ESI Mass Spectra Research Articles

Protein Conformational Heterogeneity as a Binding Catalyst: ESI-MS Study of Hemoglobin H Formation

Our previous studies of hemoglobin tetramer assembly in vitro suggested that the initial step in the oligomerization process, which ultimately dictates the high fidelity of the heterotetramer (alpha*beta*)2 assembly, is the binding of a flexible heme-free beta-globin chain to a highly ordered heme-bound alpha*-globin. In this work, we extend these studies to investigate formation of the homotetrameric hemoglobin H, whose formation in vivo is a well-documented clinical consequence of significant overexpression of beta-globin in alpha-thalassemic disorders. Upon reconstitution of the isolated beta-globin with excess heme, the predominant species in the ESI mass spectrum corresponds to the homotetramer beta*4, alongside homodimeric species and monomeric beta-globin chains in both apo and holo forms. The assembly process of the hemoglobin H homotetramer apparently follows a scenario similar to that of a normal heterodimeric hemoglobin (alpha*beta*)2 species, with the asymmetric binding event between compact and flexible polypeptide chains being the initial step. The extreme importance of large-scale chain dynamics and conformational heterogeneity for the protein assembly process is highlighted by the inability of highly structured alpha-globins to undergo ordered oligomerization to form dimers and tetramers as opposed to indiscriminate aggregation.

ICP-MS-assisted nanoHPLC-electrospray Q/time-of-flight MS/MS selenopeptide mapping in Brazil nuts

A method was developed for the identification of selenium-containing peptides issued from proteins of a Se-rich (82.9 mg kg–1) foodstuff, Brazil nut (Bertholletia excelsa). A sample purification procedure was optimized to cope with the 100-fold excess of sulfur analogues and matrix interferences. It was based on the consecutive size-exclusion fractionation of proteins and tryptic peptides, and enrichment of the Se-containing fractions, prior to nanoHPLC-ES-Q/TOF MS/MS. The characteristic isotopic patterns of selenium compounds (always minor peaks) were detected in ESI mass spectra at retention times precisely indicated by the matrix interference-free, sensitive (DL 1.3 fmol) 80Se detection by ICP collision cell MS in the same separation conditions. The potential of the method was demonstrated by the identification of 15 Se-containing peptides, from which all but one were found to originate from the selenised isoforms of the 2S protein.

Antioxidant activity of Annona crassiflora: Characterization of major components by electrospray ionization mass spectrometry

The polar components of Annona crassiflora pulp, peel and seeds ethanolic extracts were investigated by direct infusion electrospray ionization mass spectrometry (ESI-MS) both in the negative ion mode. Characteristic ESI mass spectra with many diagnostic ions were obtained for the extracts, serving for fast and reliable information. The technique provided information of component structures revealing the presence of important bioactive components widely reported as potent antioxidants such as ascorbic acid, caffeic acid, quinic acid, ferulic acid, xanthoxylin, rutin, caffeoyltartaric acid, caffeoyl glucose and [quercetin+hexose+pentose−H] −1 This is the first report on the composition by ESI-MS of araticum peel and seed ethanolic extracts demonstrating excellent antioxidant activity.

Mass spectrometric analysis of ubiquitin–platinum interactions of leading anticancer drugs: MALDI versus ESI

The protein binding of anticancer metallodrugs is regarded as an important part in their mode of action both for delivering the active moiety into the tumor but also being responsible for deactivation and/or unwanted side effects. Characterization of protein binding and release may allow new drugs to be designed which are devoid of protein interactions or capable of binding selectively to protein targets. Herein, we report the comparison of different ionization techniques, i.e. matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray ionization mass spectrometry (nESI-MS), for the analysis of small protein–platinum anticancer drug interactions. For this purpose, cisplatin, transplatin and oxaliplatin were incubated with the model proteinubiquitin (Ub) at a molar ratio of 2 : 1 (Pt : Ub) followed by MS analysis. Cisplatin, transplatin and oxaliplatin formed mainly monoadducts with Ub, but of significantly different composition. As reported earlier, cisplatin forms mainly bifunctional Ub–[Pt(NH3)2] adducts, while with transplatin the most abundant adduct was found to be a monofunctional Ub–[Pt(NH3)2Cl] species. Oxaliplatin formed exclusively bifunctional species of the formula Ub–[Pt(chxn)] (chxn = cyclohexane-1,2-diamine). The applied analysis methods provide comparable results. However, the higher resolution of the nESI-quadrupole time-of-flight (QToF)-MS allowed unambiguous characterization of a series of mono- and bis-adducts including Ub–[Pt(NH3)2(H2O)] for both cisplatin and transplatin. Applying nESI-ion trap (IT)-MS showed the advantage of higher sensitivity than the ToF instruments, allowing the detection of bisadducts of oxaliplatin after one week of incubation. In contrast to the ESI mass spectra, MALDI showed a higher degree of fragmentation of the Ub–platinum conjugates.

