MALDI Mass Research Articles

The crucial role of stability of intercalating agent for DNA binding studies in DMSO/water system

In recent years, extensive research has been directed towards understanding the interactions between various zinc complexes with DNA, specifically delving into their intercalation and binding behaviors. The binding of zinc complexes to DNA is particularly intriguing due to their distinctive intercalating capabilities. This study unveils a remarkable phenomenon observed with a specific Zn complex, ([B-Zn-N3], where B is a Schiff base ligand), during DNA intercalation investigations in the popular DMSO-Water binary solvent mixture. An unanticipated observation revealed time-dependent changes in the UV–visible absorption spectroscopic studies, coupled with the existence of an isosbestic point. This observation questions the stability of the intercalating agent itself during the intercalation process. The emergence of a decomposed product during the intercalation study has been confirmed through various analytical techniques, including CHN analysis, MALDI mass, XPS, Raman spectroscopy, and Powder XRD. The change in the chemical species on intercalation is further substantiated by theoretical studies, adding depth to our understanding of the intricate dynamics at play during DNA intercalation with the [B-Zn-N3] complex in the DMSO-Water system.

Retracted] Facile and straightforward synthesis of Hydrazone derivatives

This study was undertaken to report the swift, facile and convenient synthesis of novel hydrazones obtained by condensation reaction between 2‐Amino‐3‐formylchromone and hydrazine derivatives. Various characterization techniques such as MALDI Mass, FTIR, 1HNMR and 13CNMR spectrum analysis was done to determine the chemical structure of these novel six hydrazones. Furthermore, UV‐Vis and fluorescence spectra was studied to calculate λmax and εmax. These hydrazones are quite useful for their facile synthesis and chemical structure. Such hydrazones require separate clinical research to find their applications in biomedical fields.

Advanced development of dairy farm waste-based biocarbon-reinforced unsymmetrical structured bio-phenolic polybenzoxazine composites

In the present work, an attempt has been made to develop cow manure carbon-reinforced hybrid biophenol-based benzoxazine composites for antifouling coating applications. Bio-based benzoxazine with unsymmetrical molecular structure was synthesized using the mixture of a combination of cardanol and eugenol with diaminodiphenyl methane and paraformaldehyde, and the hybrid-benzoxazine obtained was characterized using different analytical techniques, viz., Fourier transform infrared, nuclear magnetic resonance and MALDI mass. Bio-based benzoxazine was further reinforced with varying weight percentages (1, 3 and 5 wt%) of biocarbon derived from cow manure to obtain hybrid composite coatings. The hybrid benzoxazine matrix and composites were studied for their thermal behaviour, contact angle (CA), morphology, corrosion-resistant behaviour and antifouling character to utilize them as coatings materials for different industrial applications. Results obtained from different studies inferred that the biocarbon-reinforced composites possess an enhanced value of glass transition temperature (249°C), high char yield (38.4%), improved CA (105.6°), higher efficiency of corrosion protection against mild steel surface (98%) and improved antimicrobial activity.

Synthesis and characterization of GO doped bio-resource based composites for NLO and multifaceted applications

The objective of the present work is to synthesize three different types of cardanol based benzoxazines such as cardanol-{1,4-bis(4-aminophenoxy)ethane}benzoxazine (CBAE2Bz), cardanol-{1,4-bis(4-aminophenoxy)butane} benzoxazine (CBAE4Bz) and cardanol-{1,4-bis(4-aminophenoxy)octane}benzoxazine (CBAE8Bz) by varying aliphatic ether link chain length through solventless method. The benzoxazines obtained with varying chain length were converted in to three different types of composites using paraphenylenediamine (PPDA) functionalized gaphene oxide (f-GO) as nano-reinforcement with varying weight percentage and were characterized by different analytical techniques. The molecular structure of the monomers was confirmed from Fourier transform infrared (FTIR) spectroscopy, 1HNMR, 13C NMR and MALDI mass. Data obtained from thermal studies infer that the prepared composites possess good thermal stability and enhanced values of Tg with respect to weight percent of functionalized graphene oxide content. However, the values of thermal stability, Tg, dielectric constant and dielectric loss were marginally decreased with respect to increase in length of the spacer aliphatic chain. The SEM and TEM studies confirm the homogeneous dispersion and single layer thickness of graphene sheet, respectively. The feasibility of usage of f-GO/PCAE2Bz composite in both gram positive bacteria (Bacillus subtilis, Staphylococcus aureus) and gram negative bacteria (Pseudomonas aeruginosa, Escherichia coli) environments were checked and the results obtained were discussed. Further, third-order NLO properties of open and closed aperture Z–scan results conclude that GO doped bio-resource based composites exhibit saturable absorption and self-defocusing type optical non-linearity. The third-order NLO susceptibility (χ3) of GO doped bio-resource based composites are increased with respect to quantity of reinforcement of GO to bio-based polymer. The maximum value of 1.03X 10−4 esu was observed and this nonlinearity is thermal in nature. Thus, the prepared composites may find wide range of applications in diverse surrounding conditions.

