Therapeutic potential of phosphoinositide 3-kinase inhibitors

At least one Holy Grail for many academic researchers and pharmaceutical research divisions alike has been to identify therapeutically useful selective PI3K inhibitors. There are several different but closely related PI3Ks which are thought to have distinct biological roles. Until now, however, researchers have been frustrated by poor selectivity of the available pharmacological inhibitors, which are unable to distinguish the different isoforms of PI3K adequately. Fortunately, recently published work gives cause for optimism; there are now several patent specifications published that describe new PI3K inhibitors, including some that are more selective for the delta isoform of PI3K. Given the involvement of PI3Ks in a plethora of biological settings, such isoform-selective inhibitors may have immense potential use for the treatment of patients with inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases.     

Direct observation of the autophosphorylation of insulin receptor kinase by mass spectrometry

The catalytic and signaling activities of insulin receptor kinase(IRK)are regulated by the autophosphorylation of three tyrosine residues in a cytoplasmic protein-tyrosine kinase domain at Tyro 1158,Tyro 1162 and Tyro 1163.In this study,time-course of the auphosphorylation of the core kinase(residues 978-1283)from IRK was directly investigated by online electrospray ionization mass spectrometry.It is found that two tyrosine residues were phosphorylated in reaction time range of 30 min.This study implies ...     

Inhibitory activity of flavonoids against class I phosphatidylinositol 3-kinase isoforms

Class I PI3 Kinase (PI3K) phosphorylates phosphatidylinositol 4,5-bisphophate (PIP2) to generate the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) and therefore plays an important role in fundamental cellular responses such as proliferation. There are four isoforms of class I PI3K which are known to have different functions and relate to various diseases such as cancer and inflammation. Flavonoids are abundant in fruits, vegetables and plant-derived beverages such as tea. So far, various pharmacological effects of flavonoids have been reported. We previously reported that the flavonoid baicalein exhibits potent PI3K-inhibitory activity. Recently we examined the inhibitory activity of eighteen flavonoids against PI3Ka by using an in vitro homogenous time resolved fluorescence (HTRF) kinase assay, and deduced their structure-activity relationships by comparing the activities of the analogues. Our result suggests that the number of hydroxyl groups in the A and B rings might promote the activity, while loss of C2-C3 double bond might reduce the activity. Furthermore, the activity against 4 class I PI3K isoforms of some selected flavonoids was investigated, and the results indicate that the flavonoids seem to exhibit more potent activity on PI3Ka and d isoforms compared with that on PI3Kb and g isoforms.     

Mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry

Bovine insulin was glycated under hyperglycemic reducing conditions and in nonreducing conditions. Purification through HPLC allowed isolating glycated forms of insulin and a novel triglycated form (6224.5 Da) was purified. Endoproteinase Glu-C digestion combined with mass spectrometry (MALDI-TOF/TOF) allowed determining the exact location of the glycation sites in each of the isolated glycated insulins. For the first time, a triglycated form of insulin was isolated and characterized accordingly to its glycation sites. These glucose binding sites were identified as the N-terminals of both chains (Gly1 and Phe1) and residue Lys29 of B-chain. Moreover, in diglycated insulin we found the coexistence of one specie glycated at the N-terminals of both chains (Gly1 and Phe1) and another specie containing the two glucitol adducts in B-chain (Phe1 and Lys29). Also, in monoglycated insulin generated in reducing and nonreducing conditions, one specie glycated at Phe1 and another specie glycated at Lys29, both B-chain residues coexist.     

Analysis of protein phosphorylation by mass spectrometry

Phosphorylation is one of the most frequently occurring post-translational modifications in proteins. In eukaryotic cells, protein phosphorylation on serine, threonine and tyrosine residues plays a crucial role as a modulator of protein function. A comprehensive analysis of protein phosphorylation involves the identification of the phosphoproteins, the exact localization of the residues that are phosphorylated and the quantitation of phosphorylation. In this short review we will summarize and discuss the methodologies currently available for the analysis and full characterization of phosphoproteins with special attention at mass spectrometry-based techniques. In particular, we will discuss affinity-based purification of phosphopeptides coupled to MALDI-TOF analysis, their detection using mass mapping and precursor ion scan, identification of modified sites by MS/MS and quantitation analysis     