Multiple oligomeric forms of Escherichia coli DnaB helicase revealed by electrospray ionisation mass spectrometry

The Escherichia coli DnaB protein (DnaB(6)) is the hexameric helicase that unwinds genomic DNA so it can be copied by the DNA replication machinery. Loading of the helicase onto DNA requires interactions of DnaB(6) with six molecules of its loading partner protein, DnaC. Nano-electrospray ionisation mass spectrometry (nanoESI-MS) of mutant proteins was used to examine the roles of the residues Phe102 (F102) and Asp82 (D82) in the N-terminal domain of DnaB in the assembly of the hexamer. When the proteins were prepared in 1 M ammonium acetate containing magnesium and adenosine triphosphate (ATP) at pH 7.6, both hexameric and heptameric forms of wild-type and F102W, F102E and D82N mutant DnaBs were observed in mass spectra. The spectra of the D82N mutant also showed substantial amounts of a decameric species and small amounts of a dodecamer. In contrast, the F102H DnaB mutant was incapable of forming oligomers of order higher than the hexamer. Thus, although Phe102 is not the only determinant of hexamer assembly, this residue has a role in oligomerisation. NanoESI mass spectra were obtained of mixtures of DnaB(6) with DnaC. The DnaB(6)(DnaC)(6) complex (calculated M(r) 481 164) was observed only when the two proteins were present in equimolar amounts. The data are consistent with cooperative assembly of the complex. ESI mass spectra of mixtures containing DnaC and ATP showed that DnaC slowly hydrolysed ATP to ADP as indicated by ions corresponding to DnaC/ATP and DnaC/ADP complexes. These experiments show that E. coli DnaB can form a heptameric complex and that nanoESI-MS can be used to probe assembly of large (>0.5 MDa) macromolecular complexes.

Identification of the Dinuclear and Tetranuclear Air‐Oxidized Products Derived from Labile Phenolate‐Bridged Dimanganese(II) Pyridyl‐Chelate Compounds

