Profiling proteins from azoxymethane-induced colon tumors at the molecular level by matrix-assisted laser desorption/ionization mass spectrometry.

New developments in mass spectrometry allow for the profiling of the major proteomic content of fresh tissue sections. Briefly, fresh tissue sections are sampled and blotted onto a polyethylene membrane for protein transfer and then subsequently analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Using this technology, we have compared the protein expression of normal and cancerous mouse colon tissue obtained from the same animal. By difference, several protein signals specific to cancerous tissue were observed. A protein extract obtained from the tumors was fractionated by high-performance liquid chromatography and the individual fractions analyzed by MALDI-MS. The fractions containing the targeted proteins were subjected to trypsin digestion. The resulting tryptic peptides were sequenced by tandem mass spectrometry, and based on the recovered partial amino acid sequences, three of the tumor specific protein markers were identified as calgranulin A (S100A8), calgranulin B (S100A9) and calgizzarin (S100A11).

Strain-based sequence variations and structure analysis of murine prostate specific spermine binding protein using mass spectrometry.

Mouse spermine binding protein (SBP) has been characterized using mass spectrometry, including its localization within the prostate, sequence verification, and its posttranslational modifications. MALDI (matrix-assisted laser desorption/ionization) mass spectrometry was employed for localization of proteins expressed by different lobes of the mouse prostate obtained after tissue blotting on a polyethylene membrane. The mass spectra showed complex protein profiles that were different for each lobe of the prostate. The prostate-specific spermine binding protein (SBP), primarily identified by its in-source decay fragment ion signals, was found predominantly expressed by the ventral lobe of the prostate. The MALDI in-source decay measurements combined with nanoESI (nanoelectrospay ionization) MS/MS measurements obtained after specific proteolysis of SBP, allowed the exact positioning of a single N-linked carbohydrate group, and the identification of a pyroglutamate residue at the sequence N-terminus. The N-linked carbohydrate component was further investigated and the general pattern of the N-linked carbohydrate identified. The presence of a disulfide bridge between cysteine78 and cysteine124 was also established. The full sequence characterization of SBP showed several strain-based sequence differences when compared to the published gene sequence.

Metabolism of the lipid peroxidation product, 4-hydroxy-trans-2-nonenal, in isolated perfused rat heart.

The metabolism of 4-hydroxy-trans-2-nonenal (HNE), an alpha, beta-unsaturated aldehyde generated during lipid peroxidation, was studied in isolated perfused rat hearts. High performance liquid chromatography separation of radioactive metabolites recovered from [3H]HNE-treated hearts revealed four major peaks. Based on the retention times of synthesized standards, peak I, which accounted for 20% radioactivity administered to the heart, was identified to be due to glutathione conjugates of HNE. Peaks II and III, containing 2 and 37% radioactivity, were assigned to 1, 4-dihydroxy-2-nonene (DHN) and 4-hydroxy-2-nonenoic acid, respectively. Peak IV was due to unmetabolized HNE. The electrospray ionization mass spectrum of peak I revealed two prominent metabolites with m/z values corresponding to [M + H]+ of HNE and DHN conjugates with glutathione. The presence of 4-hydroxy-2-nonenoic acid in peak III was substantiated using gas chromatography-chemical ionization mass spectroscopy. When exposed to sorbinil, an inhibitor of aldose reductase, no GS-DHN was recovered in the coronary effluent, and treatment with cyanamide, an inhibitor of aldehyde dehydrogenase, attenuated 4-hydroxy-2-nonenoic acid formation. These results show that the major metabolic transformations of HNE in rat heart involve conjugation with glutathione and oxidation to 4-hydroxy-2-nonenoic acid. Further metabolism of the GS-HNE conjugate involves aldose reductase-mediated reduction, a reaction catalyzed in vitro by homogenous cardiac aldose reductase.

Blood-brain barrier penetration of 3-aminopropyl-n-butylphosphinic acid (CGP 36742) in rat brain by microdialysis/mass spectrometry.

The detection and quantitation of the novel drug 3-aminopropyl-n-butylphosphinic acid (APBP), also known as CGP 36742, was performed in vivo using microdialysis and tandem mass spectrometry. This drug is a GABA-B antagonist with high specificity for GABA-B receptors. Animals received doses of 100, 200, 500 and 1000 mg kg-1 of the drug either intravenously or per os (p.o.). Microdialysis probes, placed by stereotaxis in either the frontal cortex or third ventricle of the rat, were used to collect dialyzate samples over several hours. Samples were then analyzed by micro-electrospray tandem mass spectrometry to achieve a molecular mass and structure specific analysis. For example, animals receiving a dose of 100 mg kg-1 p.o. showed a peak concentration of approximately 10 microM in the dialyzate. For comparison, tissue and plasma samples of the drug were measured under the same conditions using gas chromatography/mass spectrometry. This work demonstrates that the microdialysis technique in combination with the molecular specificity and high sensitivity of micro-electrospray tandem mass spectrometry can be used to study the time course of the appearance of unmodified drug in the brain of a single animal.

Recent advances in liquid chromatography-mass spectrometry and capillary zone electrophoresis-mass spectrometry for protein analysis.

The utility of the combination of separations techniques, such as liquid chromatography and capillary zone electrophoresis, with mass spectrometry in applications involving protein analysis is discussed. The use of continuous-flow fast atom bombardment and electrospray ionization mass spectrometry is compared for the analysis of tryptic digests. For liquid chromatography, both microbore and slurry-packed capillary bore columns were used to separate peptides from proteolytic digests.

Coupling capillary zone electrophoresis and continuous-flow fast atom bombardment mass spectrometry for the analysis of peptide mixtures.