Binding of DNA purine sites to dirhodium compounds probed by mass spectrometry

The adducts formed between the antitumor active compounds [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2), Rh(2)(O(2)CCH(3))(4), and Rh(2)(O(2)CCF(3))(4) with DNA oligonucleotides have been assessed by matrix-assisted laser desorption ionization (MALDI) and nanoelectrospray (nanoESI) coupled to time-of-flight mass spectrometry (TOF MS). A series of MALDI studies performed on dipurine (AA, AG, GA, and GG)-containing single-stranded oligonucleotides of different lengths (tetra- to dodecamers) led to the establishment of the relative reactivity cis-[Pt(NH(3))(2)(OH(2))(2)](2+) (activated cisplatin) approximately Rh(2)(O(2)CCF(3))(4) > cis-[Pt(NH(3))(2)Cl(2)] (cisplatin) > [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2) > Rh(2)(O(2)CCH(3))(4) approximately Pt(C(6)H(6)O(4))(NH(3))(2) (carboplatin). The relative reactivity of the complexes is associated with the lability of the leaving groups. The general trend is that an increase in the length of the oligonucleotide leads to enhanced reactivity for Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2) and Rh(2)(O(2)CCH(3))(4) (except for the case of [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](2+), which reacts faster with the GG octamers than with the dodecamers), whereas the reactivity of Rh(2)(O(2)CCF(3))(4) is independent of the oligonucleotide length. When monitored by ESI, the dodecamers containing GG react faster than the respectiveAA oligonucleotides in reactions with Rh(2)(O(2)CCF(3))(4) and Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2), whereas AA oligonucleotides react faster with Rh(2)(O(2)CCH(3))(4). The mixed (AG, GA) purine sequences exhibit comparable rates of reactivity with the homopurine (AA, GG) dodecamers in reactions with Rh(2)(O(2)CCH(3))(4). The observation of initial dirhodium-DNA adducts with weak axial (ax) interactions, followed by rearrangement to more stable equatorial (eq) adducts, was achieved by electrospray ionization; the Rh-Rh bond as well as coordinated acetate or acetonitrile ligands remain intact in these dirhodium-DNA adducts. MALDI in-source decay (ISD), collision-induced dissociation (CID) MS-MS, and enzymatic digestion studies followed by MALDI and ESI MS reveal that, in the dirhodium compounds studied, the purine sites of the DNA oligonucleotides interact with the dirhodium core. Ultimately, both MALDI and ESI MS proved to be complementary, valuable tools for probing the identity and stability of dinuclear metal-DNA adducts.     

Analysis of sulfobutyl ether-beta-cyclodextrin mixtures by ion-spray mass spectrometry and liquid chromatography-ion-spray mass spectrometry

A comparison is made of the retention properties of additives applied as positively charged pseudo-stationary phases for electrokinetic chromatography of neutral analytes. All additives have a quaternary ammonium as functional group. The polymeric additive [poly(N,N,N',N'-tetramethyl-N-trimethylenehexamethylenediammonium), Polybrene] has a concentration of 2% (w/w) in the background electrolyte (acetate, pH 5.2). Monomeric octyltrimethylammonium (OTMA) was used at a concentration below or above its critical micelle concentration (CMC) (140 mmol/l). At a concentration (259 mmol/l) above the CMC the system is that normally used for micellar electrokinetic chromatography with cationic micelles. However, even below the CMC, where OTMA is present as monomer, retention of the neutral analytes is observed as well. In all systems coating of the capillary wall with Polybrene establishes an electroosmotic flow directed towards the anode, counter-migrating to the electrophoretic movement of the additive. Based on the measurement of the mobility of the analytes (15 small, monofunctional aromatic compounds with different functional groups), their capacity factors, k(i), were determined in all systems. Low correlation of the k(i) values is observed between the particular systems, indicating their different selectivity at least for individual pairs of analytes. Based on the log k(i) values, a linear free energy relationship was applied to elucidate the main types of chemical interaction responsible for retention. As a result, cavity formation and n or pi electron interactions were found being significant for the micellar OTMA system, which agrees with findings described in the literature for other (cationic and anionic) micellar systems. For the polymeric system and for the monomeric OTMA system, the significant retention parameter is indicating n and pi electron interactions.     

Analysis of low-level 129I in brine using accelerator mass spectrometry

An improved solvent extraction procedure for iodine separation from brine samples has been applied at Xi’an Accelerator Mass Spectrometry (AMS) center. Oil in the brine sample has to be removed to avoid appearance of the third phase during solvent extraction and to improve the chemical yield of iodine. The small amount of oil remained in the water phase was first removed by phase separation through settling down sufficiently based on their immiscibility, and then by filtration through a cellulose filter, on which oil was absorbed and removed. After oil removed, extraction recovery of iodine could achieve more than 90 %. The sodium bisulfite as an effective reductant should be added before acidification to avoid loss of iodine by formation of I₂ in sample via reaction of iodate and iodide at pH 1–2, and then pH was adjusted to 1–2 to reduce the iodate to iodide followed by oxidation of iodide to I₂ and solvent extraction to separate all inorganic iodine. As a pre-nuclear era sample, ¹²⁹I/¹²⁷I ratio in brine is normally more than two orders of magnitude lower than that in present surface environmental samples, so prevention of cross-contamination and memory effect in apparatus during processing procedure are very critical for obtaining reliable results, and monitoring the procedure blank is very important for analytical quality of ¹²⁹I. The ¹²⁹I/¹²⁷I isotopic ratio in the brine samples and procedure blank of iodine reagents were measured to be (1.9–2.7) × 10^?13 and 2.08 × 10^?13, respectively, 3–4 orders of magnitudes lower than that in environmental samples in Xi’an, and the result of procedure blank is in the same level as the previous experiments in past 3 years, indicating contamination is not observed in our method.     

