Beverly B. DaGue

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).

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.

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.

Microbore HPLC / mass spectrometry for the analysis of peptide mixtures using a continuous flow interface

Microbore HPLC techniques have been combined with fast atom bombardment mass spectrometry to provide HPLC/MS capabilities for the analysis of mixtures of peptides and small proteins. The interface between the liquid chromatograph and mass spectrometer is a continuous flow direct insertion probe which contains a fused silica capillary that delivers the eluting solvent to the FAB source of the mass spectrometer at a rate of 5-10 microL/min. Data are presented for the analysis of several mixtures of peptides ranging in molecular weights from about 900 to 6000 daltons. In addition, the analysis of 100 pmol of a tryptic digest of whale myoglobin is shown where 16 of the possible 19 peptides were identified in the mass range m/z 2200-250. The advantages of this approach to HPLC/MS are a relatively high sensitivity because of the low flow rates and low background, and the ability to detect high molecular weight compounds.

Microbore high-performance liquid chromatography-mass spectrometry for the analysis of proteolytic digests by continuous-flow fast-atom bombardment mass spectrometry.

Microbore high-performance liquid chromatographic (HPLC) techniques have been combined with fast atom bombardment mass spectrometry (FAB-MS) for the mass-specific detection of mixtures of peptides produced by proteolytic hydrolysis of proteins. The continuous-flow FAB interface has been utilized for direct coupling of the microbore HPLC system and the mass spectrometer. Conditions are reported for the effective separation of 100 pmol of peptides at flow-rates of 5 microliters/min with acetonitrile gradients and 1-mm I.D. C8 columns. A comparison is also made between columns of 5 and 25 cm lengths for the separation of peptide mixtures. Data are presented for the separation of peptides on a slurry-packed C18 fused-silica capillary column with the continuous-flow HPLC-FAB-MS interface at flow-rates of 3 microliters/min.

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.

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.

Epididymal Lipocalin-Type Prostaglandin D2 Synthase: Identification Using Mass Spectrometry, Messenger RNA Localization, and Immunodetection in Mouse, Rat, Hamster, and Monkey

Abstract This study identified prostaglandin D2 synthase (PGDS) in murine epididymal fluid using a proteomic approach combining two-dimensional (2D) gel electrophoresis and mass spectrometry (MS). The caudal epididymal fluid was collected by retroperfusion, and proteins were separated by 2D gel electrophoresis followed by matrix-assisted laser desorption ionization MS analyses after trypsin digestion. The identification was based on the protein-specific peptide map as well as on sequence information generated by nano-electrospray ionization MS/MS. By in situ hybridization, the mRNA was detected in caput, corpus, and cauda, but it was not detected in the initial segment. The PGDS protein was mostly detected in the corpus and cauda by Western blot analysis and immunohistochemistry using a specific polyclonal antibody. In caudal fluid, PGDS was distributed among several isoforms (pI range, 6.5–8.8), suggesting that this protein undergoes posttranslational modification of its primary sequence. After N-glycanase digestion, the molecular mass decreased from 20–25 to 18.5 kDa, its theoretical mass. The PGDS was also detected in the epididymis of rat, hamster, and cynomolgus monkey from the caput to the cauda. In conclusion, MS is a powerful and accurate technique that allows unambiguous identification of the murine epididymal PGDS. The protein is 1) present throughout the epididymis, except in the initial segment, with an increasing luminal concentration from distal caput to cauda; 2) a major protein in caudal fluid; 3) an N-glycosylated, highly polymorphic protein; and 4) conserved during evolution.