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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.

This paper presents details of the design and fabrication of test structures specifically designed for the characterization of two distinct digital microfluidic technologies: electro-wetting on dielectric (EWOD) and surface acoustic wave (SAW). A test chip has been fabricated that includes structures with a wide range of dimensions and provides the capability to characterize enhanced droplet manipulation, as well as other integrated functions. The EWOD and SAW devices have been separately characterized first of all to determine whether integration of the technologies affects their individual performance, including device lifetime evaluation. Microfluidic functions have then been demonstrated, including combined EWOD/SAW functions. In particular, this paper details the use of EWOD to anchor droplets, while SAW excitation is applied to perform mixing. The relationship between test structure designs and the droplets anchoring performance has been studied. [ABSTRACT FROM PUBLISHER]

The article reports on the study that proposes using the term proteoform to designate different molecular forms in which the protein product of a single gene can be found. It shows that the use of the term does not present the ambiguity of various alternative terms, many with historical uses. It suggests that the term is useful to researchers, and the adoption of the term by UniProt and the wider community will improve readability and comprehension.

Reactions of the anticancer complex [(η6-bip)Ru(en)Cl]+ (where bip is biphenyl and en is ethylenediamine) with the tripeptide glutathione (γ-l-Glu-l-Cys-Gly; GSH), the abundant intracellular thiol, in aqueous solution give rise to two ruthenium cluster complexes, which could not be identified by electrospray mass spectrometry (ESI-MS) using a quadrupole mass analyzer. Here we use Fourier transform ion cyclotron mass spectrometry (nanoLC-FT-ICR MS) to identify the clusters separated by nanoscale liquid chromatography as the tetranuclear complex [{(η6-bip)Ru(GSO2)}4]2− (2) and dinuclear complex [{(η6-bip)Ru(GSO2)2}2]8− (3) containing glutathione sulfinate (GSO2) ligands. Use of 18OH2 showed that oxygen from water can readily be incorporated into the oxidized glutathione ligands. These data illustrate the power of high-resolution MS for identifying highly charged multinuclear complexes and elucidating novel reaction pathways for metallodrugs, including ligand-based redox reactions. [Copyright &y& Elsevier]

Mass spectrometry imaging (MSI) is a powerful tool in metabolomics and proteomics for the spatial localization and identification of pharmaceuticals, metabolites, lipids, peptides and proteins in biological tissues. However, sample preparation remains a crucial variable in obtaining the most accurate distributions. Common washing steps used to remove salts, and solvent-based matrix application, allow analyte spreading to occur. Solvent-free matrix applications can reduce this risk, but increase the possibility of ionisation bias due to matrix adhesion to tissue sections. We report here the use of matrix-free MSI using laser desorption ionisation performed on a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. We used unprocessed tissue with no post-processing following thaw-mounting on matrix-assisted laser desorption ionisation (MALDI) indium-tin oxide (ITO) target plates. The identification and distribution of a range of phospholipids in mouse brain and kidney sections are presented and compared with previously published MALDI time-of-flight (TOF) MSI distributions., (Copyright © 2011 John Wiley & Sons, Ltd.)