Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis

We generated a comprehensive picture of protease substrates in anti-Fas-treated apoptotic human Jurkat T lymphocytes. We used combined fractional diagonal chromatography (COFRADIC) sorting of protein amino-terminal peptides coupled to oxygen-16 or oxygen-18 differential labeling. We identified protease substrates and located the exact cleavage sites within processed proteins. Our analysis yielded 1,834 protein identifications and located 93 cleavage sites in 71 proteins. Indirect evidence of apoptosis-specific cleavage within 21 additional proteins increased the total number of processed proteins to 92. Most cleavages were at caspase consensus sites; however, other cleavage specificities suggest activation of other proteases. We validated several new processing events by immunodetection and by an in vitro assay using recombinant caspases and synthetic peptides containing presumed cleavage sites. The spliceosome complex appeared a preferred target, as 14 of its members were processed. Differential isotopic labeling further revealed specific release of nucleosomal components from apoptotic nuclei.     

A new approach for mapping sialylated N-glycosites in serum proteomes

A new approach for proteome-wide analysis of sialylated N-glycopeptides based on the diagonal chromatographic COFRADIC technology is presented here. The use of alpha(2-3,6,8,9) neuraminidase is central to isolate sialylated N-glycopeptides out of a complex peptide mixture. Two different COFRADIC techniques are introduced here, either without or with post-metabolic oxygen-18 labeling (direct versus indirect sorting), and when applied to immuno-depleted mouse serum, we herewith identified 93 sialylated glycosylation sites in 53 serum proteins.     

Kinetics of oxygen-18 exchange between inorganic phosphate and water catalyzed by myosin subfragment 1, using the 18O shift in 31P NMR

The time course of oxygen-18 exchange between [18O]Pi and normal water, catalyzed by myosin subfragment 1 in the presence of MgADP, was followed using the shift in 31P NMR caused by the presence of oxygen-18 bound to the phosphorus. Essentially all molecules of [18O]Pi that bind to the enzyme undergo complete exchange and are released as [16O4]Pi. Exchange probably occurs by formation of myosin.ATP from a myosin.ADP.Pi complex and is rapid relative to release of Pi from this complex. The kinetics of exchange give a value for the rate constant for binding Pi to myosin.ADP of 0.23 M-1 S-1 (pH 8.0, 22 degrees C). This value is consistent with exchange occurring by reversal of the ATP-ase reaction back to the myosin.ATP complex.     

Sub-equilibrium oxygen-18 and carbon-13 levels in biological carbonates: carbonate and kinetic models

Many biological carbonates contain less oxygen-18 and carbon-13 than expected for isotopic equilibrium with ambient waters. "Carbonate" explanations for the oxygen-18 deficiencies draw inspiration from McCrea's (1950) observation that dissolved inorganic carbonates (DIC), in isotopic equilibrium with water, lose oxygen-18 in proportion to the abundance of the carbonate ion. Spero et al. (1997) therefore suggested that high carbonate levels might cause foraminifera to produce isotopically lighter shells at elevated pH. Adkins et al. (2003) extended this idea to the (presumably) alkaline internal calcification sites of deep-sea corals, and related skeletal carbon-13 content to the use of molecular carbon dioxide in calcification. This review compares these "carbonate" ideas with an updated "kinetic" model, which attributes isotopic enlightenment (for carbon-13 and oxygen-18) to carbon-dioxide-based calcification, and incomplete isotopic equilibration between DIC and water.     

