1.
Evaluation of COMU as a coupling reagent for in situ neutralization Boc solid phase peptide synthesis.
Source
Institute for Molecular Bioscience, The University of Queensland, St Lucia, 4072, Australia; Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center, Department of Chemistry, Aarhus University, Langelandsgade 140, 8000, Aarhus, Denmark.
Abstract
Benzotriazole-based coupling reagents have dominated the last two decades of solid phase peptide synthesis. However, a growing interest in synthesizing complex peptides has stimulated the search for more efficient and low-cost couplingreagents, such as COMU which has been introduced as a nonexplosive alternative to the classic benzotriazole couplingreagents. Here, we present a comparative study of the coupling efficiency of COMU with the benzotriazole-based HBTU and HCTU for use in in situ neutralization Boc-SPPS. Difficult sequences, such as ACP(65-74), Jung-Redeman 10-mer, and HIV-1 PR(81-99), were used as model target peptides on polystyrene-based resins, as well as polyethylene glycol-based resins. Coupling yields obtained using fast in situ Boc-SPPS cycles were determined with the quantitative ninhydrin test as well as via LC-MS analysis of the crude cleavage products. Our results demonstrate that COMU coupling efficiency was less effective compared to HBTU and HCTU with HCTU ≥ HBTU > COMU, when polystyrene-based resins were employed. However, when the PEG resin was employed in combination with a safety catch amide (SCAL) linker, more comparable yields were observed for the three coupling reagents with the same ranking HCTU ≥ HBTU > COMU. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
- PMID:
- 22252935
- [PubMed - as supplied by publisher]
Selective binding of CD4 and CD8 T-cells to antigen presenting cells for enrichment of CMV and HIV specific T-lymphocytes.
Source
Department of Pediatric Hematology and Oncology, Bambino Gesù Children Hospital, Rome, Italy.
Abstract
Adherent antigen presenting cells (APC) pulsed with protein or peptide antigens were used to capture specific CD4 or CD8 T-cells derived from established T-cell lines or from PBMC of immune subjects based on physiological interaction between TCR and MHC-peptide complex. This method could be applied independently of epitope specificity, HLA restriction alleles, activation markers and secreted cytokines, parameters required by other methods for selection of specific T cells. Non specific T-cells were removed by applying a 1g force that did not affect binding of specific T-lymphocytes. Lymphocyte selection was specific and the average recovery was 36% for CD4 T-cells. CD8 T-cells proved trickier to purify, since solid phase APC were recognized as targets for cytotoxicity. Specificity was comparable to CD4 cells, but the average recovery for CD8 cells was 26%. No residual alloreactivity was detected in expanded T-cells. Frequency and recovery of specific T-cells were comparable to other current technologies, such as generation of T-cell lines and cytokine capture method. Since antigen and IL2 are the only reagents added to the cultures, this physiological procedure can be proposed for selection and expansion of pathogen specific T-cells not only for research purposes, but also for adoptive reconstitution of immunocompromised subjects if performed under GMP conditions.
Copyright © 2012. Published by Elsevier B.V.
- PMID:
- 22251662
- [PubMed - as supplied by publisher]
Inducible Alkylation of DNA by a Quinone Methide-Peptide Nucleic Acid Conjugate.
Abstract
The reversibility of alkylation by a quinone methide intermediate (QM) avoids the irreversible consumption that plagues most reagents based on covalent chemistry and allows for site specific reaction that is controlled by the thermodynamics rather than kinetics of target association. This characteristic was originally examined with an oligonucleotide QM conjugate but broad application depends on alternative derivatives that are compatible with a cellular environment. Now, a peptide nucleic acid (PNA) derivative has been constructed and shown to exhibit an equivalent ability to delivery the reactive QM in a controlled manner. This new conjugate demonstrates high selectivity for a complementary sequence of DNA even when challenged with an alternative sequence containing a single T/T mismatch. Alkylation of non-complementary sequences is only possible when a template strand is present to co-localize the conjugate and its target. For efficient alkylation in this example, a single-stranded region of the target is required adjacent to the QM conjugate. Most importantly, the intrastrand self adducts formed between the PNA and its attached QM remained active and reversible over more than eight days in aqueous solution prior to reaction with a chosen target added subsequently.
C-peptide and its correlation to parameters of insulin resistance in the metabolic syndrome.
