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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]

RATIONALE Intramolecular hydrogen bonds between a phosphate group and charged residues play a crucial role in the chemistry of phosphorylated peptides, driving the species to specific conformations and affecting the exposure of the phosphate moiety. The nature and extent of these interactions can be investigated by measuring the reactivity of phosphate groups toward selected substrates in the gas phase. METHODS We used Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry (MS) to perform a systematic study on the gas-phase ionic reactivity of phosphorylated amino acids and peptides with triethoxyborane (TEB). Ions of interest were generated by electrospray ionization (ESI), isolated in the cell of the FT-ICR mass spectrometer, and allowed to react with a stationary pressure of TEB. The temporal evolution of the reaction was monitored and thermal rate constants were derived. The structure of the ionic products was confirmed by Collision-Induced Dissociation (CID) tandem mass spectrometry (MS/MS). RESULTS TEB was found to react with the phosphate of protonated phosphorylated amino acids and peptides by an addition-elimination pathway. The kinetic efficiency of the reaction showed a positive correlation with the charge state of the reagent ion, suggesting the existence of charge-state-dependent exposure of the phosphate groups towards the incoming neutral during the reaction. Isomeric phosphorylated peptides, only differing for the position of the modified serine residue, showed markedly different kinetic efficiencies. CONCLUSIONS The ability of a phosphorylated species to react with TEB depends on the ease of access to the phosphate moiety in the corresponding gaseous ion. Measuring the kinetic efficiency of such reactions can represent a valuable tool to explore the accessibility of phosphate groups in biomolecules. Copyright © 2014 John Wiley & Sons, Ltd. [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]

Mass spectrometry is a vital tool for molecular characterization, and the allied technique of ion mobility is enhancing many areas of (bio)chemical analysis. Strong synergy arises between these two techniques because of their ability to ascertain complementary information about gas-phase ions. Ion mobility separates ions (from small molecules up to megadalton protein complexes) based on their differential mobility through a buffer gas. Ion mobility-mass spectrometry (IM-MS) can thus act as a tool to separate complex mixtures, to resolve ions that may be indistinguishable by mass spectrometry alone, or to determine structural information (for example rotationally averaged cross-sectional area), complementary to more traditional structural approaches. Finally, IM-MS can be used to gain insights into the conformational dynamics of a system, offering a unique means of characterizing flexibility and folding mechanisms. This Review critically describes how IM-MS has been used to enhance various areas of chemical and biophysical analysis. [ABSTRACT FROM AUTHOR]

A significant number of proteins in both eukaryotes and prokaryotes are known to be post-translationally modified by the addition of phosphate, serving as a means of rapidly regulating protein function. Phosphorylation of the amino acids serine, threonine and tyrosine are the focus of the vast majority of studies aimed at elucidating the extent and roles of such modification, yet other amino acids, including histidine and aspartate, are also phosphorylated. Although histidine phosphorylation is known to play extensive roles in signalling in eukaryotes, plants and fungi, roles for phosphohistidine are poorly defined in higher eukaryotes. Characterization of histidine phosphorylation aimed at elucidating such information is problematic due to the acid-labile nature of the phosphoramidate bond, essential for many of its biological functions. Although MS-based strategies have proven extremely useful in the analysis of other types of phosphorylated peptides, the chromatographic procedures essential for such approaches promote rapid hydrolysis of phosphohistidine-containing peptides. Phosphate transfer to non-biologically relevant aspartate residues during MS analysis further complicates the scenario. [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]

S-Nitrosocaptopril, a biologically active S-nitrosothiol, is generated as protonated species and isolated in the gas phase by electrospray ionization coupled to Fourier Transform Ion Cyclotron Resonance (FT-ICR) or ion-trap mass spectrometry. The structural and IR spectroscopic characterization of protonated S-nitrosocaptopril (SNOcapH+) is aided by the comparative study of the parent species lacking the NO feature, namely protonated captopril. The study is accomplished by methodologies based on tandem mass spectrometry, namely by energy resolved collision-induced dissociation and infrared multiple-photon dissociation (IRMPD) spectroscopy, backed by density functional theory calculations. IRMPD spectra have been obtained both in the 1000–1900 cm−1fingerprint range, using a beamline of the infrared free electron laser (IR-FEL) at the Centre Laser Infrarouge d''Orsay (CLIO), and in the O–H and N–H stretching region (2900–3700 cm−1) using the tunable IR radiation of a tabletop parametric oscillator/amplifier (OPO/OPA) laser source. The structural features of the ion have been ascertained by comparison of the experimental IRMPD spectra with the IR transitions calculated for the lowest energy isomers. Evidence is obtained that protonation occurs at the amide carbonyl oxygen which is found to be the thermodynamically most basic site. However, SNOcapH+is present as a thermally equilibrated mixture of low-energy structures, with a major contribution of the most stable isomer characterized by a transrelationship of the positively charged OH group with respect to the carboxylic acid functionality on the adjacent proline ring and by an anticonformation at the S–N (partial) double bond, though the energy difference with the analogous trans–synisomer is less than 1 kJ mol−1. The highly diagnostic N–O stretching mode has been unambiguously identified, which may be regarded as an informative probe for S-nitrosation features in more complex, biologically active molecules. [ABSTRACT FROM AUTHOR]