Synthesis and detection of oxygen-18 labeled phosphate

Phosphorus (P) has only one stable isotope and therefore tracking P dynamics in ecosystems and inferring sources of P loading to water bodies have been difficult. Researchers have recently employed the natural abundance of the ratio of (18)O/(16)O of phosphate to elucidate P dynamics. In addition, phosphate highly enriched in oxygen-18 also has potential to be an effective tool for tracking specific sources of P in the environment, but has so far been used sparingly, possibly due to unavailability of oxygen-18 labeled phosphate (OLP) and uncertainty in synthesis and detection. One objective of this research was to develop a simple procedure to synthesize highly enriched OLP. Synthesized OLP is made up of a collection of species that contain between zero and four oxygen-18 atoms and, as a result, the second objective of this research was to develop a method to detect and quantify each OLP species. OLP was synthesized by reacting either PCl(5) or POCl(3) with water enriched with 97 atom % oxygen-18 in ambient atmosphere under a fume hood. Unlike previous reports, we observed no loss of oxygen-18 enrichment during synthesis. Electrospray ionization mass spectrometry (ESI-MS) was used to detect and quantify each species present in OLP. OLP synthesized from POCl(3) contained 1.2% P(18)O(16)O(3), 18.2% P(18)O(2) (16)O(2), 67.7% P(18)O(3) (16)O, and 12.9% P(18)O(4), and OLP synthesized from PCl(5) contained 0.7% P(16)O(4), 9.3% P(18)O(3) (16)O, and 90.0% P(18)O(4). We found that OLP can be synthesized using a simple procedure in ambient atmosphere without the loss of oxygen-18 enrichment and ESI-MS is an effective tool to detect and quantify OLP that sheds light on the dynamics of synthesis in ways that standard detection methods cannot.     

N-Succinimidyl 4-[(18)F]-fluoromethylbenzoate-labeled dimeric RGD peptide for imaging tumor integrin expression

RGD peptides, radiolabeled with (18)F, have been used in the clinic for PET imaging of tumor angiogenesis in cancer patients. RGD peptides are typically labeled using a prosthetic group such as N-succinimidyl 4-[(18)F]-fluorobenzoate ([(18)F]SFB) or 4-nitrophenyl 2-[(18)F]-fluoropropionate ([(18)F]NPFP). However, the complex radiosynthetic procedures have impeded their broad application in clinical studies. We previously radiolabeled proteins and peptides with the prosthetic group, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB), which was prepared in a simple one-step procedure. In this study, we labeled a PEGylated cyclic RGD peptide dimer, PEG(3)-E[c(RGDyK)](2) (PRGD2), using [(18)F]SFMB and evaluated for imaging tumor αvβ3 integrin expression with positron emission tomography (PET). [(18)F]SFMB was prepared in one step using [(18)F]fluoride displacement of a nitrobenzenesulfonate leaving group under mild reaction conditions followed by HPLC purification. The (18)F-labeled peptide, [(18)F]FMBPRGD2 was prepared by coupling PRGD2 with [(18)F]SFMB in pH 8.6 borate buffer and purified with HPLC. The direct labeling on BMBPRGD2 was also attempted. A Siemens Inveon PET was used to image the uptake of the [(18)F]FMBPRGD2 into a U87MG xenograft mouse model. [(18)F]FMBPRGD2, was prepared with a 15% overall radiochemical yield (uncorrected) in a total synthesis time of 90?min, which was considerably shorter than the preparation of [(18)F]SFB- and [(18)F]NPFP-labeled RGD peptides. The direct labeling, however, was not successful. High quality microPET images using [(18)F]FMBPRGD2 clearly visualized tumors by 15?min with good target to background ratio. Early tracer accumulation in the bladder suggests fast renal clearance. No obvious bone uptake can be detected even at 4-h time point indicating that fluorine attachment is stable in mice. In conclusion, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB) prosthetic group can be a good alternative for labeling RGD peptides to image αvβ3 integrin expression and for labeling other peptides.     

Targeting peptides and positron emission tomography

Biologically active peptides have during the last decades made their way into conventional nuclear medicine diagnosis using single photon emission computed tomography (SPECT) and gamma-camera. Several clinical trails are also investigating the role of radiolabeled peptides for targeting radionuclide therapy. This has raised the question as to whether positron emission tomography (PET) can be used in order to obtain better quantitative information of the peptide distribution in tumor and healthy organs, i.e., to get a better dosimetry. Positron emitting analogs of the therapeutic radionuclides used have been produced and successfully applied in peptide pharmacokinetic measurements with PET. But the recent boom in (18)FDG-PET ((18)FDG = [(18)F]2-deoxy-2-fluoro-D-glucose), and with this a worldwide increasing number of PET systems, has also inspired several research groups to hunt for alternative labels to be used for peptide diagnostics and PET. The rapid kinetic of short peptides agrees well with the short half-lives of standard PET nuclides like (11)C and (18)F. Especially, (18)F appears to be excellent for labeling bioactive peptides due to its favorable physical and nuclear characteristics. However, with present techniques labeling peptides with (18)F is laborious and time-consuming, and is not yet a clinical alternative. Other halogens like (75, 76)Br and (124)I are, from the chemical point of view, easier to apply. But an even better labeling alternative may be positron emitting metal ions like (55)Co, (68)Ga, and (110m)In since they tend to give better intracellular retention and thus a better signal-to-background ratio than the halogen labels. The main drawback with these radionuclides is that they are not readily available. Some of these radionuclides also emit gamma in their decay that may affect the measuring properties of the PET equipment. This article reviews mainly the present situation of production and use of nonconventional positron emitters for peptide labeling.     