AbstractDioxygen‐sensitive dinuclear manganese complexes of the phenoxo‐hinged dinucleating ligand 2,6‐bis{[N,N′‐bis(2‐picolyl)amino]methyl}‐4‐tert‐butylphenolato (bpbp–) containing exogenous labile THF, water and perchlorato ligands are described. The manganese(II) complexes [Mn2(bpbp)(ClO4)2(THF)]+ (1) and [Mn2(bpbp)(ClO4)(H2O)2]2+ (2) have been isolated as the salts 1·ClO4·THF·3H2O, 1·B(C6H5)4·4THF and 2·(ClO4)2·H2O. Complexes 1 and 2 are spontaneously oxidised in air in solution and the solid state. The reaction products of the air oxidation in THF, water and methanol solutions are labile dinuclear MnII–MnIII, MnIII2 and MnIII–MnIV complexes containing water‐ and methanol‐derived exogenous ligands. In addition, a Mn4 complex has been isolated. Magnetic susceptibility data confirm the MnII–MnIII oxidation state assignment with an S = 2/S = 5/2 model with weak antiferromagnetic coupling (J = –3.7 cm–1) in [Mn2(bpbp)(CH3O)2(H2O)2](ClO4)2 [3·(ClO4)2]. A tetranuclear complex, [Mn4(O)4–n(OH)n(bpbp)2](ClO4)4 [n = 1 or 2; 7·(ClO4)4], recovered from THF shows a Mn4O6 adamantane‐type core with the O bridges furnished by the two phenolato groups and four hydroxide/oxide bridges. We have arrived at two feasible formulations for the core metal oxidation states and oxo‐bridge protonation states, namely [MnIII4(O)2(OH)2(bpbp)2]4+ and [MnIII3MnIV(O)3(OH)(bpbp)2]4+, for 7, on the basis of a bond valence sum analysis of the crystal structure, elemental analysis and XANES. Thus, complex 7 is at least two oxidation state levels lower than known complexes with the Mn4O6 adamantane core structure. The magnetism of 7 was fitted well to an MnIII4 three‐J model. Complex cations related to 3 by homology, and to 7 by hydration/solvation, have been identified by ESI mass spectrometry. The [Mn2(bpbp)(OH)2(H2O)2]2+ ion (4) present in aqueous solutions on dissolution of 1·ClO4·THF·3H2O in air or by simple dissolution of 3 in water‐containing solvent is isoelectronic to 3. In the presence of significant amounts of water the MnII–MnIII complexes 3 and 4 are susceptible to further metal oxidation and concomitant aquo ligand deprotonation to give ions assignable to [MnIII/IV2(bpbp)O(OCH3)2(H2O)]2+ (5) and [MnIII2(bpbp)(OH)3(H2O)]2+ (6). ESI mass spectra of water or methanol solutions of 1, 2, 3 and 7 show predominantly an ion assignable to the oxide [Mn2(bpbp)(O)]2+ (8). Cation 8 is most likely not present in solution. Using mild source conditions and MS‐MS techniques, the gas‐phase fragmentation pathways to generate 8 have been mapped. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Synthesis, spectral and biological studies of organotin(IV) complexes of heteroscorpionate

AbstractA heteroscorpionate ligand, potassium hydrobis(benzoato)(salicylaldehyde)borate (KL), has been synthesized. This was converted into organotin complexes R2SnL2 and R3SnL complexes by mixing and stirring with a methanolic solution/suspension of organotin chloride. The ligand and its complexes were characterized by elemental analyses and spectral studies (IR, 1H NMR, 13C NMR, ESI mass spectra and Thermo gravimetric analysis (TGA)). Antibacterial and antifungal studies of these compounds were evaluated by the disc diffusion method at variable concentration against three species of bacteria (Staphylococcus aureus, Klebsiella pneumonia and Bacillius subtillis) and two species of fungi (Asperjillius fiavus and Candida albicans). It was found that triorganotin derivatives (R3SnL) of the ligand were more effective as compared with diorganotin derivatives (R2SnL2). The organotin complexes of borates were tested for their algicidal activity on the cyanobacterial strains Aulosira fertilissma, Anabaena species, Anabaena variabilis and Nostoc muscorum and showed high to moderate toxicity towards the above species. The ligand and its complexes were also tested for its pH effect on soil in vitro for a duration of more than one month and it was found that they are able to kill pests without damaging the soil quality. Copyright © 2006 John Wiley & Sons, Ltd.

Folding and Assembly of Hemoglobin Monitored by Electrospray Mass Spectrometry Using an On-line Dialysis System

The native structure of hemoglobin (Hb) comprises two alpha- and two beta-subunits, each of which carries a heme group. There appear to be no previous studies that report the in vitro folding and assembly of Hb from highly unfolded alpha- and beta-globin in a "one-pot" reaction. One difficulty that has to be overcome for studies of this kind is the tendency of Hb to aggregate during refolding. This work demonstrates that denaturation of Hb in 40% acetonitrile at pH 10.0 is reversible. A dialysis-mediated solvent change to a purely aqueous environment of pH 8.0 results in Hb refolding without any apparent aggregation. Fluorescence, Soret absorption, circular dichroism, and ESI mass spectra of the protein recorded before unfolding and after refolding are almost identical. By employing an externally pressurized dialysis cell that is coupled on-line to an ESI mass spectrometer, changes in heme binding behavior, protein conformation, and quaternary structure can be monitored as a function of time. The process occurs in a stepwise sequential manner, leading from monomeric alpha- and beta-globin to heterodimeric species, which then assemble into tetramers. Overall, this mechanism is consistent with previous studies employing the mixing of folded alpha- and beta-globin. However, some unexpected features are observed, e.g., a heme-deficient beta-globin dimer that represents an off-pathway intermediate. Monomeric beta-globin is capable of binding heme before forming a complex with an alpha-subunit. This observation suggests that holo-alpha-apo-beta globin does not represent an obligatory intermediate during Hb assembly, as had been proposed previously. The on-line dialysis/ESI-MS approach developed for this work represents a widely applicable tool for studying the folding and self-assembly of noncovalent biological complexes.

