Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin-fluorescein isothiocyanate conjugates.

A protocol using enzymatic digestion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) for the investigation of the binding of the fluorescent contact allergen fluorescein isothiocyanate (FITC) to the 66 kDa large protein bovine serum albumin (BSA), as a model system for protein-hapten binding in the skin, is presented. Mass spectra of BSA-FITC digestions, using trypsin and chymotrypsin, respectively, provided sequence coverage of 97%. To investigate the number of FITC-bound peptides using CE-LIF separation, three different buffer salts at four different pH levels were evaluated. The use of 20 mM sodium citrate pH 6.5 as well as 20 mM sodium phosphate pH 6.5 or pH 7.5 as background electrolyte revealed high numbers of peptides with at least one bound FITC. The effect of the electrolyte counter ion on MALDI-MS was investigated and was found to have effect on the MALDI spectra signal-to-noise (S/N) at 50 mM but not at 10 mM. Of the 60 theoretical FITC-binding sites in BSA this MALDI-MS protocol presents 30 defined, 28 possible and 2 non-binding sites for FITC.

Specific adducts formed through a radical reaction between peptides and contact allergenic hydroperoxides.

The first step in the development of contact allergy (allergic contact dermatitis) includes the penetration of an allergy-causing chemical (hapten) into the skin, where it binds to macromolecules such as proteins. The protein-hapten adduct is then recognized by the immune system as foreign to the body. For hydroperoxides, no relevant hapten target proteins or protein-hapten adducts have so far been identified. In this work, bovine insulin and human angiotensin I were used as model peptides to investigate the haptenation mechanism of three hydroperoxide haptens: (5R)-5-isopropenyl-2-methyl-2-cyclohexene-1-hydroperoxide (Lim-2-OOH), cumene hydroperoxide (CumOOH), and 1-(1-hydroperoxy-1-methylethyl) cyclohexene (CycHexOOH). These hydroperoxides are expected to react via a radical mechanism, for which 5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride (Fe(III)TPPCl) was used as a radical initiator. The reactions were carried out in 1:1 ethanol/10 mM ammonium acetate buffer pH 7.4, for 3 h at 37 degrees C, and the reaction products were either enzymatically digested or analyzed directly by MALDI/TOF-MS, HPLC/MS/MS, and 2D gel electrophoresis. Both hydroperoxide-specific and unspecific reaction products were detected, but only in the presence of the iron catalyst. In the absence of catalyst, the hydroperoxides remained unreacted. This suggests that the hydroperoxides can enter into the skin and remain inert until activated. Through the detection of a Lim-2-OOH adduct bound at the first histidine (of two) of angiotensin I, it was confirmed that hydroperoxides have the potential to form specific antigens in contact allergy.

Mechanistic proposal for the formation of specific immunogenic complexes via a radical pathway: a key step in allergic contact dermatitis to olefinic hydroperoxides.

The widespread use of scented products causes an increase of allergic contact dermatitis to fragrance compounds in Western countries today. Many fragrance compounds are prone to autoxidation, forming hydroperoxides as their primary oxidation products. Hydroperoxides are known to be strong allergens and to form specific immunogenic complexes. However, the mechanisms for the formation of the immunogenic complexes are largely unknown. We have investigated this mechanism for (5R)-5-isopropenyl-2-methyl-2-cyclohexene-1-hydroperoxide (Lim-2-OOH) by studying the formation of adducts in the reaction between this hydroperoxide and 5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride (Fe(III)TPPCl) in the presence of protected cysteine (NAc-Cys-OMe) or glutathione (GSH). Isolated adducts originate from the addition of the thiol group of NAc-Cys-OMe over the carbon-carbon double bonds of carvone. Furthermore, adducts between NAc-Cys-OMe and carveol as well as between GSH and carvone have been identified. The formation of these adducts most likely proceeds via the radical thiol-ene mechanism. The addition of a terpene moiety to cysteine offers an explanation of the specificity of the immune response to structurally different hydroperoxides. These results also explain the lack of cross-reactivity between carvone and Lim-2-OOH. In conclusion, we propose that immunogenic complexes of olefinic hydroperoxides can be formed via the radical thiol-ene mechanism. These complexes will be specific for the individual olefinic hydroperoxides due to the inclusion of a terpene moiety derived from the hydroperoxide.

SPE and HPLC/UV of resin acids in colophonium-containing products.

A new method, involving SPE and HPLC/UV diode-array detection (DAD), was developed for the quantification of colophonium components in different consumer products, such as cosmetics. Colophonium is a common cause of contact dermatitis since its components can oxidize into allergens on exposure to air. Three different resin acids were used as markers for native and oxidized colophonium, abietic acid (AbA), dehydroabietic acid (DeA), and 7-oxodehydroabietic acid (7-O-DeA). The SPE method, utilizing a mixed-mode hydrophobic and anion exchange retention mechanism, was shown to yield very clean extracts. The use of a urea-embedded C(12) HPLC stationary phase improved the separation of the resin acids compared to common C(18). Concentrations higher than 2 mg/g of both AbA and DeA were detected in wax strips. In this product also 7-O-DeA, a marker for oxidized colophonium, was detected at a level of 28 microg/g. The LODs were in the range of 7-19 microg/g and the LOQs 22-56 microg/g. The method is simple to use and can be applied on many types of technical products, not only cosmetics. For the first time, a method for technical products was developed, which separates AbA from pimaric acid.

Off-line integration of CE and MALDI-MS using a closed-open-closed microchannel system.