Mechanism and stereochemical course at phosphorus of the reaction catalyzed by a bacterial phosphotriesterase

The reaction mechanism for the phosphotriesterase from Pseudomonas diminuta has been examined. When paraoxon (diethyl 4-nitrophenyl phosphate) is hydrolyzed by this enzyme in oxygen-18-labeled water, the oxygen-18 label is found exclusively in the diethyl phosphate product. The absolute configurations for the (+) and (-) enantiomers of O-ethyl phenylphosphonothioic acid have been determined by X-ray diffraction structural determination of the individual crystalline 1-phenylethylamine salts. The (+) enantiomer of the free acid corresponds to the RP configuration. The RP enantiomer of O-ethyl phenylphosphonothioic acid has been converted to the SP enantiomer of EPN [O-ethyl O-(4-nitrophenyl) phenylphosphonothioate]. (SP)-EPN is hydrolyzed by the phosphotriesterase to the SP enantiomer of O-ethyl phenylphosphonothioic acid. The enzymatic reaction therefore proceeds with inversion of configuration. These results have been interpreted as an indication of a single in-line displacement by an activated water molecule directly at the phosphorus center of the phosphotriester substrate. (RP)-EPN is not hydrolyzed by the enzyme at an appreciable rate.     

Enhancing the stability of O-labeled peptides through removal of immobilized trypsin by ZipTips

Trypsin-catalyzed ¹⁸O labeling is increasingly used in shotgun proteomics for relative peptide/protein quantitation. However, precise quantitative measurements are often complicated by the instability of ¹⁸O-labeled peptides caused mainly by oxygen back-exchange. Although a number of attempts have been made to reduce or prevent oxygen back-exchange, there is still room for improvement. Here we demonstrate that the removal of immobilized trypsin by filtration using ZipTips can efficiently minimize oxygen back-exchange and enhance the stability of ¹⁸O-labeled peptides under various pH conditions. The ¹⁸O-labeled peptides processed by the approach were successfully separated by immobilized pH gradient–isoelectric focusing (IPG–IEF), and no marked decrease in the extent of labeling was observed. The results also demonstrated that there was no correlation between the extent of ¹⁸O labeling and molecular weight or isoelectric point (pI). The approach presented here is especially applicable to microscale samples. Its ability to generate stably ¹⁸O-labeled samples without back-exchange should expand the application scope of the ¹⁸O-labeling technique.     

Improved method for differential expression proteomics using trypsin-catalyzed 18O labeling with a correction for labeling efficiency

Quantitative strategies relying on stable isotope labeling and isotope dilution mass spectrometry have proven to be a very robust alternative to the well established gel-based techniques for the study of the dynamic proteome. Postdigestion 18O labeling is becoming very popular mainly due to the simplicity of the enzyme-catalyzed exchange reaction, the peptide handling and storage procedures, and the flexibility and versatility introduced by decoupling protein digestion from peptide labeling. Despite recent progresses, peptide quantification by postdigestion 18O labeling still involves several computational problems. In this work we analyzed the behavior of large collections of peptides when they were subjected to postdigestion labeling and concluded that this process can be explained by a universal kinetic model. On the basis of this observation, we developed an advanced quantification algorithm for this kind of labeling. Our method fits the entire isotopic envelope to parameters related with the kinetic exchange model, allowing at the same time an accurate calculation of the relative proportion of peptides in the original samples and of the specific labeling efficiency of each one of the peptides. We demonstrated that the new method eliminates artifacts produced by incomplete oxygen exchange in subsets of peptides that have a relatively low labeling efficiency and that may be considered indicative of false protein ratio deviations. Finally using a rigorous statistical analysis based on the calculation of error rates associated with false expression changes, we showed the validity of the method in the practice by detecting significant expression changes, produced by the activation of a model preparation of T cells, with only 5 microg of protein in three proteins among a pool of more than 100. By allowing a full control over potential artifacts, our method may improve automation of the procedures for relative protein quantification using this labeling strategy.     