Polyaryl Ether Dendrimer with a 4-Phenylacetyl-5-pyrazolone-based Terbium(III) Complex as Core: Synthesis and Photopysical Properties

A series of novel dendritic beta-diketone ligands, 1-phenyl-3-[G-n]-4-phenylacetyl-5-pyrazolone (n = 0-3, G stands for polyaryl ether), were synthesized by introducing Fréchet-type dendritic branches. The corresponding Tb3+-cored dendritic complexes were characterized by X-ray crystallography, elemental analysis, ESI mass spectra, and FT-IR spectra. These dendritic complexes, prepared from aqueous solution, exhibit high stability. Interestingly, the study of photophysical properties shows that the luminescence quantum yields of the dendritic Tb-complexes increase from 0.1 to 2.26% with an increase of the dendritic generation from 0 to 3. Importantly, an "energy-reservoir effect" was observed in the dendritic system using the method based on the resonance energy transfer from these complexes to rhodamine 6G. With the increase of the dendritic generation, the metal-centered luminescence quantum yield was almost the same, and the energy transfer (phi(transfer)) from the ligand to Tb(3+) increased. Further measurements of the triplet state and oxygen quenching of these dendritic complexes verify that this enhancement of the energy transfer (phi(transfer)) is attributed to both an "antenna effect" and a "shell effect".

Peptide Ozonolysis: Product Structures and Relative Reactivities for Oxidation of Tyrosine and Histidine Residues

Angiotensin II (DRVYIHPF) and two analogs, (DRVYIAPA and DRVAIHPA), were used as model systems to study the ozonolysis of peptides containing tyrosine and histidine residues. The ESI mass spectrum of angiotensin II following exposure to ozone showed the formation of adducts containing one, three, and four oxygen atoms. CID and SID spectra of these adducts were consistent with formation of Tyr + O and His + 3O as expected from previous work with amino acids. However, several fragment ions observed in the CID and SID spectra suggested formation of a rather unexpected adduct, Tyr + 3O, and a small amount of the Phe + O adduct. These findings were confirmed by examining two angiotensin analogs. Exposure of DRVYIAPA to ozone resulted in the addition of either one or three oxygen atoms on Tyr, while DRVAIHPA showed only the addition of three oxygen atoms--all on His. Other noteworthy minor oxidation products were observed from these analogs including Tyr + 34 Da, His + 5 Da, His + 34 Da, and His + 82 Da. The reaction rates of the peptides with ozone were found to be similar: second-order rate coefficients are 274 +/- 3, 379 +/- 6, and 439 +/- 34 M(-1) s(-1) for DRVYIAPA, DRVAIHPA, and angiotensin II, respectively. The relative rates indicate (1) an isolated His residue has a slightly greater ozone reactivity than an isolated Tyr residue, and (2) the reaction rates of isolated residues are not additive when both residues are present in the same molecule.

Characteristic Fragmentation Behavior of Steroidal Phosphor‐ amidate Conjugates in Electrospray Ionization Tandem Mass Spectrometry

AbstractNovel steroidal phosphoramidate conjugates of 3′‐azido‐2′,3′‐dideoxythymidine (AZT) and amino acid esters were synthesized and determined by positive and negative ion electrospray ionization mass spectrometry. The MS fragmentation behaviors of the steroidal phosphoramidate conjugates have been investigated in conjunction with tandem mass spectrometry of ESI‐MS/MS. There were three characteristic fragment ions in the positive ion ESI mass spectra, which were the Na adduct ions with loss of steroidal moiety, amino acid ester moiety from pseudo molecular ion (M+Na)+, and the phosphoamino acid methyl ester Na adduct ion by α‐cleavage of the phosphoramidate respectively. The main fragment ions in negative ion ESI mass spectra were the ion (M−HN3)−, the ion (M−AZT−H)−, and the ion (M−steroidal moiety−H)− besides the pseudo molecular ion (M−H)−. The fragmentation patterns did not depend on the attached amino acid ester moiety.

