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Electrospray ionization in combination with Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry are used to explore the gas-phase oxygen-atom transfer chemistry, characterize and reactivities of meso-tetrakis(pentafluorophenyl)porphyrin (TPFPP) chromium complexes, as well as some other tetradentate macrocyclic ligands. The TPFPP are excellent systems to examine the effects of spin-state change on oxygen-atom transfer due to doublet nature of the [d.sup.1] [Cr.sup.V] species and quartet ground states feature of [Cr.sup.III] complexes with high-lying doublet excited states.

An oxo iron(IV) porphyrin radical cation intermediate, functional model of Compound I, is produced as a bare species. The gas-phase reactivity of this intermediate with amines including N,N-dimethylanilines is examined in order to understand the Compound I-like reactivity of high-valent oxo iron intermediates in a solvent free environment.

Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that them TORC1 pathway and the oncogenic Ras/Raf/MEK/extra cellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote fromthe N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways. [ABSTRACT FROM AUTHOR]

InfraRed Multiple Photon Dissociation (IRMPD) spectroscopy was used to assay the structural features of the fragment ions resulting from the elimination of H3PO4 in the Collision-Induced Dissociation (CID) of protonated serine. The results are interpreted with the aid of density functional theory calculations. Experiment and theory point to an aziridine-ring structure, implying participation of the vicinal amino group in the formation of this species. This finding constitutes a benchmark for investigating the same process in the CID of phosphorylated peptides. [ABSTRACT FROM AUTHOR]

Protein post-translational modifications (PTMs) are critically important in regulating both protein structure and function, often in a rapid and reversible manner. Due to its sensitivity and vast applicability, mass spectrometry (MS) has become the technique of choice for analyzing PTMs. Whilst the 'bottom-up' analytical approach, in which proteins are proteolyzed generating peptides for analysis by MS, is routinely applied and offers some advantages in terms of ease of analysis and lower limit of detection, 'top-down' MS, describing the analysis of intact proteins, yields unique and highly valuable information on the connectivity and therefore combinatorial effect of multiple PTMs in the same polypeptide chain. In this review, the state of the art in top-down MS will be discussed, covering the main instrumental platforms and ion activation techniques. Moreover, the way that this approach can be used to gain insights on the combinatorial effect of multiple post-translational modifications and how this information can assist in studying physiologically relevant systems at the molecular level will also be addressed. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:27-42, 2013 [ABSTRACT FROM AUTHOR]

Model ferris heme nitrosyl complexes, [Fe(TPP)(NO)]+ and [Fe(TPFPP)(NO)]+, where TPP is the dianion of 5,10,15,20-tetrakis-phenyl-porphyrin and TPFPP is the dianion of 5,10,15,20-tetnlis-pentafluorophenyl-porphyrin, have been obtained as isolated spedes by the gas phase reaction of NO with [FeIII(TPP)]+ and [FeIII (TPFPP)]+ ions delivered in the gas phase by electrospray ionization, respectively. The so-formed nitrosyl complexes have been characterized by vibrational spectroscopy also exploiting 15N-isotope substitution in the NO ligand. The characteristic NO stretching frequency is observed at 1825 and 1859 cm-1 for [FeIII(TPP)(NO)]+ and [FeIII(TPFPP)(NO)]+ ions, respectively, providing reference values for genuine five-coordinate FeIII(NO) porphyrin complexes differing only for the presence of either phenyl or pentafluorophenyl substituents on the meso positions of the porphyrin ligand. The vibrational assignment is aided by hybrid density functional theory (DFT) calculations ofgeometry and electronic stnlcture and frequency analysis which dearly support a singlet spin electronic state fur both [Fe(TPP)(NO)]+ and [Fe(TPFPP)(NO)]+ complexes. Both TD-DFT and CASSCF calculations suggest that the singlet ground state is best described as FeII(NO+) and that the open-shell AFC bonding scheme contribute for a high-energy excited state. The kinetics of the NO addition reaction in the gas phase are faster for [FeIII(TPFPP)]+ ions bya relatively small factor, though highly reliable because of a direct comparative evaluation. The study was aimed at gaining vibrational and reactivity data on five-coordinate FeIII(NO) porphynis complexes, typically transient species in solution, ultimately to provide insights into the nature of the Fe(NO) interaction in heme proteins. [ABSTRACT FROM AUTHOR]