Novel biologically active metallophthalocyanines as promising antioxidant-antibacterial agents: Synthesis, characterization and computational properties

This study reports on the synthesis and characterization of non-peripherally tetrasubstituted zinc(II), copper(II) and cobalt(II) phthalocyanines with 4-(trifluoromethylthio)phenoxy substituents. Synthesized compounds were characterized by FT-IR, 1H NMR, MALDI mass, elemental analysis and UV–Vis spectroscopic methods. By using some parameters obtained from quantum chemical calculations, the activity of the studied ligand (1) against metal atoms was investigated. Based on the obtained results, the NMR, IR and UV–Vis spectra of the ligand (1) in different phases were compared with experimental results. Also, theorical and experimental UV–Vis spectrum values of metallophthalocyanines (2–4) were investigated. Additionally, the possible use of all synthesized metallophthalocyanines for antioxidant and antibacterial agents were investigated. The obtained results showed that CoPc (4) had very good antioxidant activities among others. Although all of the tested metallophthalocyanines (2–4) had some antibacterial activities, ZnPc (2) had the best antibacterial effect against five different bacteria among other metallophthalocyanines tested.

Hybrid photoluminescent materials containing a benzobisthiazole core for liquid crystal and gel applications.

Tetra- and hexacatenar amide compounds containing a linear centrosymmetric benzobisthiazole core were synthesized with good yields. These compounds were characterized and their structures confirmed by elemental analysis, and FT-IR, Maldi mass and NMR spectroscopy. All compounds exhibited excellent thermal stability up to 330 °C. The tetracatenar series containing a double substitution in the meta positions did not show mesomorphic behaviour, whereas the hexacatenar and tetracatenar series having a double substitution in the meta and para positions showed liquid crystal properties with optical textures typical of columnar mesophases corroborated by POM analysis. The mesomorphic properties were dependent on the length, number and position of alkoxy chains attached at the end of the rigid core. XRD studies of the hexacatenar series showed the hexagonal columnar structure of the mesophases. Photoluminescence properties in solution were observed in the visible region, with good quantum yields. In the solid state, these compounds behave as blue emitters and they are able to change colour with acid or base addition. The hexacatenar benzobisthiazole compound with an alkoxy chain of 14 carbons presented properties of a supergelator in chloroform, leading to the formation of a fluorescent organogel material with fluorescence emission in the blue region.

Synthesis and characterization of cardanol based fluorescent composite for optoelectronic and antimicrobial applications

The present study attempted to prepare para phenylene diamine functionalized multiwalled carbon nanotube (f-MWCNT) incopoarated polymer of a new pyrene core containing cardanol based benzoxazine (PyCBz) composite (f-MWCNT-PPyCBz). For this, a new monomer of pyrene core containing cardanol based benzoxazine (PyCBz) was synthesized through solventless method and its structure was confirmed using Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance spectroscopy (1H &13C) and MALDI mass. In addition, multiwalled carbon nanotube (MWCNT) was functionalized using para phenylene diamine that acted as effective filler for preparation the composite materials. The curing behavior and thermal stability of para phenylene diamine functionalised multiwalled carbon nanotube (MWCNT) incopoarated pyrene core containing cardanol based polybenzoxazine(PPyCBz) composites were studied by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Antimicrobial studies were also carried out using Streptococcus pyogens, Bacillus cereus, Staphylococcus aureus and Escherichia coli. In short, the obtained f-MWCNT/PPyCBz nanocomposites possess high dielectric constant, good antibacterial and photo luminescent properties, which may find optical, antimicrobial and high dielectric applications.