A Deconvolution Method for the Separation of Specific Versus Nonspecific Interactions in Noncovalent Protein-Ligand Complexes Analyzed by ESI-FT-ICR Mass Spectrometry

A method to separate specific and nonspecific noncovalent interactions observed in ESI mass spectra between a protein and its ligands is presented. Assuming noncooperative binding, the specific ligand binding is modeled as a statistical distribution on identical binding sites. For the nonspecific fraction we assume a statistical distribution on a large number of "nonspecific" interacting sites. The model was successfully applied to the noncovalent interaction between the protein creatine kinase (CK) and its ligands adenosine diphosphate (ADP) and adenosine triphosphate (ATP) that both exhibit nonspecific binding in the mass spectrum. The two sequential dissociation constants obtained by applying our method are K(1,diss) = 11.8 +/- 1.5 microM and K(2,diss) = 48 +/- 6 microM for ADP. For ATP, the constants are K(1,diss) = 27 +/- 7 microM and K(2,diss) = 114 +/- 27 microM. All constants are in good correlation with reported literature values. The model should be valuable for systems with a large dissociation constant that require high ligand concentrations and thus have increased potential of forming nonspecific adducts.

Tetranuclear Complexes Containing a Luminescent Ru2M2 Core [M = CuI, (allyl)PdII]: Synthesis, Structures and Electrochemical Properties

AbstractThe reaction between [(tbbpy)2Ru(tmbiH2)](PF6)2 (tbbpy = 4,4′‐di‐tert‐butyl‐2,2′‐bipyridine; tmbiH2 = 5,5′,6,6′‐tetramethyl‐2,2′‐dibenzimidazole) and LiMe/CuI affords the tetranuclear complex 1 containing the dication [{(tbbpy)2Ru(tmbi)}2Cu2]2+. The X‐ray structure of 1 shows that the two [(tbbpy)2Ru(tmbi)] units coordinate the two Cu atoms in a twofold monodentate manner. Each copper atom is surrounded by two N‐donor atoms of different ruthenium(II) units with N–Cu–N angles of 167.3°. The Cu···Cu separation (2.72 Å) indicates intranuclear contacts. LiMe/[(allyl)PdCl]2 reacts with the starting Ru complex to form 2, which contains the tetranuclear dication [{(tbbpy)2Ru(tmbi)}2{Pd(η3‐allyl)}2]2+. Its crystal structure shows that the RuII units act as twofold monodentate ligands as well. Each PdII is in an essentially planar coordination environment containing two nitrogen atoms of two different dibenzimidazolates in the cis position with the η3‐bonded allyl group occupying the two remaining positions. In contrast to 1, where the Ru–Cu–Cu–Ru unit forms an essentially planar structure, a strongly bent structure is formed in compound 2. ESI mass spectra of 1 and 2 clearly show that the tetranuclear complexes are present in solution as well. Both complexes are luminescent. In addition, 2 catalyses the Heck coupling at 120 °C in N,N‐dimethylacetamide. In contrast to 1 and 2, the homodinuclear complex [(tbbpy)2Ru(tmbi)Ru(tbbpy)2] (3) contains the dibenzimidazolate as a twofold chelating ligand that forms two five‐membered chelate rings with the Ru centres. Electrochemical measurements of 2 show two reversible, one‐electron oxidation steps, the difference between which (ΔE = 89 mV) is distinctly larger than the pure entropic effect. Furthermore, four reduction steps are observed, the first two of which are fully reversible. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Fractionation of Methyl Cellulose According to Polarity – a Tool to Differentiate First and Second Order Heterogeneity of the Substituent Distribution

AbstractSummary: A set of four MCs (DS 1.80–1.95) has been analyzed with regard to their substituent pattern in the glucosyl units and along the polymer chain. The average heterogeneity of methylation observed for the entire material was analyzed in more detail after fractionation according to polarity. All fractions obtained were analyzed with respect to their DS, monomer composition and deviation from a random distribution of these monomers in the polymer chains. By this approach, heterogeneity of first and second order could be differentiated. While for three of the MCs only a minor DS‐gradient over the material was observed, a more pronounced heterogeneity of first order was obtained for MC 4.ESI mass spectrum of the undissolved residue of MC 2 after deuteromethylation and partial hydrolysis; DP 2 and 3 are shown in detail. Signals are assigned according to the number of CH3‐groups.magnified imageESI mass spectrum of the undissolved residue of MC 2 after deuteromethylation and partial hydrolysis; DP 2 and 3 are shown in detail. Signals are assigned according to the number of CH3‐groups.

