Identification of cytochrome P450 enzymes in human colorectal metastases and the surrounding liver: a proteomic approach.

We describe the direct identification of multiple cytochrome P450 (CYP) enzymes in healthy and cancerous tissue. CYPs in human liver colorectal metastases were compared with those in the surrounding liver using a mass spectrometry-based proteomic approach. Nano-scale reversed phase liquid chromatography combined with electrospray ionisation tandem mass spectrometry has been used to identify CYPs with no pre-selection of the proteins anticipated. Fourteen distinct CYP enzymes from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, 8B and 27A were positively identified; 13 in the liver samples and 12 in the tumour tissue. It was found that three of the colorectal metastases expressed essentially the same drug-metabolising pattern of CYPs as the surrounding liver, whilst three tumour samples from different individuals showed no CYP expression. This was likely in at least one case to be due to low sample mass. The CYP expression profile in an individual tumour is likely to be an important determinant in predicting the outcome of cancer chemotherapy.

A proteomic approach to the identification of cytochrome P450 isoforms in male and female rat liver by nanoscale liquid chromatography-electrospray ionization-tandem mass spectrometry.

Nanoscale reversed-phase liquid chromatography (LC) combined with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used as a method for the direct identification of multiple cytochrome P450 (P450) isoforms found in male and female rat liver. In this targeted proteomic approach, rat liver microsomes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel tryptic digestion of the proteins present in the 48- to 62-kDa bands. The resultant peptides were extracted and analyzed by LC-ESI-MS/MS. P450 identifications were made by searching the MS/MS data against a rat protein database containing 21,576 entries including 47 P450s using Sequest software (Thermo Electron, Hemel Hempstead, UK). Twenty-four P450 isoforms from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, CYP17, and CYP19 were positively identified in rat liver.

A preclinical pharmacokinetic study of the bioreductive drug AQ4N.

AQ4N (1,4-bis-[[2-(dimethylamino-N-oxide)ethyl]amino]5,8-dihydroxyanthracene-9,10-dione) is in a class of bioreductive agents incorporating the aliphatic N-oxide functionality and is well documented as a very effective enhancer of radiotherapy and chemotherapy. The compound is shortly to enter Phase I clinical trials in the United Kingdom, and this study describes the preclinical pharmacokinetics and metabolism of AQ4N in mice. AQ4N was administered by i.v. injection at doses of 200, 100, and 20 mg/kg and was quantified by high-performance liquid chromatography and liquid chromatography/mass spectroscopy. There was a linear increase in the maximum plasma concentration (Cmax) proportional to dose with a Cmax of 1171 microg/ml at the maximum tolerated dose of 200 mg/kg. The area under plasma concentration versus time curve (AUC) increased disproportionately with dose from 14.1 microg/h/ml at 20 mg/kg to 247 microg/h/ml at 200 mg/kg with a subsequent decrease in clearance. Terminal elimination half-lives ranged from 0.64 to 0.83 h. The spectra of the two major metabolites matched those from authentic standards with the molecular ions [M + H]+ being detected at m/z 445.4 (AQ4N), m/z 429.5 (AQ4 mono-N-oxide) and m/z 413.5 (AQ4). Only low concentrations of the toxic metabolite (AQ4) were detected in plasma at all three doses, with the AUC and Cmax at 200 mg/kg being 3.54 microg/h/ml and 3.7 microg/ml, respectively, representing <2% of AQ4N. Concentrations of the intermediate AQ4 M represented 8, 10, and 18% of those for AQ4N at the doses of 20,100, and 200 mg/kg. The concentrations necessary for a therapeutic response in vivo have been described in this pharmacokinetic study.

Characterization of fungal spores by laser desorption/ionization time-of-flight mass spectrometry.

A considerable volume of research has now been completed on the application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the analysis of bacteria; however, to date no definitive studies have been made using this technique on fungi. Preliminary studies on the application of the MALDI-MS methodology, previously developed for the analysis of bacteria, to the analysis of intact fungal spores are described here. MALDI-MS and electrospray mass spectrometry enable the high molecular weight analysis of proteins, glycoproteins, oligosaccharides and oligonucleotides. Using MALDI-MS with bacteria has demonstrated the ability to produce 'fingerprints' of the intact cells with the ions observed being associated with the proteinaceous components of the cell wall. This paper reports the adaptation of this technique to the direct analysis of fungal cells. The high percentage of carbohydrate in the fungal cell wall indicates that the ions observed in the mass spectrometric experiments may be of carbohydrate origin. Penicillium spp., Scytalidium dimidiatum and Trichophyton rubrum have been studied in this preliminary investigation and all show individually distinctive spectra which would appear to provide a profile of the cellular material with discrete peaks being observed over the mass range 2 to 13 kDa. The spectra obtained are reproducible within the method used but, as shown in our previous studies on bacteria, washing may selectively release components from the fungal cell wall.

