Rania Bakry

Analysis of protein phosphorylation by monolithic extraction columns based on poly(divinylbenzene) containing embedded titanium dioxide and zirconium dioxide nano‐powders

The potential of an organic monolith with incorporated titanium dioxide (TiO2) and zirconium dioxide (ZrO2) nanoparticles was evaluated for the selective enrichment of phosphorylated peptides from tryptic digests. A pipette tip was fitted with a monolith based on divinylbenzene (DVB) of highly porous structure, which allows sample to pass through the monolithic bed. The enrichment of phosphopeptides was enhanced by increasing the pipetting cycles during the sample preparation and a higher recovery could be achieved with adequate buffer systems. A complete automated process was developed for enrichment of phosphopeptides leading to high reproducibility and resulting in a robust method designed to minimize analytical variance while providing high sensitivity at high sample throughput. The effect of particle size on the selectivity of phosphopeptides was investigated by comparative studies with nano‐ and microscale TiO2 and ZrO2 powders. Eleven phosphopeptides from α‐casein digest could be recovered by an optimized mixture of microscale TiO2/ZrO2 particles, whereas nine additional phosphopeptides could be retained by the same mixture of nano‐structured material. When compared to conventional immobilized metal‐ion affinity chromatography and commercial phosphorylation‐enrichment kits, higher selectivity was observed in case of self fabricated tips. About 20 phosphopeptides could be retained from α‐casein and five from β‐casein digests by using TiO2 and ZrO2 based extraction tips. Further selectivity for phosphopeptides was demonstrated by enriching a digest of in vitro phosphorylated extracellular signal regulated kinase 1 (ERK1). Two phosphorylated peptides of ERK1 could be identified by MALDI‐MS/MS measurements and a following MASCOT database search.

Hepatic but not brain iron is rapidly chelated by deferasirox in aceruloplasminemia due to a novel gene mutation

Background & Aims Aceruloplasminemia is a rare autosomal recessive neurodegenerative disease associated with brain and liver iron accumulation which typically presents with movement disorders, retinal degeneration, and diabetes mellitus. Ceruloplasmin is a multi-copper ferroxidase that is secreted into plasma and facilitates cellular iron export and iron binding to transferrin. Results A novel homozygous ceruloplasmin gene mutation, c.2554+1G>T, was identified as the cause of aceruloplasminemia in three affected siblings. Two siblings presented with movement disorders and diabetes. Complementary DNA sequencing showed that this mutation causes skipping of exon 14 and deletion of amino acids 809–852 while preserving the open reading frame. Western blotting of liver extracts and sera of affected patients showed retention of the abnormal protein in the liver. Aceruloplasminemia was associated with severe brain and liver iron overload, where hepatic mRNA expression of the iron hormone hepcidin was increased, corresponding to the degree of iron overload. Hepatic iron concentration normalized after 3 and 5 months of iron chelation therapy with deferasirox, which was also associated with reduced insulin demands. During short term treatment there was no clinical or imaging evidence for significant effects on brain iron overload. Conclusions Aceruloplasminemia can show an incomplete clinical penetrance but is invariably associated with iron accumulation in the liver and in the brain. Iron accumulation in aceruloplasminemia is a result of defective cellular iron export, where hepcidin regulation is appropriate for the degree of iron overload. Iron chelation with deferasirox was effective in mobilizing hepatic iron but has no effect on brain iron.

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 μ-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.