Source
King Fahd Medical Research Center, King Abdulaziz University, P. O. Box 80216, Jeddah 21589, Saudi Arabia. ma.kamal@live.com.
Abstract
The progress of metabolic syndrome (MetS) continues with the onset of type-2 diabetes mellitus (Type-2 DM) along with linkage to other disorders such as neurodegenerative, especially Alzheimer's disease (AD), via oxidative stress and low grade systemic inflammatory process. Type-2 DM and AD are health disorders of priority research. The treatment for an individual suffering with Type-2 DM and/or AD requires monitoring by clinicians. The aim of this study was to investigate the role of C-peptide and its correlation to insulin resistance, body mass index (BMI), β cell function, insulin sensitivity, lipid profile and hemoglobin A1c (HbA1c). The study was designed to include 96 Type-2 DM individuals from India. 58.3% males and 41.7% females were selected and fasting blood samples were collected for estimation of fasting C-peptide, fasting blood sugar (FBS), postprandial blood sugar (PPBS), HbA1c and lipid profile. Analysis was done on Hitachi912 and Elecsys 2010 using Roche reagents and standard controls. Anthropometries to calculate BMI and β cell function, insulin sensitivity, and insulin resistance were obtained. The statistical tool ANOVA, followed by calculation of p-value and r-value were applied for investigating correlation of C-peptide levels to those of high density lipoprotein-C (HDL-C), low density lipoprotein-C (LDL-C), triglycerides (TGL), HbA1c, β cell function, insulin sensitivity and insulin resistance. Highly significant positive correlations were observed in different quantiles of C-peptide levels to the studied parameters of MetS, BMI and % β cell function. Lower HDL-C level was found to be significantly related to higher C-peptide levels. Similarly, TGL and C-peptide levels displayed a significant positive correlation. A significant negative correlation was observed between C-peptide quantiles and % sensitivity. Thus, insulin resistance showed a positive correlation until the fourth quantile. No significant correlation was observed between C-peptide and HbA1c levels. This study demonstrates that assessment of C-peptide levels is a useful tool to monitor the progress of MetS among patients suffering from Type-2 DM and AD, as these disorders are intertwined to each other by common metabolic pathways. Assessment of C-peptide levels, along with HDL-C levels, in patients can be used to monitor insulin resistance.
- PMID:
- 22229322
- [PubMed - as supplied by publisher]
Click Chemistry Facilitates Formation of Reporter Ions and Simplified Synthesis of Amine-Reactive Multiplexed Isobaric Tags for Protein Quantification.
Abstract
We report the development of novel reagents for cell-level protein quantification, referred to as Caltech Isobaric Tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side-chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d5-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cut-off problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.
Fast and Selective Modification of Thiol Proteins/Peptides by N-(Phenylseleno)phthalimide.
Source
Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, OH, 45701, USA.
Abstract
We previously reported that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used to selectively derivatize thiols for mass spectrometric analysis, and the introduced selenium tags are useful as they could survive or removed with collision-induced dissociation (CID). Described herein is the further study of the reactivity of various protein/peptide thiols toward NPSP and its application to derivatize thiol peptides in protein digests. With a modified protocol (i.e., dissolving NPSP in acetonitrile instead of aqueous solvent), we found that quantitative conversion of thiols can be obtained in seconds, using NPSP in a slight excess amount (NPSP:thiol of 1.1-2:1). Further investigation shows that the thiol reactivity toward NPSP reflects its chemical environment and accessibility in proteins/peptides. For instance, adjacent basic amino acid residues increase the thiol reactivity, probably because they could stabilize the thiolate form to facilitate the nucleophilic attack of thiol on NPSP. In the case of creatine phosphokinase, the native protein predominately has one thiol reacted with NPSP while all of four thiol groups of the denatured protein can be derivatized, in accordance with the corresponding protein conformation. In addition, thiolpeptides in protein/peptide enzymatic digests can be quickly and effectively tagged by NPSP following tri-n-butylphosphine (TBP) reduction. Notably, all three thiols of the peptide QCCASVCSL in the insulin peptic digest can be modified simultaneously by NPSP. These results suggest a novel and selective method for protecting thiols in the bottom-up approach for protein structure analysis.
Evaluation of a Solid-Supported Tagging Strategy for Mass Spectrometric Analysis of Peptides.