Synthesis of N-succinimidyl 4-[18F]fluorobenzoate, an agent for labeling proteins and peptides with 18F

This protocol describes the step-by-step procedure for the synthesis of N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB), an agent widely used for labeling proteins and peptides with the positron-emitting radionuclide 18F. The protocols for the synthesis of unlabeled SFB and the quaternary salt precursor 4-formyl-N,N,N-trimethyl benzenaminium trifluoromethane sulfonate also are described. For the [18F]SFB synthesis, the quaternary salt is first converted to 4-[18F]fluorobenzaldehyde. Oxidation of the latter provides 4-[18F]fluorobenzoic acid, which is converted to [18F]SFB by treatment with N,N-disuccinimidyl carbonate. Using this method, [18F]SFB can be synthesized in decay-corrected radiochemical yields of 30%-35% and a specific radioactivity of 11-12 GBq micromol(-1). The total synthesis and purification time required is about 80 min, starting from delivery of the [18F]fluoride. [18F]SFB remains an optimal reagent for labeling proteins and peptides with 18F because of good conjugation yields and metabolic stability.     

Measurement of positional isotope exchange rates in enzyme-catalyzed reactions by fast atom bombardment mass spectrometry: application to argininosuccinate synthetase

Fast atom bombardment mass spectrometry (FAB-MS) has been used to measure positional isotope exchange rates in enzyme-catalyzed reactions. The technique has been applied to the reactions catalyzed by acetyl-CoA synthetase and argininosuccinate synthetase. The FAB technique is also able to quantitatively determine the oxygen-18 or oxygen-17 content of nucleotides on as little as 10 nmol of material with no prior derivatization. Acetyl-CoA synthetase has been shown by FAB-MS to catalyze the positional exchange of an oxygen-18 of ATP from the beta-nonbridge position to the alpha beta-bridge position in the presence of acetate. These results are consistent with acetyl adenylate as a reactive intermediate in this reaction. Argininosuccinate synthetase was shown not to catalyze a positional isotope exchange reaction designed to test for the formation of citrulline adenylate as a reactive intermediate. Argininosuccinate synthetase was also found not to catalyze the transfer of oxygen-18 from [ureido-18O]citrulline to the alpha-phosphorus of ATP in the absence of added aspartate. This experiment was designed to test for the transient formation of carbodiimide as a reactive intermediate. These results suggest that either argininosuccinate synthetase does not catalyze the formation of citrulline adenylate or the enzyme is able to completely suppress the rotation of the phosphoryl groups of PPi.     

Correction Approaches for Doubly Labeled Water in Situations of Changing Background Water Abundance

Doubly labeled water (DLW) is an accurate, portable method for measuring free-living energy expenditure. However, under certain conditions shifts in baseline abundance of deuterium and oxygen-18 tracers used in the method may produce errors in derivation of both turnover (k) rates and calculated energy expenditure. Present objectives were to examine during what experimental situations baseline errors arise and to address means of correcting for such baseline shifts so that consequent errors in energy expenditure calculations are minimized. Under conditions where shifts in baseline abundance for deuterium and oxygen-18 parallel abundances corresponding to the natural meteoric water ratio, self-compensating changes in k values for both deuterium and oxygen will result in minimal error to the DLW energy expenditure calculations, provided that the dose ratio of isotopes also mimics the meteoric water line. However, in situations where relative shifts in abundance of each isotope across the measurement period are not in parallel relative to the natural meteoric water line, then the potential for larger DLW errors exists. Optimally, subjects should equilibrate with the new water source. Failing this, correction for shifting baseline can be accomplished by measuring isotopic abundance changes in a control group of subjects not given the DLW dose, but performing similar tasks and consuming the same diet as the group given DLW. Alternatively, theoretically based correction values can be calculated given knowledge of the abundances of the final drinking water and the interval time that subjects consumed the new fluid.     

