Determination of Protein Thiol Reduction Potential by Isotope Labeling and Intact Mass Measurement.

Oxidation/reduction of thiol residues in proteins is an important type of post-translational modification that is implicated in regulating a range of biological processes. The nature of the modification makes it possible to define a quantifiable electrochemical potential (E(⊕)) for oxidation/reduction that allows cysteine-containing proteins to be ranked based on their propensity to be oxidized. Measuring oxidation of cysteine residues in proteins is difficult using standard electrochemical methods, but top-down mass spectrometry recently has been shown to enable the quantification of E(⊕) for thiol oxidations. In this paper, we demonstrate that mass spectrometry of intact proteins can be used in combination with an isotopic labeling strategy and an automated data analysis algorithm to measure E(⊕) for the thiols in both E. coli Thioredoxin 1 and human Thioredoxin 1. Our methodology relies on accurate mass measurement of proteins using liquid chromatography-mass spectroscopy (LC-MS) analyses and does not necessarily require top-down fragmentation. In addition to analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol E(⊕) measurements in samples that contain mixtures of proteins. Thus, the combination of experimential methodology and data analysis regime has the potential to make such measurements in a high-throughput manner and in a manner that is more accessible to a broad community of protein scientists.

The chemical basis of serine palmitoyltransferase inhibition by myriocin.

Sphingolipids (SLs) are essential components of cellular membranes formed from the condensation of L-serine and a long-chain acyl thioester. This first step is catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) which is a promising therapeutic target. The fungal natural product myriocin is a potent inhibitor of SPT and is widely used to block SL biosynthesis despite a lack of a detailed understanding of its molecular mechanism. By combining spectroscopy, mass spectrometry, X-ray crystallography, and kinetics, we have characterized the molecular details of SPT inhibition by myriocin. Myriocin initially forms an external aldimine with PLP at the active site, and a structure of the resulting co-complex explains its nanomolar affinity for the enzyme. This co-complex then catalytically degrades via an unexpected 'retro-aldol-like' cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of SPT by covalent modification of the essential catalytic lysine. This surprising dual mechanism of inhibition rationalizes the extraordinary potency and longevity of myriocin inhibition.

Competition between glutathione and DNA oligonucleotides for ruthenium(II) arene anticancer complexes.

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.

Conductive carbon tape used for support and mounting of both whole animal and fragile heat-treated tissue sections for MALDI MS imaging and quantitation.

Analysis of whole animal tissue sections by MALDI MS imaging (MSI) requires effective sample collection and transfer methods to allow the highest quality of in situ analysis of small or hard to dissect tissues. We report on the use of double-sided adhesive conductive carbon tape during whole adult rat tissue sectioning of carboxymethyl cellulose (CMC) embedded animals, with samples mounted onto large format conductive glass and conductive plastic MALDI targets, enabling MSI analysis to be performed on both TOF and FT-ICR MALDI mass spectrometers. We show that mounting does not unduly affect small molecule MSI detection by analyzing tiotropium abundance and distribution in rat lung tissues, with direct on-tissue quantitation achieved. Significantly, we use the adhesive tape to provide support to embedded delicate heat-stabilized tissues, enabling sectioning and mounting to be performed that maintained tissue integrity on samples that had previously been impossible to adequately prepare section for MSI analysis. The mapping of larger peptidomic molecules was not hindered by tape mounting samples and we demonstrate this by mapping the distribution of PEP-19 in both native and heat-stabilized rat brains. Furthermore, we show that without heat stabilization PEP-19 degradation fragments can detected and identified directly by MALDI MSI analysis.

Ferric ion (hydr)oxo clusters in the "Venus flytrap" cleft of FbpA: Mössbauer, calorimetric and mass spectrometric studies.

Isothermal calorimetric studies of the binding of iron(III) citrate to ferric ion binding protein from Neisseria gonorrhoeae suggested the complexation of a tetranuclear iron(III) cluster as a single step binding event (apparent binding constant K(app) (ITC) = 6.0(5) × 10(5) M(-1)). High-resolution Fourier transform ion cyclotron resonance mass spectrometric data supported the binding of a tetranuclear oxo(hydroxo) iron(III) cluster of formula [Fe(4)O(2)(OH)(4)(H(2)O)(cit)](+) in the interdomain binding cleft of FbpA. The mutant H9Y-nFbpA showed a twofold increase in the apparent binding constant [K(app) (ITC) = 1.1(7) × 10(6) M(-1)] for the tetranuclear iron(III) cluster compared to the wild-type protein. Mössbauer spectra of Escherichia coli cells overexpressing FbpA and cultured in the presence of added (57)Fe citrate were indicative of the presence of dinuclear and polynuclear clusters. FbpA therefore appears to have a strong affinity for iron clusters in iron-rich environments, a property which might endow the protein with new biological functions.