Abstract
We have explored two divinylbenzene cross-linked polystyrene supports for use in a solid-supported N-terminal peptidetagging strategy. Resin-bound tags designed to be cleaved in a single step at the N-terminus of peptides have been devised and explored as peptide N-terminal tagging reagents (constructs) for subsequent mass spectrometric analysis. While the brominated tagging approach shows promise, the use of these specific solid supports has drawbacks, in terms of tagging reaction scale, for real applications in proteomics.
- PMID:
- 22220996
- [PubMed - as supplied by publisher]
Evaluation of a recombinant multi-epitope peptide for serodiagnosis of Toxoplasma gondii infection.
Source
Department of Laboratory Medicine, The Second Affiliated Hospital, Nanjing Medical University, Nanjing, 210011, China.
Abstract
Detection of Toxoplasma gondii (T. gondii) infection with sensitive and specific methods is a key step to prevent and treat the toxoplasmosis. Among the available diagnostic tests, serology is commonly used. Despite the fact that serological tests give satisfactory results, the production of reliable reagents remains laborious and expensive. There is therefore a real need to acquire specific and effective recombinant antigens for the sero-diagnosis of T. gondii infection. In this study, a multi-epitope peptide was designed and successfully expressed in E. coli, then IgG and IgM Enzyme-linked immunosorbent assay (ELISA) were developed and evaluated. Our results showed that the new multi-epitope antigen is one of the most promising recombinant antigens which could be used in routine screening of human toxoplasmosis.
Optimization of Peptide-Based ELISA for Serological Diagnostics: A Retrospective Study of Human Monkeypox Infection.
Source
1 Najít Technologies, Inc. , Portland, Oregon.
Abstract
Abstract Although smallpox has been eradicated, other diseases caused by virulent orthopoxviruses such as monkeypox virus (MPV) remain endemic in remote areas of western and central sub-Saharan Africa, and represent a potential biothreat due to international travel and/or inadvertent exposure. Unfortunately, extensive antigenic cross-reactivity among orthopoxviruses presents a challenge to serological diagnosis. We previously reported a 20mer peptide-based ELISA that identified recent MPV infection with >90% sensitivity and >90% specificity. However, the sensitivity of this approach was not determined with samples obtained at later time points after antibody titers had declined from their peak levels. To improve assay sensitivity for detecting MPV-specific antibodies at later time points, we compared diagnostic 20mer peptides to 30mer peptides. In addition, optimal 30mer peptides were tested in combination or after conjugating selected peptides to a carrier protein (bovine serum albumin) to further improve assay performance. An optimized combination of four unconjugated 30mer peptides provided 100% sensitivity for detecting MPV infection at 2-6 months post-infection, 45% sensitivity for detecting MPV infection at >2 years post-infection, and 99% specificity. However, an optimized combination of two peptide conjugates provided 100% sensitivity for detecting MPV infection at 2-6 months post-infection, 90% sensitivity for detecting MPV infection at >2 years post-infection, and 97% specificity.Peptide-based ELISA tests provide a relatively simple approach for serological detection of MPV infection. Moreover, the systematic approach used here to optimize diagnostic peptide reagents is applicable to developing improved diagnostics to a broad range of other viruses, and may be particularly useful for distinguishing between closely-related viruses within the same genus or family.
Integrated SDS Removal and Peptide Separation by Strong-Cation Exchange Liquid Chromatography for SDS-Assisted Shotgun Proteome Analysis.