Mechanism-based inactivation of dopamine beta-monooxygenase by beta-chlorophenethylamine

Functionalization of the beta-carbon of phenethylamines has been shown to produce a new class of substrate/inhibitor of dopamine beta-monooxygenase. Whereas both beta-hydroxy- and beta- chlorophenethylamine are converted to alpha-aminoacetophenone at comparable rates, only the latter conversion is accompanied by concomitant enzyme inactivation ( Klinman , J. P., and Krueger , M. (1982) Biochemistry 21, 67-75). In the present study, the nature of the reactive intermediates leading to dopamine beta-monooxygenase inactivation by beta- chlorophenethylamine has been investigated employing kinetic deuterium isotope effects and oxygen- 18 labeling as tools. Mechanistically significant findings presented herein include: 1) an analysis of primary deuterium isotope effects on turnover, indicating major differences in rate-determining steps for beta-chloro- and beta- hydroxyphenethylamine hydroxylation, Dkcat = 6.1 and 1.0, respectively; 2) evidence that dehydration of the gem-diol derived from oxygen- 18-labeled beta- hydroxyphenethylamine hydroxylation occurs in a random manner, attributed to dissociation of enzyme-bound gem-diol prior to alpha-aminoacetophenone formation; 3) the observation of a deuterium isotope effect for beta- chlorophenethylamine inactivation, Dkinact = 3.7, implicating C--H bond cleavage in the inactivation process; and 4) the demonstration that alpha-aminoacetophenone can replace ascorbic acid as exogenous reductant in the hydroxylation of tyramine. As discussed, these findings support the intermediacy of enzyme-bound alpha-aminoacetophenone in beta- chlorophenethylamine inactivation, and lead us to propose an intramolecular redox reaction to generate a ketone-derived radical cation as the dopamine beta-monooxygenase-inactivating species.     

Enzymic preparation of dioxygen-18 labelled leukotriene E4 and its use in quantitative gas chromatography—mass spectrometry

A simple and rapid method is described for the preparation of a stable isotope oxygen-18 labelled leukotriene E₄ (LTE₄). Oxygen-18 labelling of LTE₄ methyl ester in oxygen-18 water catalysed by a pig liver esterase resulted in the incorporation of two oxygen-18 atoms in the carboxylic group of LTE₄ to the extent of 89.8% ([¹⁸O₂]LTE₄) and one oxygen-18 atom to the extent of 9.4% ([¹⁶O¹⁸O]LTE₄), with only 0.7% remaining unchanged ([¹⁶O₂]LTE₄). [¹⁸O₂]LTE₄ was found not to back-exchange following incubation in acidified urine (pH 4.0) at 4°C for up to 20 h. [¹⁸O₂]LTE₄ was demonstrated to be a useful internal standard in a method for the quantitative determination of LTE₄ in human urine involving high-performance liquid chromatography and gas chromatography with negative-ion chemical ionization tandem mass spectrometry: the concentration of LTE₄ in a 24-h urine sample of a healthy subject was determined to be 68.1 pg/ml.     

Microwave‐assisted 18O labeling of Fmoc‐protected amino acids

Recently, there has been an increased interest in isotopical labeling of peptides. Although there are several techniques allowing for a complete labeling of all carboxyl groups in peptides, regioselective labeling would be beneficial in many situations. Such labeling requires the use of ¹⁸O‐labeled Fmoc amino acids. We have designed a method for such labeling that is an improvement on a technique proposed earlier. The new procedure is suitable for microscale synthesis and could be used in peptide and proteomics laboratories. Although for the majority of tested amino acids our method gives good labeling efficiency, it is time consuming. Therefore, we have decided to use microwave‐assisted procedure. This approach resulted in reduction of reaction time to 15 min and increased reaction efficiency. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Preparation of oxygen-18-labeled lipoxygenase metabolites of arachidonic acid

