Justin M. Hettick

Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral fingerprints for 12 species of fungi of the genus Aspergillus and 5 different strains of Aspergillus flavus. Prior to MALDI-TOF MS analysis, the fungi were subjected to three 1-min bead beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contain abundant peaks in the range of 5 to 20kDa and may be used to discriminate between species unambiguously. A discriminant analysis using all peaks from the MALDI-TOF MS data yielded error rates for classification of 0 and 18.75% for resubstitution and cross-validation methods, respectively. If a subset of 28 significant peaks is chosen, resubstitution and cross-validation error rates are 0%. Discriminant analysis of the MALDI-TOF MS data for 5 strains of A. flavus using all peaks yielded error rates for classification of 0 and 5% for resubstitution and cross-validation methods, respectively. These data indicate that MALDI-TOF MS data may be used for unambiguous identification of members of the genus Aspergillus at both the species and strain levels.

Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to discriminate moniliaceous fungal species; however, darkly pigmented fungi yield poor fingerprint mass spectra that contain few peaks of low relative abundance. In this study, the effect of dark fungal pigments on the observed MALDI mass spectra was investigated. Peptide and protein samples containing varying concentrations of synthetic melanin or fungal pigments extracted from Aspergillus niger were analyzed by MALDI-TOF and MALDI-qTOF (quadrupole TOF) MS. Signal suppression was observed in samples containing greater than 250ng/μl pigment. Microscopic examination of the MALDI sample deposit was usually heterogeneous, with regions of high pigment concentration appearing as black. Acquisition of MALDI mass spectra from these darkly pigmented regions of the sample deposit yielded poor or no [M+H](+) ion signal. In contrast, nonpigmented regions within the sample deposit and hyphal negative control extracts of A. niger were not inhibited. This study demonstrated that dark fungal pigments inhibited the desorption/ionization process during MALDI-MS; however, these fungi may be successfully analyzed by MALDI-TOF MS when culture methods that suppress pigment expression are used. The addition of tricyclazole to the fungal growth media blocks fungal melanin synthesis and results in less melanized fungi that may be analyzed by MALDI-TOF MS.

Haptenation: chemical reactivity and protein binding.

Low molecular weight chemical (LMW) allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed.

Tissue Binding Affects the Kinetics of Theophylline Diffusion Through the Stratum Corneum Barrier Layer of Skin

New data sets on both (i) equilibrium theophylline (TH) partitioning/binding in stratum corneum and (ii) transient TH diffusion through human epidermis are explained by an extended partition-diffusion model with reversible binding. Data conform to a linear binding isotherm within the tested concentration range (0-2000 μg/mL) with an equilibrium ratio of bound-to-free solute of approximately 1.4. The permeability coefficient for TH is 4.86 × 10(-5) cm/h, and the lag time is 20.1 h. Binding occurs as a slow process, significantly affecting the kinetics of dermal penetration.

Characterization of Cannabis sativa allergens

BACKGROUND Allergic sensitization to Cannabis sativa is rarely reported, but the increasing consumption of marijuana has resulted in an increase in the number of individuals who become sensitized. To date, little is known about the causal allergens associated with C sativa. OBJECTIVE To characterize marijuana allergens in different components of the C sativa plant using serum IgE from marijuana sensitized patients. METHODS Serum samples from 23 patients with a positive skin prick test result to a crude C sativa extract were evaluated. IgE reactivity was variable between patients and C sativa extracts. IgE reactivity to C sativa proteins in Western blots was heterogeneous and ranged from 10 to 70 kDa. Putative allergens derived from 2-dimensional gels were identified. RESULTS Prominent IgE reactive bands included a 23-kDa oxygen-evolving enhancer protein 2 and a 50-kDa protein identified to be the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. Additional proteins were identified in the proteomic analysis, including those from adenosine triphosphate synthase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and luminal binding protein (heat shock protein 70), suggesting these proteins are potential allergens. Deglycosylation studies helped refine protein allergen identification and demonstrated significant IgE antibodies against plant oligosaccharides that could help explain cross-reactivity. CONCLUSION Identification and characterization of allergens from C sativa may be helpful in further understanding allergic sensitization to this plant species.