Abstract
We report an improved shotgun method for analyzing proteomic samples containing sodium dodecyl sulfate (SDS). This method is based on the use of strong-cation exchange (SCX) liquid chromatography (LC) for SDS removal that can be integrated with peptide separation as the first dimension of the two-dimensional LC tandem mass spectrometry workflow. To optimize the performance of SDS removal, various experimental conditions, including the concentrations of chemical reagents and salts in the sample, the SDS concentration and the SCX mobile phase composition, were investigated. It was found that a peptide recovery rate of about 90% could be achieved while removing SDS efficiently. One key finding was that, by increasing the SDS concentration to a certain level (0.5%) in the digested peptide sample, the sample recovery rate could be increased. The peptide recovery rate of BSA digests was found to be 90.6±1.0% (n=3) and SDS in the SCX fractions collected was not detectable by pyrolysis GC-MS, i.e., below the detection limit of 0.00006% for the un-desalted SCX fractions. The peptide recovery rates were found to be 90.9%±2.7 (n=3) and 89.5%±0.5% (n=3) for the digests of the membrane-protein-enriched fractions of E. coli cell lysates and MCF-7 breast cancer cell line, respectively. Compared to the methods that use acid-labile surfactants, such as RapiGest and PPS, for the MCF-7 membrane fraction sample, the SDS method identified, on average (n=3), more peptides (~5%) and proteins (~16%) than the RapiGest method, while the RapiGest method identified more peptides (~21%) and proteins (~7%) from the E. coli membrane fraction than the SDS method. In both cases, the two methods identified more peptides and proteins than the PPS method. Since SCX is widely used as the first dimension of 2D-LC MS/MS, integration of SDS removal with peptide separation in SCX does not add any extra steps to the sample handling process. We demonstrated the application of this method for 2D-LC MS/MS profiling of the MCF-7 membrane protein fraction and identified 6889 unique peptides, corresponding to 2258 unique proteins or protein groups from two replicate experiments with a falsepeptide discovery rate of ~0.8%, compared to 5172 unique peptides and 1847 unique proteins identified by the RapiGest method.
iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics.
Source
Broad Institute of MIT and Harvard, United States.
Abstract
Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ or TMT allow for relative quantification ofpeptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, non-isobaric labeling with mTRAQ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly 3-fold more phosphopeptides (12,129 vs. 4,448) and nearly 2-fold more proteins (2,699 vs. 1,597) than mTRAQ labeling. While most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are co-fragmented in the same precursor isolation window, dampening observed ratios toward unity. However, due to tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of non-regulated peptides show that iTRAQ quantification is less accurate but not as variable as mTRAQ quantification.
Reusable Amperometric Biosensor for Measuring Protein Tyrosine Kinase Activity.
Source
Department of Biological Science and Technology and ‡Institute of Molecular Medicine and Bioengineering, National Chiao Tung University , Hsinchu, Taiwan, ROC.
Abstract
This work presents a simple, low-cost and reusable label-free method for detecting protein tyrosine kinase activity using a tyrosinase-based amperometric biosensor (tyrosine kinase biosensor). This method is based on the observation that phosphorylation can block the tyrosinase-catalyzed oxidation of tyrosine or tyrosyl residue in peptides. Therefore, the activity of p60c-src protein tyrosine kinase (Src) on the developed tyrosine kinase biosensor could be quickly determined when its specific peptide substrate, p60c-src substrate I, was used. The tyrosine kinase biosensor was highly sensitive to the activity of Src with a linear dynamic range of 1.9-237.6 U/mL and the lowest detection limit of 0.23 U/mL. Interestingly, the tyrosine kinase activity can be measured using the developed tyrosine kinase biosensor repetitively without regeneration. The inhibitory effect of various kinase inhibitors on the Src activity could be determined on the tyrosine kinase biosensor. Src-specific inhibitors, PP2 and Src inhibitor I, effectively suppressed Src activity, whereas PD153035, an inhibitor of the epidermal growth factor receptor, was ineffective. Staurosporine, a universal kinase inhibitor, inhibited Src activity in an ATP concentration-dependent manner. These results suggests that the activities of tyrosine kinases and their behaviors toward various reagents can be effectively measured using the developed tyrosine kinase biosensor.
Impact of immunization technology and assay application on antibody performance - a systematic comparative evaluation.
Source
Research and Development, SDIX, Newark, Delaware, United States of America.
Abstract
Antibodies are quintessential affinity reagents for the investigation and determination of a protein's expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assays (ELISA), among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein), DNA prime/protein fragment-boost ("DNA immunization"; 3 per protein), and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot). Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry), although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides) or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used.
Sequential multiplexed analyte quantification using peptide immunoaffinity enrichment coupled to mass spectrometry.
Source
Fred Hutchinson Cancer Research Center, United States.
