Anas M. Abdel Rahman

Biomolecular characterization of allergenic proteins in snow crab (Chionoecetes opilio) and de novo sequencing of the second allergen arginine kinase using tandem mass spectrometry

Snow crab (Chionoecetes opilio) proteins have been recognized as an important source of both food and occupational allergens. While snow crab causes a significant occupational allergy, only one novel allergen has recently been fully characterized. The muscle proteins from snow crab legs were profiled by SDS-PAGE. Several of these proteins were characterized using tandem mass spectrometry. Five proteins were identified; sarcoplasmic Ca-binding (20kDa), arginine kinase (40), troponin (23kDa) and α-actine (42kDa) and smooth endoplasmic reticulum Ca(2+)ATPase (113kDa). Immunoblotting using serum of sixteen allergic patients resulted in strong reactivity with the 40-kDa protein in seven patients (43%). This protein was purified by chromatography and subsequently de novo sequenced using matrix assisted laser desorption ionization and electrospray tandem mass spectrometry. We identified a second important allergen, arginine kinase, in snow crab, designated Chi o 3. Based on identity and homology analysis, using bioinformatics tools, a signature peptide was identified as a chemical surrogate for arginine kinase. The suitability of this signature peptide was tested for analytically representing the arginine kinase, by performing a multi-reaction monitoring tandem mass spectrometry approach on actual air filter samples collected from a simulated crab processing plant.

Impact of heat processing on the detection of the major shellfish allergen tropomyosin in crustaceans and molluscs using specific monoclonal antibodies.

The major heat-stable shellfish allergen, tropomyosin, demonstrates immunological cross-reactivity, making specific differentiation of crustaceans and molluscs for food labelling very difficult. The aim of this study was to evaluate the application of allergen-specific monoclonal antibodies in differential detection of shellfish-derived tropomyosin in 11 crustacean and 7 mollusc species, and to study the impact of heating on its detection. Cross-reactive tropomyosin was detected in all crustacean species, with partial detection in molluscs: mussels, scallops and snails but none in oyster, octopus and squid. Furthermore, we have demonstrated that heating of shellfish has a profound effect on tropomyosin detection. This was evident by the enhanced recognition of multiple tropomyosin variants in the analysed shellfish species. Specific monoclonal antibodies, targetting the N-terminal region of tropomyosin, must therefore be developed to differentiate tropomyosins in crustaceans and molluscs. This can help in correct food labelling practices and thus protection of consumers.

Effect of heat processing on antibody reactivity to allergen variants and fragments of black tiger prawn: a comprehensive allergenomic approach

SCOPEnPrawn allergy is one of the leading causes of IgE-mediated hypersensitivity to food. Alterations of IgE-antibody reactivity to prawn allergens due to thermal processing are not fully understood. The aim of this study was to analyze the impact of heating on prawn allergens using a comprehensive allergenomic approach.nnnMETHODS AND RESULTSnProteins from raw and heat-processed black tiger prawn (Penaeus monodon) extracts as well as recombinant tropomyosin (rPen m1) were analyzed by SDS-PAGE and immunoblotting using sera from 16 shellfish allergic patients. IgE antibody binding proteins were identified by advanced mass spectroscopy, characterized by molecular structure analysis and their IgE reactivity compared among the prepared black tiger prawn extracts. Heat processing enhanced the overall patient IgE binding to prawn extracts and increased recognition of a number of allergen variants and fragments of prawn allergens. Allergens identified were tropomyosin, myosin light chain, sarcoplasmic calcium binding protein, and putative novel allergens including triose phosphate isomerase, aldolase, and titin.nnnCONCLUSIONnSeven allergenic proteins are present in prawns, which are mostly heat-stable and form dimers or oligomers. Thermal treatment enhanced antibody reactivity to prawn allergens as well as fragments and should be considered in the diagnosis of prawn allergy and detection of crustacean allergens in processed food.

Analysis of the allergenic proteins in black tiger prawn (Penaeus monodon) and characterization of the major allergen tropomyosin using mass spectrometry

Crustaceans are the third most prevalent cause of food-induced anaphylaxis after peanuts and tree nuts. The severity of the allergenic proteins depends mainly on the amino acid sequence that induces production of IgE antibodies. In black tiger prawn (Penaeus monodon), the crude protein extract was profiled and its allergenic potency was examined against patients sera. Proteins having strong immunoreactivity with patients IgE were characterized using peptide mass fingerprinting (PMF). Tropomyosin (TM) (33 kDa), myosin light chain (20 kDa), and arginine kinase (40 kDa) were identified as allergenic proteins. Tropomyosin, the most abundant and potent allergen, was purified using ion-exchange chromatography for de novo sequencing experiments. Using bottom up tandem mass spectrometry, the full amino acid sequence was achieved by a combination of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass spectrometry (QqToF). Myosin light chain and arginine kinase were also characterized, and their related peptides were de novo sequenced using the same approach. The immunological reactivity of the crude prawn extracts and purified TM samples were analyzed using a large number of patients sera. A signature peptide was assigned for the TM protein for future quantification work of black tiger prawn TM levels in different matrices (i.e. water, air, food) in the seafood industry.