Lysophosphatidic acid triggers mast cell-driven atherosclerotic plaque destabilization by increasing vascular inflammation

Lysophosphatidic acid (LPA), a bioactive lysophospholipid, accumulates in the atherosclerotic plaque. It has the capacity to activate mast cells, which potentially exacerbates plaque progression. In this study, we thus aimed to investigate whether LPA contributes to plaque destabilization by modulating mast cell function. We here show by an imaging mass spectrometry approach that several LPA species are present in atherosclerotic plaques. Subsequently, we demonstrate that LPA is a potent mast cell activator which, unlike other triggers, favors release of tryptase. Local perivascular administration of LPA to an atherosclerotic carotid artery segment increases the activation status of perivascular mast cells and promotes intraplaque hemorrhage and macrophage recruitment without impacting plaque cell apoptosis. The mast cell stabilizer cromolyn could prevent intraplaque hemorrhage elicited by LPA-mediated mast cell activation. Finally, the involvement of mast cells in these events was further emphasized by the lack of effect of perivascular LPA administration in mast cell deficient animals. We demonstrate that increased accumulation of LPA in plaques induces perivascular mast cell activation and in this way contributes to plaque destabilization in vivo. This study points to local LPA availability as an important factor in atherosclerotic plaque stability.

Synthesis, characterization, conformational analysis of a cyclic conjugated octreotate peptide and biological evaluation of 99mTc-HYNIC-His3-Octreotate as novel tracer for the imaging of somatostatin receptor-positive tumors

Peptides are attracting increasing interest in nuclear oncology for targeted tumor diagnosis and therapy. We therefore synthesized new cyclic octapeptides conjugated with HYNIC by Fmoc solid-phase peptide synthesis. These were purified and analyzed by RP-HPLC, MALDI mass, (1)H NMR, (13)C NMR, HSQC, HMBC, COSY and IR spectroscopy. Conformational analysis of the peptides was performed by circular dichroism spectroscopy, in pure water and trifluoroethanol-water (1:1), revealed the presence of strong secondary structural features like β-sheet and random coils. Labeling was performed with (99m)Tc using Tricine and EDDA as coligands by SnCl(2) method to get products with excellent radiochemical purity >99.5 %. Metabolic stability analysis did not show any evidence of breaking of the labeled compounds and formation of free (99m)Tc. Internalization studies were done and IC(50) values were determined in somatostatin receptor-expressing C6 glioma cell line and rat brain cortex membrane, and the results compared with HYNIC-TOC as standard. The IC(50) values of (99m)Tc-HYNIC-His(3)-Octreotate (21 ± 0.93 nM) and (99m)Tc-HYNIC-TOC (2.87 ± 0.41 nM) proved to be comparable. Biodistribution and image study on normal rat under gamma camera showed very high uptake in kidney and urine, indicating kidney as primary organ for metabolism and route of excretion. Biodistribution and image study on rats bearing C6 glioma tumor found high uptake in tumor (1.27 ± 0.15) and pancreas (1.71 ± 0.03). Using these findings, new derivatives can be prepared to develop (99m)Tc radiopharmaceuticals for imaging somatostatin receptor-positive tumors.

Striatal Alterations of Secretogranin-1, Somatostatin, Prodynorphin, and Cholecystokinin Peptides in an Experimental Mouse Model of Parkinson Disease

The principal causative pathology of Parkinson disease is the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta projecting to the striatum in the brain. The information regarding the expression of neuropeptides in parkinsonism is very limited. Here we have elucidated striatal neuropeptide mechanisms in experimental parkinsonism using the unilateral 6-hydroxydopamine model to degenerate dopamine neurons. A thoroughly controlled sample preparation technique together with a peptidomics approach and targeted neuropeptide sequence collections enabled sensitive detection, identification, and relative quantitation of a great number of endogenous neuropeptides. Previously not recognized alterations in neuropeptide levels were identified in the unilateral lesioned mice with or without subchronic 3,4-dihydroxy-L-phenylalanine administration, the conventional treatment of Parkinson disease. Several of these peptides originated from the same precursor such as secretogranin-1, somatostatin, prodynorphin, and cholecystokinin. Disease-related biotransformation of precursors into individual peptides was observed in the experimental model of Parkinson disease. Several previously unreported potentially biologically active peptides were also identified from the striatal samples. This study provides further evidence that neuropeptides take part in mediating the central nervous system failure associated with Parkinson disease.

