Biomedical subjects

Rania Bakry

Publications and source records attributed to Rania Bakry.

12 recordsLinked to original sources

PubMed2007

Monolithic porous polymer layer for the separation of peptides and proteins using thin-layer chromatography coupled with MALDI-TOF-MS.

Plates for thin-layer chromatography (TLC) with an attached layer of porous polymer monolith have been prepared and used for the separation of small molecules, peptides, and proteins. The 50-200-mum. thin poly(butyl methacrylate-co-ethylene dimethacrylate) layers were prepared in situ using UV-initiated polymerization. Precise control of the reaction conditions enables the preparation of monolithic layers with a well-defined porous structure that determines the chromatographic performance. Compared to conventional TLC and high-performance TLC using precoated layers based on silica, the small layer thickness and absence of any binder is expected to improve both retention characteristics and separation efficiency of the polymer-based monolithic thin-layer chromatographic plates. Spots of the separated compounds were first detected using typical UV imaging. Since the monolithic thin layers can be also prepared directly on the stainless steel MALDI carrier plate, the separation in TLC format can be coupled with MALDI-TOF-MS. Application of a conventional MALDI matrix facilitated desorption and ionization of peptides and proteins for molecular weight determination of the separated compounds.

Chromatography, Thin Layer↗

PubMed2007

A new analytical material-enhanced laser desorption ionization (MELDI) based approach for the determination of low-mass serum constituents using fullerene derivatives for selective enrichment.

60]fullerene derivatives (dioctadecyl methano[60]fullerene, [60]fullerenoacetic acid, and IDA-[60]fullerene) were prepared and subjected to a comprehensive characterization study including protein binding properties and capacity. These fullerene derivatives were successfully applied as material-enhanced laser desorption/ionization (MELDI) carrier materials. It is shown that diverse functionalities result in characteristic human serum peak patterns (m/z 2000-20 000) in terms of signal intensity as well as the number of detectable masses. In addition, the fullerene derivatives clearly provided differences in the low molecular weight mass region (m/z 1000-4000) after elution of the adsorbed serum constituents, and [60]fullerenoacetic acid was the most effective carrier material. Novel high-speed, monolithic, high-resolution capillary columns, prepared by thermally initiated copolymerization of methylstyrene (MSt) and 1,2-bis(p-vinylphenyl)ethane (BVPE) were employed for eluate separation and target spotting. Thus, serum compounds in the low-mass range were successfully fractionated and subjected to MALDI-MS/MS analysis. This contribution, hence, proposes a new "top-down" strategy for proteome research enabling protein profiling as well as biomarker identification in the low-mass range using selective enrichment, high-resolution separation, and offline MALDI-MS/MS evaluation.

Adsorption↗

PubMed2007

Mass spectrometric identification of serum peptides employing derivatized poly(glycidyl methacrylate/divinyl benzene) particles and mu-HPLC.

Biomarkers play a key role in preclinical screening and diagnosis of a disease. Various support materials are utilized for this task, in combination with MALDI-TOF-MS. The way to effectively bind serum contents and their profiling is well-elaborated by the material-enhanced laser desorption ionization (MELDI) approach. In this particular work, focus is placed on the development of a strategy to identify low molecular weight serum peptides. Poly(GMA/DVB) is derivatized in a way to achieve an affinity termed as immobilized metal ion affinity chromatography (IMAC). Iminodiacetic acid (IDA) is used as a chelating ligand, whereas copper (Cu2+) acts as a metal ion for complexing peptides and proteins out of blood serum. Polymer binds the serum compounds over a broad mass range, which includes low mass peptides and high mass albumin (66 kDa). Bound contents are eluted from material by an acetonitrile/trifluoroacetic acid mixture, which proves the reversible nature of metal and amino acid linkage. Polystyrene/divinyl benzene (PS/DVB) monolithic capillary column is used for fractionation through RP-HPLC, prior to the target spotting. The tandem TOF fragment ion mass spectra of each fraction is acquired and used to search against the Swiss-Prot database, using the Mascot search engine for the identification of peptides.

Amino Acid Sequence↗

PubMed2006

Material-enhanced laser desorption/ionization (MELDI)--a new protein profiling tool utilizing specific carrier materials for time of flight mass spectrometric analysis.

Over the past couple of years, proteomics pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian, breast, or prostate cancer, without identification of single biomarkers. MALDI-TOF MS, for example, offers a simple approach for fast and reliable protein profiling, especially by using carrier materials with various physical and chemical properties, in combination with a MALDI matrix. This approach is referred to as material-enhanced laser desorption/ionization (MELDI). In this paper, we report the development and application of derivatized carrier materials [cellulose, silica, poly(glycidyl methacrylate/divinylbenzene) (GMA/DVB) particles, and diamond powder] for fast and direct MALDI-TOF MS protein profiling. The applicability of MELDI for rapid protein profiling was evaluated with human serum samples. These carriers, having various hydrophobicities, resulted in characteristic mass fingerprints, even if all materials were derivatized with iminodiacetic acid (IDA) to yield an immobilized metal affinity chromatography (IMAC) functionality. Our study demonstrates that analyzing complex biological samples, such as human serum, by employing different MELDI carrier materials yielded type- and size-dependent performance variation.