Isolation of lipids from photosystem I complex and its characterization with high performance liquid chromatography/electrospray ionization mass spectrometry.

A method for simultaneous analysis of lipids extracted from photosystem I complex was developed with high performance liquid chromatography/electrospray ionization mass spectrometry. The photosystem I complex was firstly solubilized and separated using deoxycholate polyacrylamide gel electrophoresis method after ultrasonic treatment of the sample (leaves of pea, Pisum sativum L.). The Photosystem I complexes were electrophoretically eluted from the deoxycholate polyacrylamide gel electrophoresis bands containing them, and the electron transport activity of the eluent measured as confirmation. Lipids, which were isolated from the complex having photosystem I activity, were separated and characterized with high performance liquid chromatography/electrospray ionization mass spectrometry. Five lipids, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylglycerol, sulphoquinovosyldiacylglycerol and phosphaditylcholine were found combining with photosystem I complex. Different species of these lipids were found in the ESI mass spectra and the compositions of the acyl groups in them were determined.

Gas-phase processes and measurements of macromolecular properties in solution: On the possibility of false positive and false negative signals of protein unfolding

Electrospray ionization mass spectrometry is increasingly applied to study protein behavior in solution, including characterization of their higher order structure, conformational dynamics and interactions with small ligands and other biopolymers. However, actual measurements of fundamental ionic parameters (mass and charge) take place in vacuum, and an array of gas phase processes occurring prior to protein ion detection and characterization may certainly affect them. While most previous studies were concerned primarily with the effect of gas phase processes on mass measurement (e.g., integrity of macromolecular complexes in the absence of solvent, non-specific interactions, etc.), the focus of our attention is the ionic charge. Charge state distributions of protein ions in ESI MS are often used to characterize large-scale dynamic processes in solution (such as protein unfolding). Formation of metastable protein aggregates either in the bulk of solution or in the electrosprayed droplets, their consequent transfer to the gas phase and asymmetric dissociation may give rise to a population of highly charged ions. Presence of such ions in ESI mass spectra usually indicates loss of native structure in solution. Therefore, studies of large-scale conformational dynamics in solution by monitoring protein ion charge state distributions should be carried out at low protein concentrations in order to minimize the occurrence of false positive signals of protein unfolding. The opposite phenomenon, absence of highly protonated ionic species in ESI spectra of unfolded proteins, does not occur even in the case of highly acidic proteins lacking a sufficient number of basic residues.

Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases

Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations.

Structural studies of HPV oncoprotein E7 and interactions with tumor suppressor pRb

Human papillomavirus (HPV) is the DNA tumor virus found in >95% of women with cervical carcinomas, which is a leading source of cancer deaths of women around the world. HPVs use their transforming oncoprotein E7 to interact with the Rb family of tumor suppressors and liberate the E2F family of transcription factors, advancing the cell cycle into S phase. The freed E2Fs transactivate genes for DNA synthesis to promote proliferation of the infected epithelial cells, while inhibiting apoptosis. Studies of the structure, dynamics and interaction among E7, pRb, and E2Fs will lend insight on the molecular details of progression of cervical cancer and suggest new strategies for interfering in E7 action. However, the difficulty of structural studies of E7 has stymied structural studies. Purification of E7 is very difficult, but we developed a successful two step strategy. E7 was found to contain 1 zinc per E7 monomer, both by ESI mass spectra and colorimetric detection of PAR titration. GST-pRb pulls down this E7 successfully. Isothermal titration calorimetry (ITC) of pRb+E7 shows HPV 1a E7 has high affinity for pRb, similar to high affinity of high risk HPV-16 E7 (They all have the same DLxCxE avid pRb-binding motif.). Apparent molecular weights of free and bound states of E7 and pRb pocket domain have been estimated using size exclusion. NMR spectra show that many amide proton peaks lie at random coil chemical shifts with overlap. E7 appears to be partly rigidly structured and partly poorly folded, a complication suited for NMR. We developed a strategy to generate acceptable NMR spectra of ill-behaved E7 and assigned 85 – 95% of its backbone 1H/15N/13C spectra. The trends in 13C chemical shifts locate β-strands and α-helix in C-terminal portion of E7. The poorly structured region is likely to confer much of the high affinity for pRb. (American Cancer Society)