Abstract A32: Proteomic signatures of deoxycholic acid and ursodeoxycholic acid treated human colon cancer cells

Abstract A32 Dietary factors play an important role in the pathogenesis of colorectal cancer (CRC), due in part to the influence of the bile acids. While the secondary bile acid, deoxycholic acid (DCA), has been associated with tumor promotion, one tertiary bile acid in particular, ursodeoxycholic acid (UDCA), has been demonstrated to exert chemopreventive properties in the colon. Although its efficacy against high-grade adenomas has already been demonstrated in phase III clinical trials, mechanisms by which UDCA suppresses colon tumorigenesis remain unclear. In order to gain further insight into the tumor modifying properties of the bile acids, we compared the effects of DCA and UDCA on human colon tumor cells (CaCo-2) using proteomic analyses. In the following study, CaCo-2 cells grown at 70% confluence were treated with either 10 µM deoxycholic acid (DCA) or 1 mM ursodeoxycholic acid (UDCA) for 72 hours. For the proteomic analysis, protein was first fractionated by PF2D isochromatofocusing on a Beckman Coulter ProteomeLab PF2D platform. Fractions corresponding to a linear gradient between pH 8.0 and 4.0 were collected and processed through an automated autoloader for further separation by HPRP-PF2D reverse phase chromatography. Two dimensional protein expression maps displaying isoelectric point (pI) versus hydrophobicity were generated by the ProteoView/DeltaVue software package. Pairwise analysis of these complex protein chromatograms demonstrated common peak profiles (signatures) as well as unique peaks (fingerprints) characteristic of UDCA or DCA treatment. One of these protein peaks that exhibited a differential expression pattern between the two drug treatments was identified by MALDI and tandem mass spectrometry sequencing as heat-shock protein 60 (Hsp60). Furthermore, this protein was found to be less oxidized upon treatment with UDCA, suggesting the attenuation of oxidative stress present in cancer cells. Further analyses on the role of Hsp60 as well as resultant diminished oxidative stress will enable the discovery of potential mechanisms by which DCA and UDCA control cellular pathways. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A32.

142 POSTER Interaction of chlorambucil and intercalating aniline mustards with defined DNA sequences using MALDI and ESI mass spectrometry

142 POSTER Interaction of chlorambucil and intercalating aniline mustards with defined DNA sequences using MALDI and ESI mass spectrometry

Monitoring Mouse Prostate Development by Profiling and Imaging Mass Spectrometry

Mass spectrometry-based tissue profiling and imaging are technologies that allow identification and visualization of protein signals directly on thin sections cut from fresh frozen tissue specimens. These technologies were utilized to evaluate protein expression profiles in the normal mouse prostate during development (1-5 weeks of age), at sexual maturation (6 weeks of age), and in adult prostate (at 10, 15, or 40 weeks of age). The evolution of protein expression during normal prostate development and maturation were subsequently compared with 15-week prostate tumors derived from genetically engineered mice carrying the Large T antigen gene under regulation of the prostate-specific probasin promoter (LPB-Tag mouse model for prostate cancer). This approach identified proteins differentially expressed at specific time points during prostate development. Furthermore expression of some of these proteins, for example probasin and spermine-binding protein, were associated with prostate maturation, and prostate tumor formation resulted in their loss of expression. Cyclophilin A, a protein found in other cancers, was differentially alpha-acetylated on the N terminus, and both isoforms appeared during normal prostate and prostate tumor development. Imaging mass spectrometry localized the protein signals to specific prostatic lobes or regions. Thus, tissue profiling and imaging can be utilized to analyze the ontogeny of protein expression during prostate morphogenesis and tumorigenesis and identify proteins that could potentially serve as biomarkers for prostate cancer.