Abstract
Peptide immunoaffinity enrichment coupled to selected reaction monitoring (SRM) mass spectrometry (immuno-SRM) has emerged as a technology with great potential for quantitative proteomic assays. One advantage over traditional immunoassays is the tremendous potential for concurrent quantification of multiple analytes from a given sample (i.e. multiplex analysis). We sought to explore the capacity of the immuno-SRM technique for analyzing large numbers of analytes by evaluating the multiplex capabilities and demonstrating the sequential analysis of groups of peptides from a single sample. To evaluate multiplex analysis, immuno-SRM assays were arranged in groups of 10, 20, 30, 40, and 50peptides using a common set of reagents. The multiplex immuno-SRM assays were used to measure syntheticpeptides added to plasma covering several orders of magnitude concentration. Measurements made in large multiplex groups were highly correlated (r2 ≥ 0.98) and featured good agreement (bias ≤ 1%) compared to single plex assays or a 10-plex configuration. The ability to sequentially enrich sets of analyte peptides was demonstrated by enriching groups of 10 peptides from a plasma sample in a sequential fashion. The data show good agreement (bias ≤ 1.5%) and similar reproducibility regardless of enrichment order. These significant advancements demonstrate the utility of immuno-SRM for analyzing large numbers of analytes, such as in large biomarker verification experiments or in pathway-based targeted analysis.
Nef Decreases HIV-1 Sensitivity to Neutralizing Antibodies that Target the Membrane-proximal External Region of TMgp41.
Source
Section of Infectious Diseases, Imperial College London, London, United Kingdom.
Abstract
Primate lentivirus nef is required for sustained virus replication in vivo and accelerated progression to AIDS. While exploring the mechanism by which Nef increases the infectivity of cell-free virions, we investigated a functional link between Nef and Env. Since we failed to detect an effect of Nef on the quantity of virion-associated Env, we searched for qualitative changes by examining whether Nef alters HIV-1 sensitivity to agents that target distinct features of Env. Nef conferred as much as 50-fold resistance to 2F5 and 4E10, two potent neutralizing monoclonal antibodies (nAbs) that target the membrane proximal external region (MPER) of TMgp41. In contrast, Nef had no effect on HIV-1 neutralization by MPER-specific nAb Z13e1, by the peptide inhibitor T20, nor by a panel of nAbs and other reagents targeting gp120. Resistance to neutralization by 2F5 and 4E10 was observed with Nef from a diverse range of HIV-1 and SIV isolates, as well as with HIV-1 virions bearing Env from CCR5- and CXCR4-tropic viruses, clade B and C viruses, or primary isolates. Functional analysis of a panel of Nef mutants revealed that this activity requires Nef myristoylation but that it is genetically separable from other Nef functions such as the ability to enhance virus infectivity and to downregulate CD4. Glycosylated-Gag from MoMLV substituted for Nef in conferring resistance to 2F5 and 4E10, indicating that this activity is conserved in a retrovirus that does not encode Nef. Given the reported membrane-dependence of MPER-recognition by 2F5 and 4E10, in contrast to the membrane-independence of Z13e1, the data here is consistent with a model in which Nef alters MPER recognition in the context of the virion membrane. Indeed, Nef and Glycosylated-Gag decreased the efficiency of virion capture by 2F5 and 4E10, but not by other nAbs. These studies demonstrate that Nef protects lentiviruses from one of the most broadly-acting classes of neutralizing antibodies. This newly discovered activity for Nef has important implications for anti-HIV-1 immunity and AIDS pathogenesis.
Polymeric Dibromomaleimides As Extremely Efficient Disulfide Bridging Bioconjugation and Pegylation Agents.
Source
Department of Chemistry, University of Warwick , Coventry CV4 7AL, U.K.
Abstract
A series of dibromomaleimides have been shown to be very efficacious at insertion into peptidic disulfide bonds. This conjugation proceeds with a stoichiometric balance of reagents in buffered solutions in less than 15 min to give discrete products while maintaining the disulfide bridge and thus peptide conformation. The insertion is initiated by disulfide reduction using a water-soluble phosphine, tris(2-carboxyethyl)phosphine (TCEP) which allows for subsequent substitution of the two maleimide bromides by the generated thiols. Reaction of salmon calcitonin (sCT) with 2,3-dibromomaleimide (1.1 excess) in the presence of TCEP (1.1 equiv) in aqueous solution at pH 6.2 gives complete production of a single conjugate which requires no workup. A linear methoxy poly(ethylene glycol) (PEG) was functionalized via a Mitsunobu reaction and used for the successful site-specific and rapid pegylation of sCT. This reaction occurs in 15 min with a small stoichiometry excess of the pegylating agent to give insertion at the disulfide with HPLC showing a single product and MALDI-ToF confirming conjugation. Attempts to use the group in a functional ATRP polymerization initiator led to polymerization inhibition. Thus, in order to prepare a range of functional polymers an indirect route was chosen via both azide and aniline functional initiators which were converted to 2,3-dibromomaleimides via appropriate reactions. For example, the azide functional polymer was reacted via a Huisgen CuAAC click reaction to an alkyne functional 2,3-dibromomaleimide. This new reagent allowed for the synthesis of conjugates of sCT with comb polymers derived from PEG methacrylic monomers which in addition gave appropriate cloud points. This reaction represents a highly efficient polymer conjugation method which circumvents problems of purification which normally arise from having to use large excesses of the conjugate. In addition, the tertiary structure of the peptide is efficiently maintained.