Characterization and de novo sequencing of snow crab tropomyosin enzymatic peptides by both electrospary ionization and matrix-assisted laser desorption ionization QqToF tandem mass spectrometry

The protein tropomyosin (TM) is a known major allergen present in shellfish causing frequent food allergies. TM is also an occupational allergen generated in the working environment of snow crab (Chionoecetes opilio) processing plants. The TM protein was purified from both claw and leg meats of snow crab and analyzed by electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) using hybrid quadruple time-of-flight tandem mass spectrometry (QqToF-MS). The native polypeptide molecular weight of TM was determined to be 32,733 Da. The protein was further characterized using the bottom-up MS approach. A peptide mass fingerprinting was obtained by two different enzymatic digestions and de novo sequencing of the most abundant peptides performed. Any post-translational modifications were identified by searching their calculated and predicted molecular weights in precursor ion spectra. The immunological reactivity of snow crab extract was evaluated using specific antibodies and allergenic reactivity assessed with serum of allergic patients. Subsequently, a signature peptide for TM was identified and evaluated in terms of identity and homology using the basic local alignment search tool (BLAST). The identification of a signature peptide for the allergen TM using MALDI-QqToF-MS will be critical for the sensitive and specific quantification of this highly allergenic protein in the work place.

Absolute quantification method and validation of airborne snow crab allergen tropomyosin using tandem mass spectrometry.

Measuring the levels of the major airborne allergens of snow crab in the workplace is very important in studying the prevalence of crab asthma in workers. Previously, snow crab tropomyosin (SCTM) was identified as the major aeroallergen in crab plants and a unique signature peptide was identified for this protein. The present study advances our knowledge on aeroallergens by developing a method of quantification of airborne SCTM by using isotope dilution mass spectrometry. Liquid chromatography tandem mass spectrometry was developed for separation and analysis of the signature peptides. The tryptic digestion conditions were optimized to accomplish complete digestion. The validity of the method was studied using international conference on harmonization protocol, Where 2-9% for CV (precision) and 101-110% for accuracy, at three different levels of quality control. Recovery of the spiked protein from PTFE and TopTip filters was measured to be 99% and 96%, respectively. To further demonstrate the applicability and the validity of the method for real samples, 45 kg of whole snow crab were processed in an enclosed (simulated) crab processing line and air samples were collected. The levels of SCTM ranged between 0.36-3.92 μg m(-3) and 1.70-2.31 μg m(-3) for butchering and cooking stations, respectively.

Targeted metabolomics in cultured cells and tissues by mass spectrometry: Method development and validation

Metabolomics is the identification and quantitation of small bio-molecules (metabolites) in biological samples under various environmental and genetic conditions. Mass spectrometry provides the unique opportunity for targeted identification and quantification of known metabolites by selective reaction monitoring (SRM). However, reproducibility of this approach depends on careful consideration of sample preparation, chemical classes, and stability of metabolites to be evaluated. Herein, we introduce and validate a targeted metabolite profiling workflow for cultured cells and tissues by liquid chromatography-triple quadrupole tandem mass spectrometry. The method requires a one-step extraction of water-soluble metabolites and targeted analysis of central metabolites that include glycolysis, amino acids, nucleotides, citric acid cycle, and the hexosamine biosynthetic pathway. The sensitivity, reproducibility and molecular stability of each targeted metabolite were assessed under experimental conditions. Quantitation of metabolites by peak area ratio was linear with a dilution over a 4 fold dynamic range with minimal deviation R(2)=0.98. Inter- and intra-day precision with cells and tissues had an average coefficient of variation <15% for cultured cell lines, and somewhat higher for mouse liver tissues. The method applied in triplicate measurements readily distinguished immortalized cells from malignant cells, as well as mouse littermates based on their hepatic metabolic profiles.