Analysis of tellurium by spark source mass spectrometry

Summary Three tellurium standards were analyzed by both spark source mass spectrometry (SSMS) and graphite furnace atomic absorption spectrophotometry (GFAAS). From these results relative sensitivity coefficients (RSC) for spark source mass spectrometry were derived for 24 elements.With these RSC's SSMS results within a factor 1.5 from the GFAAS values could be obtained for the determination of various impurities in tellurium. A comparison is made between SSMS, GFAAS and glow discharge mass spectrometry (GDMS) for the analysis of 4–6 N tellurium samples.

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Analyse von Tellurium mit Hilfe der Funkenionisations-Massenspektrometrie

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Dedicated to Prof, Dr. G. Tölg on the occasion of his 60th birthday     

Identifying sites of protein modification by using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and on-line membrane preconcentration-capillary electrophoresis-tandem mass spectrometry

A strategy for rapidly identifying the number and sites of chemical or posttranslational modification of proteins is described. The use of matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry to determine the molecular weight of the adducted protein as well as map the proteolytic digest of peptides offers a rapid method to screen for the possible site of adduction. To unequivocally determine the amino acid sequence of the peptide bearing the adduct as well as structurally characteize the covalent modification, the peptide mixture is subjected to membrane preconcentration-capillary electrophoresis-mass spectrometry and tandem mass spectrometry (mPC-CE-MS/MS). The high resolving separation capability of capillary electrophoresis (CE) afford a chromatograhic step that lends itself to separation of complex mixtures of peptides with minimal sample loss. The membrane preconcentration-CE cartridge allows sample loading volumes 10,000-fold greater than conventional CE. In this work the binding site of the fluorescent label acrylodan to the intestinal fatty binding protein is characterized and shown to be covalently bound at lysine-27, by using mPC-CE-MS/MS.     

Analysis of carotenoids by field desorption mass spectrometry

The mass spectra of a variety of authentic carotenoids covering a range of structural types have been studied using the field desorption technique. All the compounds examined show the molecular ion as the base peak, even where this ion is of very low intensity in the electron-impact spectra. Little fragmentation is observed, but thermal decomposition can be induced in certain cases by using higher emitter currents. Application to a simple mixture of authentic compounds and a more complex mixture of sedimentary origin shows the potential of the method in the rapid qualitative screening of carotenoids in crude extracts.     

Analysis of oxysterols by electrospray tandem mass spectrometry

Oxysterols are oxygenated derivatives of cholesterol. They are intermediates in cholesterol excretion pathways and may also be regarded as transport forms of cholesterol. The introduction of additional hydroxyl groups to the cholesterol skeleton facilitates the flux of oxysterols across the blood brain barrier, and oxysterols have been implicated in mediating a number of cholesterol-induced metabolic effects. Oxysterols are difficult to analyze by atmospheric pressure ionization mass spectrometry on account of the absence of basic or acidic functional groups in their structures. In this communication, we report a method for the derivatization and analysis of oxysterols by electrospray mass spectrometry. Oxysterols with a 3beta-hydroxy-Delta5 structure were converted by cholesterol oxidase to 3-oxo-Delta4 steroids and then derivatized with the Girard P reagent to give Girard P hydrazones, which were subsequently analyzed by tandem mass spectrometry. The improvement in sensitivity for the analysis of 25-hydroxycholesterol upon oxidation and derivatization was over 1000.     

Characterization of O-glycosylation sites in MUC2 glycopeptides by nanoelectrospray QTOF mass spectrometry

Sequencing of eight O-glycosylated peptides by nanoESI-QTOF-MS/MS was carried out to provide a sensitive general characterization method for determination of glycosylation site(s) and of the type of the attached carbohydrate moiety in a single experiment. The glycopeptide structures were chosen to demonstrate the feasibility of this sensitive and accurate approach, where isobaric peptide structures either (i) with the same number of attachment sites in different position in the peptide backbone, and (ii) with the same number of sugar moieties distributed on different attachment sites in the peptide backbone, can be clearly distinguished. Beside the B-type carbohydrate sequence ions of high abundance, it is possible to register diagnostic b- and y-type glycosylated peptide ions of lower abundance due to high dynamic range of the QTOF analyser. The applicability of this approach for detailed analysis of highly clustered O-glycan structures as found in biological mucin samples is discussed.     

