can automatically examine the data files, alert the user to significant discrepancies, and correct the scan times accordingly. This eliminates errors in the recorded scan times.
sedimentation velocity; hydrodynamic modeling
ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anti-cancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug–drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.
Efflux inhibition; ABCB1; ABCC1; ABCG2; Fluorescent substrate; Flow cytometry
Topoisomerase 1 (TOP1) generates transient nicks in the DNA to relieve torsional stress encountered during the cellular processes of transcription, replication, and recombination. At the site of the nick there is a covalent linkage of TOP1 with DNA via a tyrosine residue. This reversible TOP1-cleavage complex intermediate can become trapped on DNA by TOP1 poisons such as camptothecin, or by collision with replication or transcription machinery, thereby causing protein-linked DNA single- or double-strand breaks and resulting in cell death. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a key enzyme involved in the repair of TOP1-associated DNA breaks via hydrolysis of 3′-phosphotyrosine bonds. Inhibition of TDP1 is therefore an attractive strategy for targeting cancer cells in conjunction with TOP1 poisons. Existing methods for monitoring the phosphodiesterase activity of TDP1 are generally gel based or of high cost. Here we report a novel, oligonucleotide-based fluorescence assay that is robust, sensitive, and suitable for high-throughput screening of both fragment and small compound libraries for the detection of TDP1 inhibitors. We further validated the assay using whole cell extracts, extending its potential application to determine of TDP1 activity in clinical samples from patients undergoing chemotherapy.
TDP1; Assay; High-throughput screen; Fragment; Inhibitor; Topoisomerase
Since the discovery of tubulin as the major component of microtubules over 40 years ago, its diversity of forms has raised a continuum of fundamental questions about its regulation and functions in a variety of organisms across phyla. Its high abundance in the brain or in specialized organelles such as cilia has allowed early characterization of this important target for anticancer drugs. However, it was only when matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry technologies became available in the late 1980's that the full complexity of tubulin expression patterns became more obvious. This contributed in a major way to the idea that due to increasing and conserved tubulin heterogeneity during evolution, a tubulin code read by microtubule associated proteins might exist and be of functional significance. We review here the merging of recent genetic and cell biology studies with proteomics to decipher this code and illustrate some of the tubulin proteomic approaches with new data generated in our laboratories.
tubulin; microtubule; proteomics; mass spectrometry
A simple, accurate and robust quantitative CE method for the determination of oversulfated chondroitin sulfate (OSCS) as a contaminant in heparin (Hep) preparations is described. After degradation of the polysaccharides by acidic hydrolysis, the hexosamines produced, i.e., GlcN from Hep and GalN from OSCS, were derivatized with anthranilic acid (AA) and separated by means of CE in approx. 10 min with high sensitivity detection at 214 nm (limit of detection (LOD) of approx. 200 pg). Furthermore, AA-derivatized GlcN and GalN showed quite similar molar absorptivity allowing for direct and simple quantification of OSCS in Hep samples. Moreover, a preliminary step of specific enzymatic treatment by using chondroitin ABC lyase may be applied for the specific elimination of interference in the analysis due to the possible presence in Hep samples of natural chondroitin sulfate and dermatan sulfate impurities, making this analytical approach highly specific for OSCS contamination, since chondroitin ABC lyase is unable to act on this semi-synthetic polymer. The CE method was validated for specificity, linearity, accuracy, precision, LOD and limit of quantification (LOQ). Due to the very high sensitivity of CE, as little as 1% OSCS contaminant in Hep sample could be detected and quantified. Finally, a contaminated raw Hep sample was found to contain 38.9% OSCS while a formulated contaminated Hep was calculated to have 39.7% OSCS.
Oversulfated chondroitin sulfate; Heparin; Glucosamine; Galactosamine; Capillary Electrophoresis; Anthranilic acid
A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl labeled lipids; energy donors and perylenoyl labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.
Glycosphingolipid; Lipid transfer protein; Phospholipid bilayers; Galactosylceramide; Anthrylvinyl; Perylenoyl
The budding yeast Saccharomyces cerevisiae expresses different isoforms of glucose transporters (HXTs) in response to different levels of glucose. Here, we constructed reporter strains in which the nourseothricin (NAT) resistance gene is expressed under the control of the HXT1, 2, or 3 promoter. The resulting HXT-NAT reporter strains exhibited a strict growth dependence on glucose, and their growth could be easily controlled and optimized by adjusting glucose concentration, demonstrating the value of the reporter strains for studying the molecular basis of differential expression of HXT genes, as well as for screening drugs that inhibit glucose uptake and glycolysis.