Protein profiling for cancer biomarker discovery using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and infrared imaging: A review

Cancer biomarker refers to a substance or process that is indicative of the presence of cancer in the body. A biomarker might be either a molecule secreted by a tumor or it can be a specific response of the body to the presence of cancer. Cancer biomarker-based diagnostics have applications for establishing disease predisposition, early detection, cancer staging, therapy selection, identifying whether or not a cancer is metastatic, therapy monitoring, assessing prognosis, and advances in the adjuvant setting. Full adoption of cancer biomarkers in the clinic has to date been slow, and only a limited number of cancer biomarker products are currently in routine use. Among proteomic technologies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) is a technique that has allowed rapid progress in cancer biology. Different further developed methods including e.g. SELDI (surface-enhanced laser desorption/ionization) and MELDI (material-enhanced laser desorption/ionization) are simple and high-throughput techniques that analyze with high sensitivity and specificity intact proteins expressed in complex biological mixtures, such as serum, urine, and tissues. The combination of mass spectrometry (MS) with infrared (IR) spectroscopic imaging is an attempt to combine different technologies in systems analytics. Both MALDI-TOF and infrared tissue imaging enable studying proteins distribution in tissue samples with a resolution down to 50 and 5 μm, respectively. In this review, we summarize recent applications and the synergistic combination of these new technologies to proteomic profiling for cancer biomarker discovery.

Evaluation of extraction methods for the simultaneous analysis of simple and macrocyclic trichothecenes

The article is concerned with the simultaneous determination of simple and macrocyclic trichothecenes using high-performance liquid chromatography (LC) coupled to UV and mass spectrometric (MS) detection. Emphasis is put on the liquid-liquid extraction (LLE) and solid phase extraction (SPE) procedure from plant material such as wheat, comparing SPE cartridges packed with different stationary phases based on silica and polymer sorbents. In this coherence a polymeric material on the basis of poly(glycidyl methacrylate-divinylbenzene) (GMA-DVB) is developed with special regard on synthesis procedures to enhance the extraction recovery of trichothecenes in a broad polarity range. Evaluation of extraction techniques showed that the introduced material is competitive with commercially available high quality SPE materials. Percentage recovery is 82% for polar compounds, 89% for medium polar compounds and 98% for lipophilic compounds.

Near Infrared Spectroscopy for Polymer Research, Quality Control and Reaction Monitoring:

This review covers recent applications of near infrared (NIR) spectroscopy in the determination of physico-chemical and morphological parameters of polymeric materials. Near infrared measurements in the diffuse reflection mode are highlighted, which analyse the structural parameters such as porosity, surface area and particle size. Fundamentals and applications of the technique are discussed and examples of quantitative and qualitative analysis are explained. Various approaches like on- and in-line techniques, bulk measurements and kinetic studies for recording spectra are discussed. Furthermore, this review addresses the development of calibrations, which allow for the differentiation and quantification of materials with varying physical and morphological properties. Parameters like constitution, composition and crystallinity have a strong affect on the material characteristics. Therefore, chemical, physical and mechanical properties of synthetic as well as natural substances, such as polymeric composites and cotton or wool, need to be studied in-depth. To sum up, NIR spectroscopy has been developed as a flexible, robust and high-throughput analytical method that can be combined with chemometric and multivariate data analysis for fast and reliable screening in material science.

Laser desorption/ionization mass spectrometric analysis of small molecules using fullerene-derivatized silica as energy-absorbing material

In spite of the growing acceptance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the analysis of a wide variety of compounds, including polymers and proteins, its use in analyzing low-molecular-weight molecules (<1000 m/z) is still limited. This is mainly due to the interference of matrix molecules in the low-mass range. Here the derivatized fullerenes covalently bound to silica particles with different pore sizes are applied as thin layer for laser desorption/ionization (LDI) mass spectrometric analysis. Thus, an interference of intrinsic matrix ions can be eliminated or minimized in comparison with the state-of-the-art weak organic acid matrices. The desorption/ionization ability of the developed fullerene-silica materials depends on the applied laser power, sample preparation and pore size of the silica particles. Thus, fullerene-silica serves as an LDI support for mass spectrometric analysis of molecules (<1500 Da). The performance of the fullerene-silica is demonstrated by the mass analysis of variety of small molecules such as carbohydrates, amino acids, peptides, phospholipids and drugs.