Indicators and Reagents↗

PubMed2006

Progress in capillary electrophoresis of biomarkers and metabolites between 2002 and 2005.

Biomarker discovery and metabolite research is a fast-growing and extremely important domain not only for the early detection of certain diseases but also for controlling its progress as well as in pharmaceutical investigations. For the analytical separation and identification, CE plays an indisputable role. Capillary systems enhancing different selectivity are applied and connected to different kind of detection systems. As the choice of buffer and its composition is responsible for a successful separation, special emphasis is put on solvent effects in this review. Altogether the most important capillary electrophoretic techniques applied for biomarker and metabolites analysis published between 2002 and 2005 are summarized and discussed.

Animals↗

PubMed2006

Progress in capillary electrophoresis coupled to matrix-assisted laser desorption/ionization - time of flight mass spectrometry.

In this review the most important techniques developed to hyphenate CE to MALDI-TOF-MS are summarized. The principles of the different interfaces and ways to solve the hyphenation problem are explained and discussed in detail. The most important applications especially in the proteomic field are reviewed, and the advantages of CE-MALDI-TOF-MS for the analysis of these compounds compared to other techniques such as ESI-MS are exhaustingly discussed from a critical point of view. CE coupled to MALDI-TOF-MS has started to overpass traditionally used CE-coupling techniques, especially CE-ESI-MS, offering the possibility to analyze samples of interest even weeks after CE analysis and using multiplexing systems for high-sample throughput.

Electrophoresis, Capillary↗

PubMed2006

Monolithic poly(glycidyl methacrylate-co-divinylbenzene) capillary columns functionalized to strong anion exchangers for nucleotide and oligonucleotide separation.

In the present work, poly(glycidyl methacrylate-co-divinylbenzene) monoliths were synthesized and further derivatized to obtain strong anion exchange supports. Capillary monoliths (65 x 0.2 mm id) were prepared in situ by copolymerization of glycidyl methacrylate and divinylbenzene, employing 1-decanol and tetrahydrofuran as porogens. The free epoxy groups were derivatized in a two step synthesis to obtain quaternary ammonium functionalities. On testing the pressure stability of the synthesized monolith, a highly linear dependence between flow rate and pressure drop was obtained, indicating the high stability of the material even at high flow rates. The morphology of the copolymer was investigated by scanning electron microscopy. Mercury intrusion porosimetry showed a narrow pore size distribution, having a maximum at 439 nm. On recording a van Deemter plot the number of theoretical plates per meter was found to be 59324. The produced strong anion exchange monoliths turned out to be highly suitable for the separation of nucleotides and oligonucleotides.

Benzene↗

PubMed2006

Ultra-fast mass fingerprinting by high-affinity capture of peptides and proteins on derivatized poly(glycidyl methacrylate/divinylbenzene) for the analysis of serum and cell lysates.

The development of support materials in mass fingerprinting is an important task required for diagnostic markers in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The material-based approach, which we introduced as material-enhanced laser desorption/ionization (MELDI), focuses not only on different functionalities, but also emphasizes the morphology, i.e. porosity or particle size of the carrier material. As a result, it provides a quick and sensitive platform for effective binding of peptides and proteins out of different biofluids, e.g. serum, spinal fluid, urine or cell lysates, and to subsequently analyze them with MALDI-TOF MS. This approach includes a built-in desalting step for serum protein profiling and is sensitive enough to detect proteins and peptides down to 100 fmol/microL. Here we co-polymerized glycidyl methacrylate (GMA) with divinylbenzene (DVB) using thermal polymerization to yield a GMA/DVB polymer for further modifications. Different affinities have been created, such as immobilized metal ion affinity (IDA-Cu2+), reversed-phase (RP) and anion-exchanger (AX) chromatography. The diverse derivatizations and the dispersity of the particles created by different chemical synthetic approaches were confirmed by characteristic infrared (IR) peaks. The polymerization carried out by non-stirring yielded an average pore radius of 6.1 microm (macro-pores) that enhanced the binding capacity enormously by offering enlarged surface areas. Moreover, atomic absorption spectrometry (AAS) provided the metal content loaded on iminodiacetic acid (IDA) in the case of poly(GMA/DVB)-IDA-Cu2+. To summarize, the optimized MELDI approach is sensitive in its performance, extremely fast and can be adapted to robotic systems for routine analysis, allowing sample preparation in less than 5 min in contrast to the conventional surface-enhanced laser desorption/ionization (SELDI) methods.