Synthesis and crystal structure of 2,4-dihydro-4-[(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)imino]-5-methyl-2-phenyl-3H-pyrazol-3-one and its copper(II) complex

2-Diazo-3-methyl-1-phenyl-5-pyrazolone ( 2) and 2,4-dihydro-4-[(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)imino]-5-methyl-2-phenyl-3H-pyrazol-3-one ( 3) were obtained in a one-pot reaction from 3-methyl-1-phenylpyrazol-5-one ( 1), mesyl azide, and K 2CO 3 in MeOH or CH 2Cl 2 solution. NMR, IR and ESI mass spectra as well as the crystal structure are reported for red colored 3. The crystal structure determined at 150 K reveals that the corresponding bonds in the two phenylpyrazol-5-one moieties in each of the two independent “U” shaped molecules have essentially the same lengths, with an extensive delocalization in the central O–C–C–N–C–C–O fragment of the molecule. In particular, the hydrogen atom, formally a part of a hydroxyl group, is found near-midway [O1–HY and O11–HY = 1.24(4) and 1.19(4) Å, in molecule 1, and O21–HX and O31–HY = 1.203(4) and 1.19(4) Å, in molecule 2] between the two oxygen atoms (formally in a carbonyl and a hydroxy group), with near linear O–H–O bond angles of 173.8(4)° and 172.7(4)° in the two independent molecules. All C–O bond lengths are in the narrow range of 1.271(3)–1.288(3) Å. Further, since this compound shows different coordinating groups, its binding ability towards copper(II) ions was investigated. A dark red complex 4, obtained by reaction of 3 with copper(II) ions at stoichiometric ratio, was characterized as a neutral 1:1 species, [ 3:CuCl 2], involving tautomeric forms, by ESI-MS, ESI-MS/MS, IR, UV/Vis and EPR spectra. Interestingly, copper coordination to ligand 3 occurs solely by oxygen atoms, in spite of the presence of nitrogen donor centers. The deprotonation of 4 was monitored by UV/Vis, and a corresponding pKa value was determined as (5.6 ± 0.2).

Use of solid-phase extraction, reverse osmosis and vacuum distillation for recovery of aromatic sulfonic acids from aquatic environment followed by their determination using liquid chromatography-electrospray ionization tandem mass spectrometry

Three different sample preparation techniques (i) solid-phase extraction, (ii) reverse osmosis and (iii) vacuum distillation have been investigated and the recoveries were compared for determination of highly water-soluble benzene and stilbene sulfonic acids in aqueous environment by liquid chromatography with photodiode array (PDA) and electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). The recoveries were quite high using vacuum distillation (>90%) compared to solid phase extraction and reverse osmosis. The negative ion ESI mass spectra containing the peaks of quasimolecular ion [M − H] − allow the molecular mass determination of unknown compounds whereas the structures were proposed using fragments obtained from MS/MS analysis of [M − H] − ions. At lower fragmentation voltages only the quasimolecular ion [M − H] − was observed and as fragmentation voltages increased, it led to the formation of fragment ions corresponding to [M − H–SO 3] −, [M − H–SO 2] −, and SO 3 −. The detection limits were 1–28 μg/L with LC–ESI-MS. The sample collected from wastewater treatment plant was found to contain 21.1, 13.3, 12.1, 41.8 and 9.9 μg/L of cis-4,4 l-diaminostilbene-2,2 l-disulfonic acid ( cis-DASDA), trans-4,4 l-diaminostilbene-2,2 l-disulfonic acid ( trans-DASDA), 3-amino acetanilide-4-sulfonic acid (3-AASA), 4-chloroaniline-2-sulfonic acid (4-CASA), 2-chloroaniline-5-sulfonic acid (2-CASA), respectively.