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We have developed an automated quench-flow microreactor which interfaces directly to an electrospray ionization (ES1) mass spectrometer. We have used this device in conjunction with ESI Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to demonstrate the potential of this approach for studying the mechanistic details of enzyme reactions. For the model system chosen to test this device, namely, the pre-steady-state hydrolysis of p-nitrophenyl acetate by the enzyme chymotrypsin, the kinetic parameters obtained are in good agreement with those in the literature. To our knowledge, this is the first reported use of online quench-flow coupled with FrICR MS. Furthermore, we have exploited the power of FTICR MS to interrogate the quenched covalently bound enzyme intermediate using top-down fagmentafion. The accurate mass capabilities of FTICR MS permitted the nature of the intermediate to be assigned with high confidence. Electron capture dissociation (ECD) fragmentation allowed us to locate the intermediate to a five amino acid section of the protein-which includes the known catalytic residue, [Ser.sub.195]. This experimental approach, which uniquely can provide both kinetic and chemical details of enzyme mechanisms, is a potentially powerful tool for studies of enzyme catalysis. 10.1021/ac9026302

We describe a quantitative Fourier transform ion cyclotron resonance mass spectrometric (FTICR MS) analysis of the relative proportions of post-translational modification states (PTMs) of core histones in cultured cells and tissues. A novel preseparation process using a monolithic column interfaced to a 12 T FTICR MS equipped with electron capture dissociation (ECD) yields very high mass accuracy spectra, allowing direct assignment of the PTMs present in the dominant modification states of intact H4, resolving a well recognized ambiguity between trimethylation and acetylation states. By eliminating preseparation, we also obtain a highly quantitative analysis of the distribution of H4 PTMs. Rapid, extensive, and reversible effects on PTMs induced by a histone deacetylase inhibitor indicate that H4 and other core histones are accessible to modification throughout the chromatin, not just in regions of active transcription. These methods provide tools for analysis of the histone code and its role in chromatin function.

Studies were conducted for the BCP homologue of Burkholderia cenocepacia (BcBCP) and bacterioferritin comigratory protein (BCP) of Escherichia coli to clarify the catalytic mechanism related to the active and resolving cysteine and unresolving cysteine of BCP peroxiredins. The characterization supported the subdivision of the BCP family of peroxiredoxins into two classes based on their catalytic activity.

Combination of high-resolution Fourier transform ion cyclotron resonance mass spectrometry and top-down fragmentation techniques were used to analyze the mechanistic details of hydrogen peroxide reduction by Escherichia coli bacterioferritin comigratory protein (BCP). The mutant BCP enzyme adopted a different and novel mechanistic pathway and reacted with peroxide to form a sulfenic acid on Cys-45 and is also a substrate for reduction by thioredoxin.

A proteomic technological approach to characterize anterior gradient-2 protein as p53 inhibitor which is over expressed in human cancer is described. The method could be applied to develop clinical biomarker assay in an aptamer microarray format.

Zinc exchange reactions of Zn4-SmtA by a new method using stable isotope labeling combined with Fourier transform ion cyclotron resonance mass spectrometry is investigated. The Zn4 cluster of SmtA contains a kinetically inert Zn site, a feature that could be related to its secondary and tertiary structure.