Glycolysis; Glucose uptake; Glucose transporters; Yeast; HXT-NAT reporter strains
Random peptide libraries are displayed on filamentous bacteriophage as fusions to either the minor coat protein, pIII, or the major coat protein, pVIII. We have devised a means of isolating the peptide displayed on a phage clone by transferring it to the N-terminus of the maltose-binding protein (MBP) of Escherichia coli encoded by malE. Transfer of a peptide sequence to monomeric MBP eliminates phage-encoded amino acids downstream of the insert peptide as well as avidity effects caused by multivalent display on phage. Peptide:MBP fusions are also easily affinity purified on amylose columns. The pMal-p2 vector was engineered to accept phage DNA encoding pIII- and pVIII-displayed peptides fused to their respective leader sequences. Both types of leader sequence were shown to target the peptide:MBP fusions to the periplasm of E. coli. A streamlined procedure for transferring peptides to MBP was applied to clones that had been isolated from a panel of pVIII-displayed peptide libraries by screening with an HIV-1-specific monoclonal antibody (Ab). By enzyme-linked immunosorbent assay, the Ab bound each of the peptide:MBP fusions and required the presence of a disulfide bridge within each peptide. Some of the peptide:MBP fusions were also analyzed using surface plasmon resonance. Thus, our study shows the value of malE fusion vectors in characterizing phage-displayed peptides.
The DNA of all organisms is persistently damaged by endogenous reactive molecules. Most of the single-base endogenous damage is repaired through the base excision repair (BER) pathway that is initiated by members of the DNA glycosylase family. Although the BER pathway is often considered to proceed through a common abasic site intermediate, emerging evidence indicates that there are likely distinct branches reflected by the multitude of chemically different 3’- and 5’-ends generated at the repair site. In this paper, we have applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to the analysis of model DNA substrates acted upon by recombinant glycosylases. We examine the chemical identity of several possible abasic site and nicked intermediates generated by monofunctional and bifunctional glycosylases. Our results suggest that the intermediate from endoIII/Nth might not be a simple β-elimination product as previously described. Upon the basis of 18O incorporation experiments, we propose a new mechanism for the endoIII/Nth family of glycosylases that may resolve several of the previous controversies. We further demonstrate that the use of an array of lesion-containing oligonucleotides can be used to rapidly examine the substrate preferences of a given glycosylase. Some of the lesions examined here can be acted upon by more than one glycosylase, resulting in a spectrum of damaged intermediates for each lesion, suggesting that the sequence and coordination of repair activities that act upon these lesions may influence the biological outcome of damage repair.
DNA glycosylase; MALDI-TOF-MS; oxidative damage; substrate specificity
Protein tyrosine phosphatases (PTPs) are key signal-transduction regulators and have emerged as potential drug targets for inhibitor design. Here we report a yeast-based assay that provides a general means of assessing the activity and/or inhibition of essentially any classical PTP in living cells. The assay uses the activity of an exogenously expressed PTP to counter the activity of a co-expressed and toxic tyrosine kinase, such that only active PTPs are capable of rescuing growth. PTP activity gives rise to both increased growth and decreased phosphotyrosine levels; cellular PTP activity can therefore be monitored by either yeast-growth curves or anti-phosphotyrosine western blots. We show that four PTPs (TCPTP, Shp2, PEST, PTPα) are capable of rescuing the effects of v-Src toxicity. Since these PTPs are chosen from four distinct sub-families, it is likely that biologically and medicinally important PTPs from other subfamilies can similarly function in the cellular PTP assay. Because many small-molecule PTP inhibitors fail to penetrate cell membranes effectively, this cell-based assay has the potential to serve as a useful screening tool for determining the cellular efficacy of candidate inhibitors in a more biologically relevant context than can be provided by an in vitro PTP assay.