Probing the Hexosamine Biosynthetic Pathway in Human Tumor Cells by Multitargeted Tandem Mass Spectrometry

Cancer progression is accompanied by increases in glucose and glutamine metabolism, providing the carbon and nitrogen required in downstream anabolic pathways. Fructose-6P, glutamine, and acetyl-CoA are central metabolites and substrates of the hexosamine biosynthesis pathway (HBP) to UDP-N-acetylglucosamine (UDP-GlcNAc), an essential high-energy donor for protein glycosylation. Golgi and cytosolic glycosylation pathways are sensitive to UDP-GlcNAc levels, which in turn regulates metabolic homeostasis in a poorly understood manner. To study the hexosamine biosynthesis pathway in cancer cells, we developed a targeted approach for cellular metabolomics profiling by liquid chromatography-tandem mass spectrometry. Human cervical (HeLa) and prostate cancer (PC-3) cell lines were cultured in medium with increasing concentrations of glucose, glutamine, or GlcNAc to perturb the metabolic network. Principal component analysis indicated trends that were further analyzed as individual metabolites and pathways. HeLa cell metabolism was predominantly glycolytic, while PC-3 cells showed a greater dependency on extracellular glutamine. In both cell lines, UDP-GlcNAc levels declined with glucose but not glutamine starvation, whereas glutamine abundance increased UDP-GlcNAc levels 2-3-fold. GlcNAc supplementation increased UDP-GlcNAc 4-8-fold in both HeLa and PC-3 cells. GlcNAc supplementation in HeLa cells induced nonmonotonic changes in NADH/NAD+, NADPH/NADP+, reactive oxygen species, and reduced/oxidized glutathione. In PC-3 cells, GlcNAc supplementation also increased glucose and glutamine uptake and catabolism. Our results suggest that stimulation of the HBP in cancer cells regulates metabolism and redox potential, which might be exploited to target cancer cells.

Comprehensive Proteomics Approach in Characterizing and Quantifying Allergenic Proteins from Northern Shrimp: Toward Better Occupational Asthma Prevention

Occupational asthma is a major chronic health dilemma among workers involved in the seafood industry. Several proteins notoriously known to cause asthma have been reported in different seafood. This work involves the application of an allergenomics strategy to study the most potent allergens of northern shrimp. The proteins were extracted from shrimp tissue and profiled by gel electrophoresis. Allergenic proteins were identified based on their reactivity to patient sera and were structurally identified using tandem mass spectrometry. Northern shrimp tropomyosin, arginine kinase, and sarcoplasmic calcium-binding protein were found to be the most significant allergens. Multiple proteolytic enzymes enabled 100% coverage of the sequence of shrimp tropomyosin by tandem mass specrometry. Only partial sequence coverage was obtained, however, for the shrimp allergen arginine kinase. Signature peptides, for both tropomyosin and arginine kinase, were assigned and synthesized for use in developing the multiple reaction monitoring tandem mass spectrometric method. Subsequently, air samples were collected from a shrimp processing plant and two aerosolized proteins quantified using tandem mass specrometry. Allergens were detected in all areas of the plant, reaching levels as high as 375 and 480 ng/m(3) for tropomyosine and arginine kinase, respectively. Tropomyosine is much more abundant than arginine kinase in shrimp tissues, so the high levels of arginine kinase suggest it is more easily aerosolized. The present study shows that mass spectrometric analysis is a sensitive and accurate tool in identifying and quantifying aerosolized allergens.

N-Glycan Remodeling on Glucagon Receptor Is an Effector of Nutrient Sensing by the Hexosamine Biosynthesis Pathway

Background: The hexosamine biosynthesis pathway to UDP-GlcNAc has been implicated in glucose homeostasis. Results: UDP-GlcNAc and Golgi N-acetylglucosaminyltransferases modify the N-glycans on glucagon receptor, which increases sensitivity to glucagon in vivo. Conclusion: The hexosamine biosynthesis pathway contributes to glucose homeostasis, in part through N-glycan branching on glucagon receptor. Significance: Hepatic Mgat5 and the N-glycan branching pathway may be a therapeutic target for control of glycemia. Glucose homeostasis in mammals is dependent on the opposing actions of insulin and glucagon. The Golgi N-acetylglucosaminyltransferases encoded by Mgat1, Mgat2, Mgat4a/b/c, and Mgat5 modify the N-glycans on receptors and solute transporter, possibly adapting activities in response to the metabolic environment. Herein we report that Mgat5−/− mice display diminished glycemic response to exogenous glucagon, together with increased insulin sensitivity. Glucagon receptor signaling and gluconeogenesis in Mgat5−/− cultured hepatocytes was impaired. In HEK293 cells, signaling by ectopically expressed glucagon receptor was increased by Mgat5 expression and GlcNAc supplementation to UDP-GlcNAc, the donor substrate shared by Mgat branching enzymes. The mobility of glucagon receptor in primary hepatocytes was reduced by galectin-9 binding, and the strength of the interaction was dependent on Mgat5 and UDP-GlcNAc levels. Finally, oral GlcNAc supplementation rescued the glucagon response in Mgat5−/− hepatocytes and mice, as well as glycolytic metabolites and UDP-GlcNAc levels in liver. Our results reveal that the hexosamine biosynthesis pathway and GlcNAc salvage contribute to glucose homeostasis through N-glycan branching on glucagon receptor.