CE coupled to MALDI with novel covalently coated capillaries

CE offers the advantage of flexibility and method development options. It excels in the area of separation of ions, chiral, polar and biological compounds (especially proteins and peptides). Masking the active sites on the inner surface of a bare fused silica capillary wall is often necessary for CE separations of basic compounds, proteins and peptides. The use of capillary surface coating is one of the approaches to prevent the adsorption phenomena and improve the repeatability of migration times and peak areas of these analytes. In this study, new capillary coatings consisting of (i) derivatized polystyrene nanoparticles and (ii) derivatized fullerenes were investigated for the analysis of peptides and protein digest by CE. The coated capillaries showed excellent run‐to‐run and batch‐to‐batch reproducibility (RSD of migration time ≤0.5% for run‐to‐run and ≤9.5% for batch‐to‐batch experiments). Furthermore, the capillaries offer high stability from pH 2.0 to 10.0. The actual potential of the coated capillaries was tested by combining CE with MALDI‐MS for analysing complex samples, such as peptides, whereas the overall performance of the CE‐MALDI‐MS system was investigated by analysing a five‐protein digest mixture. Subsequently, the peak list (peptide mass fingerprint) generated from the mass spectra of each fraction was entered into the Swiss‐Prot database in order to search for matching tryptic fragments using the MASCOT software. The sequence coverage of analysed proteins was between 36 and 68%. The established technology benefits from the synergism of high separation efficiency and the structure selective identification via MS.

Use of fullerene‐, octadecyl‐, and triaconthyl silica for solid phase extraction of tryptic peptides obtained from unmodified and in vitro glycated human serum albumin and fibrinogen

SPE plays a crucial role in bioanalytical research. In the present work a novel fullerene(C60)-derivatised silica material is compared with octadecyl(C18) - and triaconthyl(C30)-silicas regarding recoveries of peptides and sequence coverage of HSA and fibrinogen digests. C30- and C60(30 nm)-SPE materials were found to be the two most prominent SPE materials. At low peptide concentrations C60-material prepared from a silica gel with a pore size of 30 nm has proven to be the best material with regards to recoveries. By increasing the amount of loaded peptides recoveries decrease due to its relative low binding capacity in contrast to C30-silica particles, showing no changes. The best sequence coverages of Aalpha- and Bbeta-chains of 20 pmol fibrinogen digest can also be achieved using these two SPE materials, C60 (30 nm) demonstrates an outstanding value of sequence coverage (62.15%) achieved for the gamma-chain. After nonenzymatic glycation the digests of fibrinogen and HSA were also separated. This makes the detection of a considerably higher number of glycated peptides possible compared to the unfractionated digests and the use of boronate affinity chromatography in the case of fibrinogen. For HSA, ten new sites of glycation at lysine and arginine residues have been explored. Using the detailed SPE/off-line MALDI method the glycation sites on fibrinogen are first described in this paper.

Alternative profiling platform based on MELDI and its applicability in clinical proteomics

The presence of numerous proteomics data and their results in literature reveal the importance and influence of proteins and peptides on human cell cycle. For instance, the proteomic profiling of biological samples, such as serum, plasma or cells, and their organelles, carried out by surface-enhanced laser desorption/ionization mass spectrometry, has led to the discovery of numerous key proteins involved in many biological disease processes. However, questions still remain regarding the reproducibility, bioinformatic artifacts and cross-validations of such experimental set-ups. The authors have developed a material-based approach, termed material-enhanced laser desorption/ionization mass spectrometry (MELDI-MS), to facilitate and improve the robustness of large-scale proteomic experiments. MELDI-MS includes a fully automated protein-profiling platform, from sample preparation and analysis to data processing involving state-of-the-art methods, which can be further improved. Multiplexed protein pattern analysis, based on material morphology, physical characteristics and chemical functionalities provides a multitude of protein patterns and allows prostate cancer samples to be distinguished from non-prostate cancer samples. Furthermore, MELDI-MS enables not only the analysis of protein signatures, but also the identification of potential discriminating peaks via capillary liquid chromatography mass spectrometry. The optimized MELDI approach offers a complete proteomics platform with improved sensitivity, selectivity and short sample preparation times.