protein tyrosine phosphatases (PTPs); yeast; cell-based assay; TCPTP; Shp2; PEST; PTPα
Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of structural damage on DNA structure and function. In contrast to the synthesis of oligonucleotides with normal DNA bases, oligonucleotide synthesis with modified bases often requires modified synthetic or deprotection conditions. Furthermore, several modified bases of biological interest are prone to further damage during synthesis and oligonucleotide isolation. In this paper, we describe the application of MALDI-TOF-MS to the characterization of a series of modified synthetic oligonucleotides. The potential for and limits in obtaining high mass accuracy for confirming oligonucleotide composition are discussed. Examination of the isotope cluster is also proposed as a method for confirming oligonucleotide elemental composition. MALDI-TOF-MS analysis of the unpurified reaction mixture can be used to confirm synthetic sequence and to reveal potential problems during synthesis. Analysis during and after purification can yield important information on depurination and base oxidation. It can also reveal unexpected problems that can occur with non-standard synthesis, deprotection or purification strategies. Proper characterization of modified oligonucleotides is essential for the correct interpretation of experiments performed with these substrates, and MALDI-TOF-MS analysis provides a simple yet extensive method of characterization that can be used at multiple stages of oligonucleotide production and utilization.
MALDI-TOF; base analogs; synthetic oligonucleotides; oxidative damage; isotope cluster
Globoid-cell leukodystrophy (GLD) or Krabbe disease is a lysosomal disease caused by β-galactocerebrosidase (GALC) deficiency resulting in a rapidly progressive neurodegenerative disorder. Unfortunately, the only available treatment is hematopoietic bone marrow transplantation, which prevents its fulminant manifestation but without treating further neurological manifestations. Here we describe the development of a cellular high-throughput screening (HTS) assay using GLD patient fibroblasts to screen for small molecules that enhance the residual mutant GALC enzymatic activity. Small molecules have substantial therapeutic potential in GLD as they are more prone to cross the blood-brain barrier, reaching the neuronal affected cells. The transformation of primary skin fibroblasts with SV40 large T antigen showed to maintain the biochemical characteristics of the GLD cells and generates sufficient cells for the HTS. Using a specific fluorescent substrate, residual GALC activity from a SV40-transformed GLD patient fibroblast was measurable in high-dense microplates plates. The pilot quantitative HTS against a small compound collection showed robust statistics. The small molecules that showed active concentration-response curves were further studied in primary GLD fibroblasts. This cell-based HTS assay demonstrates the feasibility of employing live-GLD patient cells to identify therapeutic agents that can be potentially be used for the treatment of this progressive neurodegenerative disease.
β-galactocerebrosidase; high-throughput screening; small molecules; Krabbe Disease
With the recent discovery of a unique class of dual-specificity phosphatases that dephosphorylate glucans, we report an in vitro assay tailored for the detection of phosphatase activity against phosphorylated glucans. We demonstrate that in contrast to a general phosphatase assay utilizing a synthetic substrate, only phosphatases that possess glucan phosphatase activity liberate phosphate from the phosphorylated glucan amylopectin using the described assay. This assay is simple and cost-effective, providing reproducible results that clearly establish the presence or absence of glucan phosphatase activity. The assay described will be a useful tool in characterizing emerging members of the glucan phosphatase family.
Glucan; Phosphatase; Malachite green; Amylopectin; Glycogen; Starch
The surface lipopolysaccharide of gram-negative bacteria is both a virulence factor and a B cell antigen. Antibodies against O-antigen of lipopolysaccharide may confer protection against infection, and O-antigen conjugates have been designed against multiple pathogens. Here, we describe a simplified methodology for extraction and purification of the O-antigen core portion of Salmonella lipopolysaccharide, suitable for large-scale production. Lipopolysaccharide extraction and delipidation are performed by acetic acid hydrolysis of whole bacterial culture and can take place directly in a bioreactor, without previous isolation and inactivation of bacteria. Further O-antigen core purification consists of rapid filtration and precipitation steps, without using enzymes or hazardous chemicals. The process was successfully applied to various Salmonella enterica serovars (Paratyphi A, Typhimurium, and Enteritidis), obtaining good yields of high-quality material, suitable for conjugate vaccine preparations.
