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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]

We present a detailed analysis of the relative yields in dissociation products of doubly protonated polypeptide cations obtained via electron capture dissociation (ECD). These experimental studies are complemented by molecular dynamics force field modelling, using the AMBER force field, to correlate with putative gas-phase conformations for these peptides. It is shown that the highest gas-phase basicity amino acid residue (i.e. arginine) is included in all the charged fragments. This is of particular use in determining the primary structure tryptic digest peptides, which will ordinarily posses a high basicity C-terminal residue (i.e. arginine or lysine). Further, these results suggest that the relative ECD dissociation pattern is related to the secondary structure of the peptide. In particular, the ECD fragmentation pattern in gonadatropin releasing hormone (GnRH) variants appears to depend on whether a ß-turn or an extended a-helical structure is formed. In the peptide bradykinin, modelling suggests that the C-terminal arginine engages in much more extended solvation of the backbone than the N-terminal arginine. This strongly correlates with the observed dominance of c over z fragments. This work forms the first attempt at a systematic qualitative correlation of the low-energy structures of modelled gas-phase polypeptides, and their corresponding ECD dissociation pattern. [ABSTRACT FROM AUTHOR]

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.

Abstract: Oxidation of cysteine is now known to serve as a fundamental mechanism to control protein function or activity. Many redox-regulated proteins do not oxidize to homogeneity, resulting in a mixture of reduced and oxidized species which cannot be separated chromatographically. Here we describe a protocol for the separation of reduced and oxidized forms of the tumor suppressor protein p53. This purification method relies on the reversible labeling of thiol groups with biotin and exploitation of the ultrastrong biotin–avidin interaction. This purification procedure can be applied to other cysteine-containing proteins where enrichment of the oxidized form is required. [Copyright &y& Elsevier]

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]

The organometallic anticancer complex [(η(6)-bip)Ru(en)Cl](+) (1; bip = biphenyl, en = ethylenediamine) selectively binds to N7 of guanine bases of oligonucleotides and native DNA. However, under physiologically relevant conditions (micromolar Ru concentrations, pH 7, 22 mM NaCl, 310 K), the tripeptide glutathione (γ-L-Glu-L-Cys-Gly; GSH) is kinetically competitive with guanine (as guanosine 3',5'-cyclic monophosphate, cGMP) for coordination with complex 1, and gives rise to a ruthenium thiolato adduct. This thiolato adduct can subsequently undergo oxidation to a sulfenate intermediate, providing a facile route for the formation of a final cGMP adduct via the displacement of S-bound glutathione by G N7 (F. Y. Wang, J. J. Xu, A. Habtemariam, J. Bella and P. J. Sadler, J. Am. Chem. Soc., 2005, 127, 17734). In this work, the competition between GSH and the single-stranded 14-mer oligonucleotide 5'-TATGTACCATGTAT-3' (I) and duplex III (III = I + II, II = 5'-ATACATGGTACATA) for complex 1 and its analogue [(η(6)-tha)Ru(en)Cl](+) (2, tha = tetrahydroanthracene) under physiologically relevant conditions was investigated using conventional ESI-MS and high resolution ESI-FTICR-MS coupled to conventional HPLC and nanoscale HPLC, respectively. The results indicate that whether there was high excess of GSH or not in the reaction mixtures, the reaction of complex 1 or 2 with single-stranded oligonucleotide I always gave rise to mono-ruthenated oligonucleotide, and the reaction of complex 1 or 2 with duplex III gave rise to the mono-ruthenated duplex oligonucleotide. Furthermore, the ruthenation of duplex III by complex 1 showed no significant discrimination between the complementary strands I and II, but complex 2 appeared to bind preferentially to strand II compared to strand I as revealed by the high resolution FTICR-MS analysis. GSH is highly abundant in cells at millimolar concentrations and is well known to be involved in the deactivation of the clinical drug cisplatin and in platinum resistance. Our findings reveal a potentially contrasting role for GSH in the mechanism of action of these ruthenium anticancer complexes that may contribute to the lack of cross-resistance with platinum drugs.

The high mass accuracy of FT-ICR MS combined with (15)N-labelling shows that mono- and di- platinated products from the reaction of erythrocyte superoxide dismutase with the anticancer drug cisplatin in solution retain their ammine ligands, in contrast to a recent X-ray crystallographic study.