Blood Proteins↗

PubMed2005

Quantitative detection of phosphoproteins by combination of two-dimensional difference gel electrophoresis and phosphospecific fluorescent staining.

Here we combine a standard two-dimensional difference gel electrophoresis (DIGE) protocol with subsequent post-staining of gels with phosphospecific fluorescent Pro-Q Diamond dye. The combination of these two methods for fluorescence detection of proteins allows quantitative detection of phosphoproteins in 2-DE-gels. We established this protocol within a functional proteomics experiment. Mammary epithelial cells (EpH4) were stimulated in culture by epidermal growth factor (EGF), endosomal fractions prepared after subcellular fractionation and phosphorylated proteins successfully detected on endosomes. For instance, Endo A cytokeratin, known as phosphoprotein and differentiation marker inducible by MAPK signaling, was identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). With this protocol, all steps of combined proteome and phosphoproteome profiling experiments are significantly simplified and accelerated, taking full advantage of both methods in terms of specificity, sensitivity and accuracy of quantification.

Animals↗

PubMed2005

Sample pretreatment and determination of non steroidal anti-inflammatory drugs (NSAIDs) in pharmaceutical formulations and biological samples (blood, plasma, erythrocytes) by HPLC-UV-MS and micro-HPLC.

The article discusses the qualitative and quantitative determination of non-steroidal anti-inflammatory drugs like salicin, salicylic acid, tenoxicam, ketorolac, piroxicam, tolmetin, naproxen, flurbiprofen, diclofenac and ibuprofen by reversed phase high performance liquid chromatography (RP-HPLC) and micro-HPLC (micro-HPLC) hyphenated with UV-absorbance and mass spectrometric detection. Both detection methods delivered calibration plots with good linearity (r(2) > 0.9800), limits of detection in the low nanogram range and recovery rates between 94 and 104 %. For the analysis of biological samples such as blood, plasma and erythrocytes liquid-liquid extraction (LLE) and solid phase extraction (SPE) on the basis of new synthesized glycidylmethacrylate/divinylbenzene copolymer (GMA/DVB) particles and commercially available material on the basis of poly(divinylbenzene-co-N-vinylpyrrolidone) copolymer were investigated. Finally the use of a micro-HPLC system with separation columns in the range of 8 cm x 200 microm I.D. for the determination of non-steroidal anti-inflammatory drugs (NSAIDs) is presented, emphasizing on the type of column and sample amount needed.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2003

Recent progress in high-performance capillary bioseparations.

Bioanalysis is a fast growing domain with new technological developments in the field of mass spectrometry, separation science, and bioinformatics. The central role of separation prior to detection and evaluation of analytes is indisputable. In fact, since the decipherment of the human genome via multichannel capillary electrophoresis in combination with laser-induced fluorescence [1], the analysis of the proteome, i.e., the type and amount of all proteins expressed at a certain time by the genome in a cell or tissue [2] became of central interest. Therefore, new fast and sensitive capillary liquid chromatographic, electrophoretic, and electrochromatographic separation techniques, suitable for high-throughput analysis of proteins, peptides and nucleic acids, are required.

Base Sequence↗

PubMedDate not supplied

Poly(glycidyl methacrylate/divinylbenzene)-IDA-FeIII in phosphoproteomics.

The study of protein phosphorylation has grown exponentially in recent years, as it became evident that important cellular functions are regulated by phosphorylation and dephosphorylation of proteins on serine, threonine and tyrosine residues. The use of immobilized metal affinity chromatography (IMAC) to enrich phosphopeptides from peptide mixtures has been shown to be useful especially prior to mass spectrometric analysis. For the selective enrichment applying solid-phase extraction (SPE) of phosphorylated peptides, we introduce poly(glycidyl methacrylate/divinylbenzene) (GMD) derivatized with imino-diacetic acid (IDA) and bound Fe(III) as a material. GMD is rapidly synthesized and the resulting free epoxy groups enable an easy access to further derivatization with, e.g., IDA. Electron microscopy showed that the synthesized GMD-IDA-Fe(III) for SPE has irregular agglomerates of spherical particles. Inductively coupled plasma (ICP) analysis resulted in a metal capacity of Fe(III) being 25.4 micromol/mL. To enable on-line preconcentration and desalting in one single step, GMD-IDA-Fe(III) and Silica C18 were united in one cartridge. Methyl esterification (ME) of free carboxyl groups was carried out to prevent binding of nonphosphorylated peptides to the IMAC function. The recovery for a standard phosphopeptide using this SPE method was determined to be 92%. The suitability of the established system for the selective enrichment and analysis of model proteins phosphorylated at different amino acid residues was evaluated stepwise. After successful enrichment of beta-casein deriving phosphopeptides, the established system was extended to the analysis of in vitro phosphorylated proteins, e.g. deriving from glutathione-S-transferase tagged extracellular signal regulated kinase 2 (GST-ERK2).

Amino Acid Sequence↗