O-antigen; Lipopolysaccharide; Salmonella
Styrene and 1,3-butadiene are important intermediates used extensively in the plastics industry. They are metabolized mainly through cytochrome P450-mediated oxidation to the corresponding epoxides, which are subsequently converted to diols by epoxide hydrolase or through spontaneous hydration. The resulting styrene glycol and 3-butene-1,2-diol have been suggested as biomarkers of exposure to styrene and 1,3-butadiene, respectively. Unfortunately, poor ionization of the diols within electrospray mass spectrometers becomes an obstacle to the detection of the two diols by liquid chromatography/electrospray ionization–mass spectrometry (LC/ESI–MS). We developed an LC/ESI–MS approach to analyze styrene glycol and 3-butene-1,2-diol by means of derivatization with 2-bromopyridine-5-boronic acid (BPBA), which not only dramatically increases the sensitivity of diol detection but also facilitates the identification of the diols. The analytical approach developed was simple, quick, and convincing without the need for complicated chemical derivatization. To evaluate the feasibility of BPBA as a derivatizing reagent of diols, we investigated the impact of diol configuration on the affinity of a selection of diols to BPBA using the established LC/ESI–MS approach. We found that both cis and trans diols can be derivatized by BPBA. In conclusion, BPBA may be used as a general derivatizing reagent for the detection of vicinal diols by LC/MS.
Mass spectrometry; Styrene glycol; 3-Butene-1; 2-diol; Boronic acid; Derivatization
The aggregation of amyloidogenic proteins/peptides has been closely linked to the neuropathology of several important neurological disorders. In Alzheimer's disease (AD), amyloid beta (Aβ) peptides and their aggregation are believed to be at least partially responsible for the etiology of AD. The aggregate-inflicted cellular toxicity can be inhibited by short peptides whose sequence are homologous to segments of the Aβ(1–42) peptide responsible for β-sheet stacking (referred to as the β-sheet breaker peptides). Herein a water-soluble ferrocene (Fc)-tagged β-sheet breaker peptide (Fc-KLVFFK6) is used as an electrochemical probe for kinetic studies of the inhibition of the Aβ(1–42) fibrillation process and for determination of the optimal concentration of β-sheet breaker peptide for efficient inhibition. Our results demonstrated that Fc-KLVFFK6 interacts with the Aβ aggregates instantaneously in solution, and sub-stoichiometric amount of Fc-KLVFFK6 is sufficient to inhibit the formation of the Aβ oligomers and fibrils and to reduce the toxicity of Aβ(1–42). The interaction between Fc-KLVFFK6 and Aβ(1–42) follows a pseudo-first-order reaction, with a rate constant of 1.89 ± 0.05 × 10−4 s−1. Tagging β-sheet breaker peptides with a redox label facilitates design, screening, and rational use of peptidic inhibitors for impeding/altering Aβ aggregation.
amyloid beta; aggregation; inhibition kinetics; ferrocene tag; β-sheet breaker; cytotoxicity
A principal challenge in microarray experiments is to facilitate hybridization between probe strands on the array with complementary target strands from solution while suppressing any competing interactions that the probes and targets may experience. Synthetic DNA analogs, whose hybridization to targets can exhibit qualitatively different dependence on experimental conditions than for nucleic acid probes, open up an attractive alternative for improving selectivity of array hybridization. Morpholinos (MOs), a class of uncharged DNA analogs, are investigated as microarray probes instead of DNA. Morpholino microarrays were fabricated by contact printing of amino-modified probes onto aldehyde slides. In addition to covalent immobilization, MOs were found to efficiently immobilize through physical adsorption; such physically adsorbed probes could be removed by post-printing washes with surfactant solutions. Hybridization of double-stranded DNA targets to MO microarrays revealed a hybridization maximum at intermediate ionic strengths. The decline in hybridization at lower ionic strengths was attributed to an electrostatic barrier accumulated from hybridized DNA targets, while at higher ionic strengths it was attributed to stabilization of target secondary structure in solution. These trends, which illustrate ionic strength tuning of forming on-array relative to solution secondary structure, were supported by a stability analysis of MO/DNA and DNA/DNA duplexes in solution.
surface hybridization; microarrays; morpholinos; DNA; immobilization; PNA
The separation and quantification of glycosaminoglycan (GAG) chains, with different levels of sulfation, from cells, media and prepared through chemoenzymatic synthesis or metabolic engineering, poses a major challenge in glycomics analysis. A method for microscale separation and quantification of heparin, heparan sulfate and heparosan from cells is reported. This separation relies on a mini-strong anion exchange spin column eluted stepwise with different concentrations of sodium chloride. Disaccharide analysis by LC-MS was used to monitor the chemical structure of the different GAG chains that were recovered.
heparosan; heparan sulfate; heparin; glycosaminoglycan; anion exchange chromatography; disaccharide analysis