Simultaneous measurement of nineteen binding constants of peptides to vancomycin using affinity capillary electrophoresis-mass spectrometry.

On-line affinity capillary electrophoresis-electrospray ionization-mass spectrometry (ACE-MS) was used for the simultaneous measurement of multiple binding constants of an all-D-tetrapeptide library to the model receptor, vancomycin. Determination of Kd values for the 19 peptides of the form Fmoc-DXYA is demonstrated. The data are compared with the results obtained for individual compounds using ACE-UV, and good correlation between the two detection methods is shown. Simultaneous determination of multiple Kd values by ACE-MS is achieved in one set of experiments, whereas only one Kd value can be obtained by ACE-UV during the same time. ACE-MS measures multiple binding constants in solution in a fast and reliable manner using femtomole amounts of samples.

Microscale epitope mapping by affinity capillary electrophoresis-mass spectrometry.

Using beta-endorphin as a model system, a new microscale solution-based approach for linear epitope mapping based on affinity capillary electrophoresis-mass spectrometry (ACE-MS) is demonstrated. Tryptic peptides are separated in a neutral coated capillary and monitored by ultraviolet absorbance and electrospray mass spectrometry. Then, following injection of the peptide digest mixture, anti-beta-endorphin antibody is injected. The peptide, which binds to the antibody, is captured and disappears from its migration time. Following this subtraction-screening procedure, the binding of the individually synthesized or isolated immunoreactive peptide is examined by the ACE-MS procedure to confirm that the epitope resides on the peptide. A series of truncated peptides can then be made and the precise epitope determined by ACE-MS. The method utilizes low femtomole amounts of antibody and peptide digest per run and is rapid and easily automatable.

Multichannel microchip electrospray mass spectrometry.

Microfabricated multiple-channel glass chips were successfully interfaced to an electrospray ionization mass spectrometer (ESI-MS). The microchip device was fabricated by standard photolithographic, wet chemical etching, and thermal bonding procedures. A high voltage was applied individually from each buffer reservoir for spraying sample sequentially from each channel. With the sampling orifice of the MS grounded, it was found that a liquid flow of 100-200 nL/min was necessary to maintain a stable electrospray. The detection limit of the microchip MS experiment for myoglobin was found to be lower than 6 x 10(-8) M. Samples in 75% methanol were successfully analyzed with good sensitivity, as were aqueous samples. The parallel mutliple-channel microchip system allowed ESI-MS analysis of different samples of standard peptides and proteins in one chip.

Integrated multichannel microchip electrospray ionization mass spectrometry: analysis of peptides from on-chip tryptic digestion of melittin.

In continuation of our work to develop an integrated multichannel microchip interfaced to electrospray mass spectrometry (ESI-MS), this paper demonstrates one of several applications of this approach in monitoring tryptic digestion products. The multichannel microchip allowed integration of sample preparation onto the microchip to facilitate the analysis process. Melittin was selected as a model oligopeptide because it possesses a cluster of four adjacent basic residues which enable probing the site specificity of trypsin as a function of digest times. Reactions were performed on-chip in different wells for specific time periods and then analyzed by infusion from the microchip by ESI-MS, using leucine enkephalin as internal standard. The rate of formation and disappearance of the molecular ion and individual fragments was followed for a melittin to trypsin concentration ratio of 300:1. The results indicate the potential of integrating enzymatic reactions with multichannel microchip ESI-MS for automated optimization of reaction condition while consuming only small amounts of sample.

Application of capillary electrophoresis-electrospray ionization mass spectometry in the determination of molecular diversity.

By means of capillary electrophoresis coupled online to electrospray ionization MS, a library of theoretically 171 disubstituted xanthene derivatives was analyzed. The method allowed the purity and makeup of the library to be determined: 160 of the expected compounds were found to be present, and 12 side-products were also detected in the mixture. Due to the ability of capillary electrophoresis to separate analytes on the basis of charge, most of the xanthene derivatives could be resolved by simple capillary electrophoresis-MS procedures even though 124 of the 171 theoretical compounds were isobaric with at least one other molecule in the mixture. Any remaining unresolved peaks were resolved by MS/MS experiments. The method shows promise for the analysis of small combinatorial libraries with fewer than 1000 components.

Characterization of DNA adducts formed by anti-benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide.

The anti-11,12-dihydrodiol 13,14-epoxide of benzo[g]chrysene, a fjord-region-containing hydrocarbon, was found to react with DNA in vitro to yield, as the major product, an adduct in which the epoxide of the 11R, 12S, 13S, 14R enantiomer was opened trans by the amino group of deoxyadenosine. The structures of this adduct and other deoxyadenosine and deoxyguanosine adducts were established by spectroscopic methods. In reactions with deoxyguanylic acid, a product tentatively identified as a 7-substituted guanine was also detected. The mutagenic properties of this dihydrodiol epoxide in shuttle vector pSP189 showed that mutation at AT pairs accounted for 39% of base change mutations whereas chemical findings indicated that about 60% of adducts formed in calf thymus DNA involved adenines. Since calf thymus DNA is 56% AT and the target supF gene is 41% AT, the findings represent a fairly close relationship between adduct formation and mutagenic response. Overall, the chemical and mutagenic selectivities for the two purine bases in DNA were similar, though not identical, to those for the only other fjord-region-containing hydrocarbon studied in depth, i.e., benzo[c]phenanthrene. A major difference for these two hydrocarbon derivatives, however, is that benzo[c]phenanthrene dihydrodiol epoxides react to much higher extents (approximately 4-fold) with DNA than did the benzo[g]chrysene derivative.

New promise in combinatorial chemistry: synthesis, characterization, and screening of small-molecule libraries in solution.

BACKGROUND: The increasing interest in combinatorial chemistry as a tool for the development of therapeutics has led to many new methods of creating molecular libraries of potential lead compounds. Current methods have made it possible to develop libraries of several million compounds. As a result, the limiting factor in the screening of libraries has become the identification and characterization of active species. We have recently described a method for generating libraries of water-soluble compounds containing mixtures of 10(4) to 10(5) different small organic molecules by using generally applicable solution phase chemistry. We set out to develop new methods to characterize and decode these libraries. RESULTS: Libraries were generated by condensing a multi-acid-chloride core molecule with various amines, producing molecules with functional groups about a rigid backbone. Composition and complexity of the libraries was evaluated using electrospray mass spectrometry to analyze model libraries containing up to 55 different molecules. The number of peaks obtained in mass spectrometry is directly correlated with the complexity of the library, and we were therefore able to deduce which of the expected compounds had in fact been formed in the library, and which of the building blocks in the library were not efficiently used. An iterative selection procedure was developed using this information, which allowed the screening of libraries of up to 50,000 chemical species to produce a competitive inhibitor of the enzyme trypsin. CONCLUSIONS: Our strategy for the identification of active species should be broadly applicable to other methods of generating complex libraries of small molecules. The selection from the library of a compound with desired biological properties augurs well for the potential value of generating and screening complex mixtures of small molecules in solution.