Plasma pseudocholinesterase and porcine liver esterase were used to catalyse the incorporation of the stable isotope oxygen-18 into the carboxyl moiety of lipoxygenase metabolites of arachidonic acid. This simple method produces eicosanoid products containing two oxygen-18 atoms; but the enzymes studied were found to display large substrate specificity in the efficiencies at which oxygen-18 could be incorporated into the lipoxygenase metabolites. Furthermore, [18O2]LTB4 was found not to back exchange during in vitro incubation with human neutrophils. The methods involved for stable isotope incorporation are simple, efficient and produce highly enriched species in a short time. By varying the type of esterase, the amount of esterase or the length of incubation highly enriched species of all eicosanoids tested could be prepared.     

Examination of the mechanism of sucrose synthetase by positional isotope exchange

The mechanism of the sucrose synthetase reaction has been probed by the technique of positional isotope exchange. [beta-18O2, alpha beta-18O]UDP-Glc has been synthesized starting from oxygen-18-labeled phosphate and the combined activities of carbamate kinase, hexokinase, phosphoglucomutase, and uridine diphosphoglucose pyrophosphorylase. The oxygen-18 at the alpha beta-bridge position of the labeled UDP-Glc has been shown to cause a 0.014 ppm upfield chemical shift in the 31P NMR spectrum of both the alpha- and beta-phosphorus atoms in UDP-Glc relative to the unlabeled compound. The chemical shift induced by each of the beta-nonbridge oxygen-18 atoms was 0.030 ppm. Incubation of [beta-18O2, alpha beta-18O]UDP-Glc with sucrose synthetase in the presence and absence of 2,5-anhydromannitol did not result in any significant exchange of an oxygen-18 from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. It can thus be concluded that either sucrose synthetase does not catalyze the cleavage of the scissile carbon-oxygen bond of UDP-Glc in the absence of fructose or, alternatively, the beta-phosphoryl group of the newly formed UDP is rotationally immobilized.     

18F]azadibenzocyclooctyne ([18F]ADIBO): a biocompatible radioactive labeling synthon for peptides using catalyst free [3+2] cycloaddition

N-Terminally azido-modified peptides were labeled with the novel prosthetic labeling synthon [(18)F]azadibenzocyclooctyne ([(18)F]ADIBO) using copper-free azide-alkyne [3+2]-dipolar cycloaddition in high radiochemical yields (RCYs). (18)F-Labeled [(18)F]ADIBO was prepared by nucleophilic substitution of the corresponding tosylate in 21% overall RCY (EOB) in a fully automated synthesis unit within 55 min. [(18)F]ADIBO was incubated with azide-containing peptides at room temperature in the absence of toxic metal catalysts and the formation of the triazole conjugate was confirmed. Finally, the azide-alkyne [3+2]-dipolar cycloaddition was shown to proceed with 95% radiochemical yield in ethanol within 30 min, allowing for a development of a kit-like peptide labeling approach with [(18)F]ADIBO.     

Study of spin-lattice and spin-spin relaxation times of 1H, 2H, and 17O in muscular water.

Spin-lattice (T1) and spin-spin (T2) relaxation times of proton, deuteron, and oxygen-17 in muscle water have been measured at 9.21 MHz in the temperature range of 0 degree--40 degrees C. The values of the apparent activation energy for the three nuclei are (in kJ . mol-1) 9.1, 19, and 18 for 1/T1, and -1.3, 4.2, and 14 for 1/T2, respectively. The relatively small values for T2 for 1H and 2H and their low apparent activation energies are attributed to hydrogen exchange between water and proteins; this exchange does not affect the 17O relaxation. Quantitative calculations on deuteron T1 and oxygen-17 T1 and T2 have been made. The effect of surface-induced anisotropy on a minor fraction of water molecules is considered in some detail, and a new expression for its spectral density similar to that of liquid crystalline systems is applied in the calculation. It is suggested that water on the surfaces of macromolecules has a rotational correlation time of tau c approximately 1 x 10(-9) S, with a time constant of tau x approximately 3 x 10(-7) S, which is characteristic of the relaxation of the local structure.