The intrinsic reactivity of a manganese(V)-oxo porphyrin complex, a typically fleeting intermediate in catalytic oxidation reactions in solution, has been elucidated in a study focused on its gas-phase ion-chemistry. The naked high-valent Mn(V)-oxo porphyrin intermediate 1 ([(tpfpp)Mn(V)O](+); tpfpp=meso-tetrakis(pentafluorophenyl)porphinato dianion), has been obtained by controlled treatment of [(tpfpp)Mn(III)]Cl (2-Cl) with iodosylbenzene in methanol, delivered in the gas phase by electrospray ionization and assayed by FT-ICR mass spectrometry. A direct kinetic study of the reaction with selected substrates, each containing a heteroatom X (X=S, N, P) including amines, sulfides, and phosphites, was thus performed. Ionic products arising from electron transfer (ET), hydride transfer (HT), oxygen-atom transfer (OAT), and formal addition (Add) may be observed, with a predominance of two-electron processes, whereas the product of hydrogen-atom transfer (HAT), [(tpfpp)Mn(IV)OH](+), is never detected. A thermochemical threshold for the formation of the product radical cation allows an evaluation of the electron-transfer ability of a Mn(V)-oxo complex, yielding a lower limit of 7.85 eV for the ionization energy of gaseous [(tpfpp)Mn(IV)O]. Linear free-energy analyses of the reactions of para-substituted N,N-dimethylanilines and thioanisoles indicate that a considerable amount of positive charge is developed on the heteroatom in the oxidation transition state. Substrates endowed with different heteroatoms, but similar ionization energy display a comparable reaction efficiency, consistent with a mechanism initiated by ET. For the first time, the kinetic acidity of putative hydroxo intermediates playing a role in catalytic oxidations, [(tpfpp)Fe(IV)OH](+) and [(tpfpp)Mn(IV)OH](+), has been investigated with selected reference bases, revealing a comparatively higher basicity for the ferryl, [(tpfpp)Fe(IV)O], with respect to the manganyl, [(tpfpp)Mn(IV)O], unit. Finally, the neat association reaction of 2 has been studied with various ligands showing that harder ligands are more strongly bound., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One of the main goals of comparative cell signaling analyses is the characterization of protein changes between different biological samples, either globally or by targeting specific proteins of interest. Highly accurate and precise strategies are thus required for the relative quantification of proteins extracted from two or more different cell populations. Stable isotope labeling with amino acids in cell culture (SILAC) is a general method for mass spectrometric quantitative proteomics based on metabolic incorporation of stable isotope-labeled amino acids into the cellular protein pool. This method has been applied with great success to a variety of quantitative proteomics problems aimed at gaining further insight into cell signaling pathways. In this chapter, we describe how SILAC can be used for the elucidation of cellular mechanisms, including temporal proteome profiling and the quantitative analysis of the extent of specific posttranslational modifications., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Building a mathematical model of a biological system requires input of experimental data for each networked component, ultimately generating a model that can be used to test scientific hypotheses. A fundamental requirement in the computation of these systems is that the total amount of each component can be specified precisely. An added level of complexity occurs because a vast number of protein posttranslational modifications modulate protein function. Each of these modified forms therefore needs to be considered as a separate system component, and must therefore be quantified individually. In this chapter, we describe how designer QconCAT proteins can be used to determine the absolute amounts of both the polypeptide components and their covalently modified derivatives in both yeast and mammalian extracts derived from living cell populations., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Protein degradation is a fundamental biological process, which is essential for the maintenance and regulation of normal cellular function. In humans and animals, proteins can be degraded by a number of mechanisms: the ubiquitin-proteasome system, autophagy and intracellular proteases. The advances in contemporary protein analysis means that proteomics is increasingly being used to explore these key pathways and as a means of monitoring protein degradation. The dysfunction of protein degradative pathways has been associated with the development of a number of important diseases including cancer, muscle wasting disorders and neurodegenerative diseases. This review will focus on the role of proteomics to study cellular degradative processes and how these strategies are being applied to understand the molecular basis of diseases arising from disturbances in protein degradation., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Anion-pi interactions between a pi-acidic aromatic system and an anion are gaining increasing recognition in chemistry and biology. Herein, the binding features of an electron-deficient aromatic system (1,3,5-trinitrobenzene (TNB)) and selected anions (OH-, Br-, and I-) are examined in the gas phase by using the combined information derived from collision-induced dissociation experiments at variable energy, infrared multiple-photon dissociation spectroscopy, and quantum chemical calculations. We provide spectroscopic evidence for two different structural motifs of anion-arene complexes depending on the nature of the anion. The TNB-OR- complexes (R=H, or alkyl groups which were studied earlier) adopt an anionic sigma-complex structure whereby RO- attacks the aromatic ring with covalent bond formation, and develops a tetrahedral ring carbon bound to H and OR. The halide complexes rather conform to a structure in which the TNB moiety is hardly altered, and the halogen is placed on an unsubstituted carbon atom over the periphery of the ring at a C-X distance that is appreciably longer than a typical covalent bond length. The ensuing structural motif, previously characterized in the solid state and named weak sigma interaction, is now confirmed by an IR spectroscopic assay in the gas phase, in which the sampled species are unperturbed by crystal packing or solvation effects.