Discovery of competing anaerobic and aerobic pathways in umpolung amide synthesis allows for site-selective amide 18O-labeling.
Source
Department of Chemistry and Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37235-1822.
Abstract
The mechanism of umpolung amide synthesis was probed by interrogating potential sources for the oxygen of the product amide carbonyl that emanates from the α-bromo nitroalkane substrate. Using a series of (18)O-labeled substrates and reagents, evidence is gathered to advance two pathways from the putative tetrahedral intermediate. Under anaerobic conditions, a nitro-nitrite isomerization delivers the amide oxygen from nitro oxygen. The same homolytic nitro-carbon fragmentation can be diverted by capture of the carbon radical intermediate with oxygen gas (O(2)) to deliver the amide oxygen from O(2). This understanding was used to develop a straightforward protocol for the preparation of (18)O-labeled amides in peptides by simply performing the umpolung amide synthesis reaction under an atmosphere of .
Optimization of Electron Transfer Dissociation via Informed Selection ofReagents and Operating Parameters.
Source
Department of Chemistry, University of Virginia , McCormick Rd., Charlottesville, Virginia 22904, United States.
Abstract
Electron transfer dissociation (ETD) has improved the mass spectrometric analysis of proteins and peptides with labile post-translational modifications and larger intact masses. Here, the parameters governing the reaction rate of ETD are examined experimentally. Currently, due to reagent injection and isolation events as well as longer reaction times, ETD spectra require significantly more time to acquire than collision-induced dissociation (CID) spectra (>100 ms), resulting in a trade-off in the dynamic range of tandem MS analyses when ETD-based methods are compared to CID-based methods. Through fine adjustment of reaction parameters and the selection of reagents with optimal characteristics, we demonstrate a drastic reduction in the time taken per ETD event. In fact, ETD can be performed with optimal efficiency in nearly the same time as CID at low precursor charge state (z = +3) and becomes faster at higher charge state (z > +3).
A rapid method for cross-species quantitation of apolipoproteins A1, B48 and B100 in plasma by ultra-performance liquid chromatography/tandem mass spectrometry.
Source
Merck Research Laboratories, 126 E. Lincoln Ave., Rahway, NJ, 07065, USA. michael_lassman@merck.com.
Abstract
Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible,peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 μL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner. Copyright © 2011 John Wiley & Sons, Ltd.
Copyright © 2011 John Wiley & Sons, Ltd.
Enzyme-linked immunosorbent assay for okadaic acid: investigation of analytical conditions and sample matrix on assay performance.
Source
Agricultural University of Hebei, Faculty of Food Science and Technology, Baoding 071001, People's Republic of China.
Abstract
Okadaic acid (OA) is a lipophilic marine biotoxin. In this study, OA was coupled with the carrier proteins keyhole limpet hemocyanin and bovine serum albumin as immunity and detection antigens by an active ester method. The polyclonal antibody against OA was prepared successfully, an indirect competitive ELISA (ciELISA) developed for the detection of OA in shellfish, and the reactive conditions of ciELISA optimized. The LOD (15% inhibition concentration) for the microwell plates was 1.28 +/- 0.38 ng/mL, corresponding to 12.8 +/- 3.8 ng/g. Two extraction methods were used to remove shellfish matrix interference with high recovery of spiked samples, and the methanol extraction of shellfish mussel was analyzed after dilution in phosphate-buffered saline. For validation of the optimized ciELISA, spiked and natural samples were analyzed by ciELISA, and HPLC with fluorescence detection. The correlation of linear regression equation was y = 1.0064x - 10.234, and the correlation coefficient was 0.9347. From the results of the comparative study, the established ciELISA assay using polyclonal antibody against OA could be used in preliminary screening of suspicious shellfish samples.
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