Platelet Factor-4 Is an Indicator of Blood Count Recovery in Acute Myeloid Leukemia Patients in Complete Remission

To investigate whether serum biomarkers can be used to indicate the responsiveness of acute myeloid leukemia to remission induction chemotherapy, we performed MALDI-TOF protein profile analysis of patient sera. The resulting spectra revealed a protein (or peptide) peak at m/z 7764 that varied in intensity; its intensity was much higher in samples from patients in complete remission than in those from patients with resistant disease or in samples taken prior to treatment (at the time of diagnosis). Using fractionation, trypsin digestion, MS/MS, and protein molecular weight analyses, we identified the m/z 7764 protein as platelet factor-4 (PF4). This identification was confirmed by a magnetic bead-based MALDI immunoassay. Statistical comparison of PF4 levels and platelet counts in patient sera revealed a significant positive correlation between the two variables. This study demonstrates that PF4 protein levels are a good indicator for the recovery of blood count in the complete remission of acute myeloid leukemia. The linear positive correlation curve indicates that blood count recovery of platelets to >100,000/mm(3) is equivalent to a serum PF4 recovery level of >2.492 microg/ml.

Highly Efficient Classification and Identification of Human Pathogenic Bacteria by MALDI-TOF MS

Accurate and rapid identification of pathogenic microorganisms is of critical importance in disease treatment and public health. Conventional work flows are time-consuming, and procedures are multifaceted. MS can be an alternative but is limited by low efficiency for amino acid sequencing as well as low reproducibility for spectrum fingerprinting. We systematically analyzed the feasibility of applying MS for rapid and accurate bacterial identification. Directly applying bacterial colonies without further protein extraction to MALDI-TOF MS analysis revealed rich peak contents and high reproducibility. The MS spectra derived from 57 isolates comprising six human pathogenic bacterial species were analyzed using both unsupervised hierarchical clustering and supervised model construction via the Genetic Algorithm. Hierarchical clustering analysis categorized the spectra into six groups precisely corresponding to the six bacterial species. Precise classification was also maintained in an independently prepared set of bacteria even when the numbers of m/z values were reduced to six. In parallel, classification models were constructed via Genetic Algorithm analysis. A model containing 18 m/z values accurately classified independently prepared bacteria and identified those species originally not used for model construction. Moreover bacteria fewer than 10(4) cells and different species in bacterial mixtures were identified using the classification model approach. In conclusion, the application of MALDI-TOF MS in combination with a suitable model construction provides a highly accurate method for bacterial classification and identification. The approach can identify bacteria with low abundance even in mixed flora, suggesting that a rapid and accurate bacterial identification using MS techniques even before culture can be attained in the near future.

Identification of Glioma Neovascularization-related Proteins by Using MALDI-FTMS and Nano-LC Fractionation to Microdissected Tumor Vessels

The identification of angiogenesis-related proteins is important for the development of new antiangiogenic therapies, and such proteins are potential new biomarkers for gliomas. The aim of this study was to identify proteins that are exclusively present in glioma neovasculature and not in the vasculature of normal brain. We combined advanced proteomics techniques to compare the expression profiles of microdissected blood vessels from glioma with blood vessels of normal control brain samples. We measured the enzymatic generated peptide profiles from these microdissected samples by MALDI-FTMS. Subsequently, the samples were fractionated by nano-LC prior to MALDI-TOF/TOF. This combined approach enabled us to identify four proteins that appeared to be exclusively expressed in the glioma blood vessels. Two of these proteins, fibronectin and colligin 2, were validated on tissue sections using specific antibodies. We found that both proteins are present in active angiogenesis in glioma, other neoplasms, and reactive conditions in which neoangiogenesis takes place. This work proves that gel-free mass spectrometric techniques can be used on relatively small numbers of cells generated by microdissection procedures to successfully identify differentially expressed proteins.