Determination of protein phosphorylation sites by mass spectrometry: a novel electrospray-based method

Several methods are used to identify protein phosphorylation sites. We report a novel electrospray-based method for the determination of phosphorylation sites by mass spectrometry, using two different declustering potential values. This method allows one to obtain, with a single liquid chromatography/mass spectrometry (LC/MS) run, the pattern with either the phosphorylated or the unphosphorylated species of a protein tryptic digest, that can be further analyzed by tracing back the origin of each HPO3-deprived form using the capabilities of tandem mass spectrometers.     

Determination of calcium binding sites in gas-phase small peptides by tandem mass spectrometry

Low-energy (LE) and high-energy (HE) collisionally activated decompositions (CAD) of calcium/peptide complexes of the form [M-H+Ca]⁺ and [M+Ca]²⁺ reflect the site of calcium binding in various gas-phase peptides that are models of the calcium binding site III of rabbit skeletal troponin C. The Ca²⁺ binding sites involve an aspartic acid, glutamic acid, and asparagine, which are in the metal-binding loops of calcium-binding proteins. Both fast atom bombardment (FAB) and electrospray ionization (ESI) were used to generate the metal/peptide complexes. When submitted to LE CAD, ESI-produced Ca²⁺/peptide complexes undergo fragmentations that are controlled by Ca²⁺ binding and provide information on the Ca²⁺ binding site. The LE CAD spectra are simple, indicating that Ca²⁺ binding involves specific oxygen ligands including acidic side chains and that only a few low-energy fragmentation channels exist. The HE CAD spectra of FAB-produced Ca²⁺/peptide complexes are more complex, owing to the introduction of high internal energy into the precursor ion. Interactions of the other alkaline-earth metal ions Mg²⁺ and Ba²⁺ with these peptides reveal that the ligand preferences of these metal ions are slightly different than those of Ca²⁺.     

Identification of phosphorylation sites in proteins by nanospray quadrupole ion trap mass spectrometry

A method is described for identifying serine phosphorylation sites in proteins, based on conventional (32)P labeling followed by electrophoretic separation, 'in-gel' digestion with a protease, peptide extraction, reversed-phase high-performance liquid chromatographic separation and collection and off-line analysis of the radioactive fractions by nanospray ion trap mass spectrometry. The method was successfully applied to the identification of three phosphorylation sites in two proteins which were subjected to in vitro phosphorylation under physiological conditions. Different combinations of the various scanning modes of the ion trap, including high-resolution, multiple subfragmentation (or MS(n)) and fast scan analysis, were employed to identify the phosphopeptides, determine their sequence and localize the exact site of phosphorylation. 'Blind' fragmentation using fast scans was used to analyze a phosphopeptide which was undetectable in other scanning modes. The sequence, phosphorylation site and double cysteine modification of the potassium adduct of a peptide containing 35 residues were also determined by multiple fragmentation. The results not only support the validity of the proposed method for routine identification of phosphorylation sites, but also demonstrate the exceptional capability of off-line ion trap mass spectrometry in combination with nanospray ionization for performing very detailed studies on the structure of peptides.     

Analysis of estrogens in river sediments by liquid chromatography-electrospray ionisation mass spectrometry. Comparison of tandem mass spectrometry and time-of-flight mass spectrometry

A method has been developed and optimised for the determination of two natural estrogens, estrone (E1) and 17beta-estradiol (E2), and one synthetic estrogen, 17alpha-ethynylestradiol (EE2), in river sediments at the sub-ng/g level. This procedure includes microwave-assisted solvent extraction (MASE), solid-phase extraction and high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionisation. Using sediments spiked with the three estrogens at 10 ng/g wet weight, efficient extraction (>92%) of all the three analytes was achieved by MASE, and whole-procedure recoveries ranged from 82 to 98%. Optimisation of the LC separation allowed for substantial reduction of ionisation suppression in the electrospray source to a final level of <18% suppression. Time-of-flight mass spectrometry (TOF-MS) and MS/MS were compared for the analysis of sediment extracts, with the latter technique proving to be the most selective. The method detection limits achieved by LC-MS/MS were 15, 30 and 40 pg/g for E1, E2 and EE2, respectively, which were 13-fold lower than those obtained by LC-TOF-MS. Analysis of river sediments collected from the River Ouse, UK, showed the presence of the natural estrogens at sub-ng/g level. E1 levels ranged from 0.40 ng/g (dry weight) to 3.30 ng/g while E2 levels ranged from <0.03 to 1.20 ng/g and EE2 was never detected (<0.04 ng/g).