In this work, a new technique for off-line hyphenation between CE and MALDI-MS is presented. Two closed fused-silica capillaries were connected via a silicon chip comprising an open microcanal. The EOF in the system was evaluated using mesityloxide or leucine-enkephalin as a sample and with a running buffer that rendered the analyte neutrally charged. Comparison was made between the EOF in a closed system (first capillary solely included in the electrical circuit) and in a closed-open system (first capillary and microcanal included in the electrical circuit). It was concluded that the experimental values of the EOF agreed with the theory. The influence of the capillary outer diameter on the peak dispersion was investigated using a closed-open-closed system (first capillary, microcanal and second capillary included in the electrical circuit). It was clearly seen that a capillary with 375 microm od induced considerably higher peak dispersion than a 150 microm od capillary, due to a larger liquid dead volume in the connection between the first capillary outlet and the microcanal. Mass spectrometric analysis has also been performed following CE separation runs in a closed-open-closed system with cytochrome c and lysozyme as model proteins. It was demonstrated that a signal distribution profile of the separated analytes could be recorded over a 30 mm long microcanal.

Capillary electrophoretic separation and fractionation of hydrophobic peptides onto a pre-structured matrix assisted laser desorption/ ionization target for mass spectrometric analysis.

A CE separation of hydrophobic peptides followed by fractionation onto a prestructured MALDI target and off-line MS analysis was performed. An improved and partially automated manufacturing procedure of the previously described MALDI target is presented. This target is structurally coated with silicone and especially developed for hydrophobic peptides and proteins. Here, the target plate was designed specifically for the CE fraction collection. Different solvents were evaluated to meet the requirements of peptide solubility and compatibility to both the CE and MALDI methods and to the fractionation procedure. CE-MALDI-MS analysis of nine highly hydrophobic peptides from cyanogen bromide-digested bacteriorhodopsin is demonstrated.

A screening procedure for the solubilization of chloroplast membrane proteins from the marine green macroalga Ulva lactuca using RP-HPLC-MALDI-MS.

A protocol for purification and analysis of chloroplast membrane proteins in the green macroalga Ulva lactuca has been developed, including reversed phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Five different solvents were evaluated for extraction of membrane proteins by three methods. The highest protein yield was achieved when proteins were extracted directly from the chloroplasts using the solvent hexafluoroisopropanol. A range of proteins of increasing hydrophobicity was separated by HPLC. Analysis of both HPLC fractions and non-separated samples by MALDI-TOF-MS revealed proteins with molecular weights spanning between 1 and 376 kDa.

Membrane protein and peptide sample handling for MS analysis using a structured MALDI target.

Different sample handling methods for hydrophobic proteins and peptides were evaluated in association with the utilization of a structured matrix-assisted laser/desorption ionization (MALDI) target for increased sensitivity. The fluorinated organic solvent hexafluoroisopropanol (HFIP) was used for the solubilization of both the full-length protein bacteriorhodopsin (BR) and a cyanogen bromide digest thereof, and compared to the performance of the non-ionic detergents octyl-beta-D-glucopyranoside (OG), dodecyl-beta-D-maltoside (DM), and Triton X-100. A concentrating effect was seen when using the structured MALDI plate for BR dissolved in all the different detergents, of which OG generated the best-quality spectra for the full-length integral membrane protein as well as for the hydrophobic peptides. However, the uneven analyte distribution obtained with the detergent preparations required selective and thus time-consuming acquisition of spectra. When instead HFIP was used as sample solvent, a tenfold increase in sensitivity was achieved for full-length BR. Addition of acids to the HFIP-solubilized sample, or to the MALDI matrix solution, improved the signals for a few of the peptides, while degrading the spectra of others. Consequently, the addition of acid could be used as a complementary sample preparation method for hydrophobic peptides. On-target washing to remove contaminants (e.g., salt) was performed, and a recrystallization protocol for signal improvement specifically suited for hydrophobic peptides is described. Results from digestion and solubilization in different micro centrifuge tubes were examined to determine the influence of different materials on the possible sample loss due to wall adhesion. Studies of sample solution storage times suggest immediate analysis after solubilization to obtain best results.

Simple fabrication of a structured matrix-assisted laser desorption/ionization target coating for increased sensitivity in mass spectrometric analysis of membrane proteins.

A new prestructured target plate for matrix-assisted laser desorption/ionization (MALDI) was developed specifically for hydrophobic integral membrane proteins. This sample support contains predefined concentrating sample spots with a focusing effect on droplets with a high content of hexafluoroisopropanol (HFIP). This fluorinated organic solvent is advantageous for solubilizing hydrophobic proteins that are not soluble in water or the organic solvents normally used in sample preparation protocols for MALDI-MS. The prestructured plate was constructed by coating a regular steel plate with a thin layer of a silicone polymer, leaving sample spots of bare steel. Fabrication of the concentrating silicone structure was fast and very straightforward, without expensive or complicated equipment. Removing the layer, and thus regenerating the steel plate, was done by a simple washing procedure. The application and cleaning procedure are not constrained by a particular design of sample support or to any specific brand of mass spectrometer. When using the prestructured MALDI plate with HFIP as the sample solvent for 17 pmol of a cyanogen bromide digest of the highly hydrophobic membrane protein bacteriorhodopsin, an improved focusing effect and an increase of more than five-fold in average sensitivity were observed, compared with a regular steel target. Experimental results show a two-fold increase in average sensitivity when the new prestructured target plate was used, compared with a commercially available concentrating support.