Combined capillary zone electrophoresis (CZE)-continuous-flow fast atom bombardment (CF-FAB) mass spectrometry is described for the analysis of mixtures of peptides. A 90 cm x 50 microns I.D. fused-silica capillary column was used for electrophoretic separations and was connected to the CF-FAB probe via an interface which allows a total flow into the mass spectrometer of about 5 microliters/min. Solutions of peptides were pneumatically loaded onto the CZE capillary, providing sample amounts of 0.1-20 pmol. The magnetic mass spectrometer was scanned over the desired mass range, usually between m/z 500 and 2500. Results are shown for separation and analysis of mixtures of synthetic peptides and also for protease digests of recombinant human growth hormone and horse heart cytochrome c.

Carcinogenicity and metabolic activation of hexestrol.

The carcinogenic activity of the synthetic estrogen hexestrol was measured in male Syrian hamsters. Between 90% and 100% of the animals treated with hexestrol or with 3',3",5',5"-tetradeuteriohexestrol, implanted subcutaneously as 25-mg pellets, were found with renal carcinoma after 6-7 months. In vitro hexestrol metabolism, mediated by phenobarbital-induced rat liver microsomes, led to the formation of 3'-hydroxyhexestrol. This metabolite was identified by comparison with authentic reference material synthesized by oxidation of hexestrol with Fremy's salt. Diethylstilbestrol could not be detected as a metabolite. In urine of male Syrian hamsters, 3'-hydroxyhexestrol, 3'-methoxyhexestrol, 1-hydroxyhexestrol, and other hydroxylated and/or methoxylated hexestrol metabolites were identified. Again, diethylstilbestrol was not detectable as a hexestrol metabolite in vivo. The reactivity of 3'-hydroxyhexestrol was then studied to determine if this catechol estrogen played a role in hexestrol carcinogenicity. Horseradish peroxidase catalyzed the oxidation of 3'-hydroxyhexestrol to 3',4'-hexestrol quinone. This oxidation reaction could also be carried out non-enzymatically using silver oxide or silver carbonate on celite as oxidants. The quinone was unstable (t1/2 in methylene chloride: 53 min). It reacted with sulfur-containing compounds such as mercaptoethanol by Michael addition to form 3'-(2-hydroxyethylthio)-5'-hydroxyhexestrol. 3',4'-Hexestrol quinone reacted with simple amines such as ethylamine to form N-ethyl-aminohexestrol. The chemical reactions described above were carried out to test the reactivity of identified or suspected metabolic intermediates of hexestrol. It was concluded that carcinogenicity of hexestrol was not based on its conversion to diethylstilbestrol. Rather, catechol estrogen formation may be necessary for the carcinogenic action of hexestrol in analogy to events observed earlier with estradiol.

Reactivity of 4',4"-diethylstilbestrol quinone, a metabolic intermediate of diethylstilbestrol.

In a search for the carcinogenic metabolite of diethylstilbestrol, the interactions of 4',4"-diethylstilbestrol quinone with peptides and nucleic acids were investigated. Nonextractable binding of 4',4"-diethylstilbestrol quinone to calf thymus DNA or poly G were observed. However, adduct nucleosides could not be isolated subsequent to enzymatic digestion of nucleic acids. Binding to dGMP or dAMP also occurred, but the initially bound stilbene estrogen could mostly be extracted with 18 extractions using various organic solvents. Non-covalent interactions of 4',4"-diethylstilbestrol quinone with calf thymus DNA were observed spectrally only after exhaustive dialysis of the DNA versus water, but not with native DNA. In chemical reactions of 4',4"-diethylstilbestrol quinone and nucleosides, nucleotides, and amines such as n-pentyl amine, only Z,Z-dienestrol could be identified as reaction product. The quinone did react with mercaptoethanol via Michael addition to the unsaturated carbonyl system to form a stable adduct, 4-(2-hydroxyethylthio)-3,4-di(p-hydroxyphenyl)-2-hexene. It also reacted covalently with sulfur-containing peptides such as reduced glutathione or bovine serum albumin. Partially purified rat liver cytochrome P-450 reductase reduced 4',4"-diethylstilbestrol quinone to E- and Z-diethylstilbestrol. It is proposed that 4',4"-diethylstilbestrol quinone forms unstable adduct intermediates with DNA which decompose with time. Also, covalent binding of 4',4"-diethylstilbestrol quinone to important proteins via thioether linkages may play a role in carcinogenesis.

Diethylstilbestrol (DES) quinone: a reactive intermediate in DES metabolism.

The quinone of E-diethylstilbestrol (DES), a postulated metabolic intermediate derived from DES, has been synthesized by oxidation of DES in chloroform using silver oxide. The reaction product was structurally characterized by infrared, ultraviolet, nuclear magnetic resonance, and mass spectrometry. The product of oxidation of DES by hydrogen peroxide, catalyzed by horseradish peroxidase and also by rat uterine peroxidase, was shown to be identical with synthetic DES quinone based on identical u.v. spectra and on identical decomposition products. DES quinone was stable only in non-protic solvents such as chloroform. In acids, bases or protic solvents, DES quinone rearranged to Z,Z-dienestrol (beta-DIES). The half-life of DES quinone in water was approximately 40 min; in methanol it was approximately 70 min. Bacterial mutagenicity (Ames) tests did not indicate that DES quinone had mutagenic or genotoxic activity. However, DES quinone was found to bind to calf thymus DNA without any enzyme mediation at levels significantly above the binding of DES under the same conditions. Based on the binding of DES quinone to DNA, this intermediate must be considered as a possible carcinogenic metabolite of DES.