Proteomics in cardiovascular surgery

Proteomics describes, analogous to the term genomics, the study of the complete set of proteins present in a cell, organ, or organism at a given time. The genome tells us what could theoretically happen, whereas the proteome tells us what does happen. Therefore, a genomic-centered view of biologic processes is incomplete and does not describe what happens at the protein level. Proteomics is a relatively new methodology and is rapidly changing because of extensive advances in the underlying techniques. The core technologies of proteomics are 2-dimensional gel electrophoresis, liquid chromatography, and mass spectrometry. Proteomic approaches might help to close the gap between traditional pathophysiologic and more recent genomic studies, assisting our basic understanding of cardiovascular disease. The application of proteomics in cardiovascular medicine holds great promise. The analysis of tissue and plasma/serum specimens has the potential to provide unique information on the patient. Proteomics might therefore influence daily clinical practice, providing tools for diagnosis, defining the disease state, assessing of individual risk profiles, examining and/or screening of healthy relatives of patients, monitoring the course of the disease, determining the outcome, and setting up individual therapeutic strategies. Currently available clinical applications of proteomics are limited and focus mainly on cardiovascular biomarkers of chronic heart failure and myocardial ischemia. Larger clinical studies are required to test whether proteomics may have promising applications for clinical medicine. Cardiovascular surgeons should be aware of this increasingly pertinent and challenging field of science.

Identification of phenolic compounds isolated from pigmented rices and their aldose reductase inhibitory activities

Two anthocyanins (cyanidin-3- O-β-glucoside and peonidin-3- O-β-glucoside) and other phenolic (ferulic acid) were, respectively isolated from black and pigmented brown rices ( Oryza sativa L. japonica) and their complete structures were determined by spectroscopic analyses (H NMR, C NMR and MALDI MASS). The HPLC profile of anthocyanins extracted from black rice showed cyanidin-3- O-β-glucoside as the first peak (85%) and peonidin 3- O-β- d-glucoside as the second (15%), while that of pigmented brown rice showed ferulic acid as the first peak (85.7%) and tocols as the second (14.3%). Several tocols were isolated and identified from the unsaponifiable fractions of both rices having some difference on their structures and amounts. The aldose reductase inhibitory activity of isolated compounds was in the following decreasing order: cyanidin-3-glucoside > quercetin > ferulic acid > peonidin-3-glucoside > tocopherol. All isolated compounds showed significant inhibitory activity against aldose reductase suggesting that both pigmented rices might contribute significantly in combating diabetic complications as health-promoting food ingredients in food processing.

Functional Screening of Serine Protease Inhibitors in the Medical Leech Hirudo medicinalis Monitored by Intensity Fading MALDI-TOF MS

The blood-feeding invertebrates are a rich biological source of drugs and lead compounds to treat cardiovascular diseases because they have evolved highly efficient mechanisms to feed on their hosts by blocking blood coagulation. In this work, we focused our attention on the leech Hirudo medicinalis. We performed, by "intensity fading" MALDI-TOF mass spectrometry, a comprehensive detection and functional analysis of pre-existent peptides and small proteins with the capability of binding to trypsin-like proteases related to blood coagulation. Combining "intensity fading MS" and off-line LC prefractionation allowed us to detect more than 75 molecules present in the leech extract that interact specifically with a trypsin-like protease over a sample profile of nearly 2,000 different peptides/proteins in the 2-20-kDa range. Moreover we resolved 232 individual components from the complex mixture, 13 of which have high sequence homology with previously described serine protease inhibitors. Our findings indicate that such extracts are much more complex than expected. Additionally, intensity fading MS, when complemented with LC separation strategies, seems to be a useful tool to investigate complex biological samples, establishing a new bridge between profiling, functional peptidomics, and subsequent drug discovery.

Synthesis of hyperbranched carbosiloxane macromolecule with triallyloxysilane and applications to the dendritic core

A hyperbranched carbosiloxane macromolecule (AB 3-type with Si–O bonds) was prepared by the self-hydrosilation of triallyloxysilane (H–Si(OCH 2CH=CH 2) 3) in the presence of a platinum catalyst. The prepared AB 3-type hyperbranched carbosilane was used for the carbosilane dendrimer as the core, which was progressed until the second generation by the use of iterative controlled methods such as hydrosilation and alcoholysis. NMR, MALDI mass and GPC identified the triallyloxysilane-based hyperbranched carbosilanes and dendrimers. To cite this article: C. Kim, H. Kim, C. R. Chimie 7 (2004) .