An affinity purification procedure to isolate oxidized p53.

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.

Mapping a noncovalent protein-peptide interface by top-down FTICR mass spectrometry using electron capture dissociation.

Noncovalent protein-ligand and protein-protein complexes are readily detected using electrospray ionization mass spectrometry (ESI MS). Furthermore, recent reports have demonstrated that careful use of electron capture dissociation (ECD) fragmentation allows covalent backbone bonds of protein complexes to be dissociated without disruption of noncovalent protein-ligand interactions. In this way the site of protein-ligand interfaces can be identified. To date, protein-ligand complexes, which have proven tractable to this technique, have been mediated by ionic electrostatic interactions, i.e., ion pair interactions or salt bridging. Here we extend this methodology by applying ECD to study a protein-peptide complex that contains no electrostatics interactions. We analyzed the complex between the 21 kDa p53-inhibitor protein anterior gradient-2 and its hexapeptide binding ligand (PTTIYY). ECD fragmentation of the 1:1 complex occurs with retention of protein-peptide binding and analysis of the resulting fragments allows the binding interface to be localized to a C-terminal region between residues 109 and 175. These finding are supported by a solution-phase competition assay, which implicates the region between residues 108 and 122 within AGR2 as the PTTIYY binding interface. Our study expands previous findings by demonstrating that top-down ECD mass spectrometry can be used to determine directly the sites of peptide-protein interfaces. This highlights the growing potential of using ECD and related top-down fragmentation techniques for interrogation of protein-protein interfaces.

Identification of two reactive cysteine residues in the tumor suppressor protein p53 using top-down FTICR mass spectrometry.

The tumor suppressor p53 is a redox-regulated transcription factor involved in cell cycle arrest, apoptosis and senescence in response to multiple forms of stress, as well as many other cellular processes such as DNA repair, glycolysis, autophagy, oxidative stress and differentiation. The discovery of cysteine-targeting compounds that cause re-activation of mutant p53 and the death of tumor cells in vivo has emphasized the functional importance of p53 thiols. Using a combination of top-down and middle-down FTICR mass spectrometry, we show that of the 10 Cys residues in the core domain of wild-type p53, Cys182 and Cys277 exhibit a remarkable preference for modification by the alkylating reagent N-ethylmaleimide. The assignment of Cys182 and Cys277 as the two reactive Cys residues was confirmed by site-directed mutagenesis. Further alkylation of p53 beyond Cys182 and Cys277 was found to trigger co-operative modification of the remaining seven Cys residues and protein unfolding. This study highlights the power of top-down FTICR mass spectrometry for analysis of the cysteine reactivity and redox chemistry in multiple cysteine-containing proteins.

Matrix-free mass spectrometric imaging using laser desorption ionisation Fourier transform ion cyclotron resonance mass spectrometry.

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.

Identification of clusters from reactions of ruthenium arene anticancer complex with glutathione using nanoscale liquid chromatography Fourier transform ion cyclotron mass spectrometry combined with (18)O-labeling.

Reactions of the anticancer complex [(eta(6)-bip)Ru(en)Cl](+) (where bip is biphenyl and en is ethylenediamine) with the tripeptide glutathione (gamma-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 [{(eta(6)-bip)Ru(GSO(2))}(4)](2-) (2) and dinuclear complex [{(eta(6)-bip)Ru(GSO(2))(2)}(2)](8-) (3) containing glutathione sulfinate (GSO(2)) ligands. Use of (18)OH(2) 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.

Platination of superoxide dismutase with cisplatin: tracking the ammonia ligands using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS).

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.

Inert site in a protein zinc cluster: isotope exchange by high resolution mass spectrometry.

Using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, we show that one Zn2+ ion in the Zn4 cluster of cyanobacterial metallothionein is inert to exchange with 67Zn2+. We suggest that this is zinc in site A, which together with the surrounding alpha and beta secondary structure forms a zinc-finger fold.

Electron capture dissociation of polypeptides using a 3 Tesla Fourier transform ion cyclotron resonance mass spectrometer.

Electron capture dissociation (ECD) of polypeptides has been demonstrated using a commercially available 3 Tesla Fourier transform ion cyclotron resonance (FTICR) instrument. A conventional rhenium filament, designed for high-energy electron impact ionisation, was used to effect ECD of substance P, bee venom melittin and bovine insulin, oxidised B chain. A retarding field analysis of the effective electron kinetic energy distribution entering the ICR cell suggests that one of the most important parameters governing ECD for this particular instrument is the need to employ low trapping plate voltages. This is shown to maximise the abundance of low-energy electrons. The demonstration of ECD at this relatively low magnetic field strength could offer the prospect of more routine ECD analysis for the wider research community, given the reduced cost of such magnets and (at least theoretically) the greater ease of electron/ion cloud overlap at lower field.