The effect of solvent and matrix combinations on the analysis of bacteria by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

The ability to rapidly identify the taxonomic class of the wide variety of microorganisms involved in human and animal disease is becoming increasingly important, especially with the increasing development of resistance to the antibiotics which form the main defence against them. A number of groups have recognised the utility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry MALDI-TOF in the analysis of these microorganisms. However, no consistent methodology has been developed which is in general use. In particular the use of different solvent extraction systems and mass spectrometric matrices can have significant effects on the quality of the data obtained. We have now studied a number of the commonly used matrices and a range of solvent systems of widely varying polarity in an attempt to devise an optimum analytical strategy for the rapid characterisation of these organisms by MALDI-TOFMS. The E. coli ATCC 9637 organisms were initially washed to remove growth medium contaminants, followed by extraction with one of a range of solvents prior to admixing with a number of different single matrices or binary and ternary combinations of these matrices. The results obtained indicate that a binary combination of 2-(4-hydroxyphenylazo)benzoic acid and 2-mercaptobenzothiazole (1:1) as matrix provides the best data after the proteinaceous material from the organism cell surface was extracted with 17% formic acid, 33% isopropyl alcohol and 50% water, (solvent 2 in this work).

The characterization of micro-organisms by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A variety of gram-positive and gram-negative intact bacterial cells have been analysed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and shown to provide fingerprint mass spectra with discrete peaks being observed over the mass range from 3 to 40 kDa. The spectra show both more peaks and peaks at a higher mass/charge ratio than have hitherto been reported for these micro-organisms and would appear to provide a profile of cellular proteinaceous material. The spectra are shown to be reproducible over variable time periods of up to three months and factors affecting reproducibility are discussed. The procedure, which requires minimal sample preparation, yields results in 30-40 minutes and allows visual identification of species- and strain-specific biomarkers for the characterization of the organisms. The importance of accurately defining sample preparation methodologies is central to the ability of the technique to generate reliable and reproducible data.

Two-dimensional high-performance liquid chromatographic method for assaying S-adenosyl-L-methionine and its related metabolites in tissues.

S-Adenosyl-L-methionine (SAM) is a methyl-donor compound which is actively involved in a variety of biochemical reactions. An assay has been developed permitting the quantitative measurement of SAM and its related metabolites (S-adenosylhomocysteine, decarboxylated SAM, methylthioadenosine, adenosine and adenine) in liver and cell cultures. As gradient reversed-phase chromatographic or cation-exchange chromatographic methods often resulted in overlapping peaks, a two-dimensional high-performance liquid chromatographic (HPLC) procedure was developed involving gradient reversed-phase chromatographic separation followed by ion-exchange chromatography. After precipitating large molecules in the sample by perchloric acid, gel permeation was carried out on a Sephadex G 25 column to separate small water-soluble metabolites from proteins and membrane fragments. The freeze-dried sample was injected onto an ODS column and a 0-10% acetonitrile gradient in 10 mM ammonium formate buffer (pH 2.9) (20 min, linear) was applied. The relevant fractions were collected and injected onto a cation-exchange column (Partisil SCX, 10 microns, 250 mm x 4.6 mm I.D.). Elution and quantification were carried out using ammonium formate buffers of various concentration (15-400 mM), pH 2.9. The detector response (254 nm) as a function of concentration was linear over the concentration range 30-500 pmol. The detection limits of the compounds after the two-dimensional chromatographic procedure ranged from 10 to 60 pmol and the recovery was higher than 70%. The reproducibility of the results obtained from given samples was within 9-22% for rat liver and 6-24% for mast cells.

On the purity of 3X-recrystallized bovine alpha-chymotrypsin.

The results of an electrospray-mass spectrometric analytical study of aqueous solutions of fifteen commercial samples of 3X-recrystallized bovine alpha-chymotrypsin are presented and discussed. It was found that only six samples were predominantly alpha-chymotrypsin and that two samples contained no alpha-chymotrypsin at all. The remaining seven samples were found to be mixtures of alpha-chymotrypsin with other chymotrypsins and, in some cases, neochymotrypsinogens. The majority of the results are rationalised in terms of previously postulated and/or observed products of proteolytic activation of bovine chymotrypsinogen A. However, evidence is also presented for the presence in many of the samples of three new serine proteases, of significantly lower molecular masses than alpha-chymotrypsin, which cannot at present be explained. The paper is concluded with a brief discussion of the implications of the analytical findings for enzymological studies.

Some electrospray mass spectrometric evidence for the existence of covalent O-acyl enzyme intermediates.

Electrospray mass spectrometry has been used to measure the masses of the species present in solutions of three serine proteases (alpha-chymotrypsin, subtilisin Carlsberg and subtilisin BPN') before, during and after completion of the hydrolytic reaction with cinnamoyl imidazole and indole acryloyl imidazole. The masses measured during the reaction demonstrated that covalent O-acyl enzyme intermediates had been formed.

High-performance liquid chromatographic-mass spectrometric assay of high-value compounds for pharmaceutical use from plant cell tissue culture: Cinchona alkaloids.

The use of high-performance liquid chromatography (HPLC) interfaced with thermospray (TSP) mass spectrometry is described for the separation and identification of various alkaloids from Cinchona ledgeriana extracts. The use of water-acetonitrile-acetic acid (71:25:4) with 0.01 M ammonium acetate (pH 3.0) as the mobile phase gave good HPLC separation and good TSP sensitivity. The specificity obtained by single-ion monitoring allowed the analysis of commercially important alkaloids such as quinine and quinidine in plant material, transformed roots and in cells from tissue culture, with relatively simple extraction and work-up procedures. TSP gave protonated species with few fragment ions but collision-induced dissociation offers the promise of increased analytically specificity from the fragment ion data. This work has important implications for the biotechnological production of pharmaceuticals normally obtained from plant sources.