Determination of the toluene diisocyanate binding sites on human serum albumin by tandem mass spectrometry

Diisocyanates are highly reactive chemical compounds widely used in the manufacture of polyurethanes. Although diisocyanates have been identified as causative agents of allergic respiratory diseases, the specific mechanism by which these diseases occur is largely unknown. To better understand the chemical species produced when diisocyanates react with protein, tandem mass spectrometry was employed to unambiguously identify the binding sites of the industrially important isomers, 2,4- and 2,6-toluene diisocyanate, on human serum albumin at varying diisocyanate/protein ratios. The 2,4-isomer results in approximately 2-fold higher conjugation product ion abundances than does the 2,6-isomer, suggesting that the 2,4-isomer has a higher reactivity toward albumin. Both isomers preferentially react with the N-terminal amine of the protein and the ε-NH(2) of lysine. At a low (1:2) diisocyanate/protein ratio, five binding sites are identified, whereas at a high (40:1) ratio, near-stoichiometric conjugation is observed with a maximum of 37 binding sites identified. Binding sites observed at the lowest conjugation ratios are conserved at higher binding ratios, suggesting a subset of 5-10 preferential binding sites on albumin. Diisocyanate-protein conjugation results in a variety of reaction products, including intra- and intermolecular crosslinking, diisocyanate self-polymerization, and diisocyanate hydrolysis.

Occupational sensitization to soy allergens in workers at a processing facility

Background Exposure to soy antigens has been associated with asthma in community outbreaks and in some workplaces. Recently, 135 soy flake processing workers (SPWs) in a Tennessee facility were evaluated for immune reactivity to soy. Allergic sensitization to soy was common and was five times more prevalent than in health care worker controls (HCWs) with no known soy exposure.

Structural Elucidation of Isocyanate-Peptide Adducts Using Tandem Mass Spectrometry

Diisocyanates are highly reactive chemical compounds widely used in the manufacture of polyurethanes. Although diisocyanates have been identified as causative agents of allergic respiratory diseases, the specific mechanism by which these diseases occur is largely unknown. To better understand the chemical species produced when isocyanates are reacted with model peptides, tandem mass spectrometry was employed to unambiguously identify the binding site of four commercially-relevant isocyanates on model peptides. In each case, the isocyanates react preferentially with the N-terminus of the peptide. No evidence of side-chain/isocyanate adduct formation exclusive of the N-terminus was observed. However, significant intra-molecular diisocyanate crosslinking was observed between the N-terminal amine and a side-chain amine of arginine, when Arg was located within two residues of the N-terminus. Addition of multiple isocyanates to the peptide occurs via polymerization of the isocyanate at the N-terminus, rather than via addition of multiple isocyanate molecules to varied residues within the peptide. The direct observation of isocyanate binding to the N-terminus of peptides under these experimental conditions is in good agreement with previous studies on the relative reaction rate of isocyanate with amino acid functional groups.

Peptide sequencing by MALDI 193-nm photodissociation TOF MS.

Ultraviolet photodissociation time-of-flight (TOF) mass spectrometry (MS) is described as a method for determination of peptide ion primary structure. Monoisotopic selection and bond-specific activation, combined with the rapidity of TOF MS analysis, render this technique invaluable to the rapidly expanding field of proteomics. Photofragment ion spectra of model peptides acquired using both post-source decay (PSD) focusing and TOF-TOF experimental methods are exhibited. Advantages of 193-nm photodissociation for de novo sequencing of peptide ions are discussed.

Hexamethylene diisocyanate (HDI) vapor reactivity with glutathione and subsequent transfer to human albumin

INTRODUCTION Airway fluid glutathione (GSH) reactivity with inhaled vapors of diisocyanate, a common occupational allergen, is postulated to be a key step in exposure-induced asthma pathogenesis. METHODS A mixed (vapor/liquid) phase exposure system was used to model the in vivo reactivity of inhaled HDI vapor with GSH in the airway fluid. HDI-GSH reaction products, and their capacity to transfer HDI to human albumin, were characterized through mass spectrometry and serologic assays, using HDI-specific polyclonal rabbit serum. RESULTS HDI vapor exposure of 10mM GSH solutions resulted in primarily S-linked, bis(GSH)-HDI reaction products. In contrast, lower GSH concentrations (100μM) resulted in mainly mono(GSH)-HDI conjugates, with varying degrees of HDI hydrolysis, dimerization and/or intra-molecular cyclization, depending upon the presence/absence of H2PO4(-)/HPO4(2-) and Na(+)/Cl(-) ions. The ion composition and GSH concentration of the fluid phase, during HDI vapor exposure, strongly influenced the transfer of HDI from GSH to albumin, as did the pH and duration of the carbamoylating reaction. When carbamoylation was performed overnight at pH 7, 25 of albumins lysines were identified as potential sites of conjugation with partially hydrolyzed HDI. When carbamoylation was performed at pH 9, more rapid (within 3h) and extensive modification was observed, including additional lysine sites, intra-molecular cross-linkage with HDI, and novel HDI-GSH conjugation. CONCLUSIONS The data define potential mechanisms by which the levels of GSH, H2PO4(-)/HPO4(2-), and/or other ions (e.g. H(+)/OH(-), Na(+), Cl(-)) affect the reactivity of HDI vapor with self-molecules in solution (e.g. airway fluid), and thus, might influence the clinical response to HDI respiratory tract exposure.