Decision peptide-driven: A free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry

The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse ¹⁸O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse ¹⁸O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse ¹⁸O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using ¹⁸O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase.     

Accelerated 18O-labeling in urinary proteomics

Proteolytic (18)O-labeling of peptides has been studied and optimized in order to improve the labeling efficiency and to accelerate the process without increasing the degree of incomplete labeling. Using peptides generated from tryptic digested bovine serum albumin (BSA) and cytochrome c as model proteins, it was shown that complete labeling was achieved after 2 h at pH 6. To increase the sample throughput in a bottom-up proteomic setup, tryptic digestion of proteins in-solution was replaced with tryptic digestion using immobilized trypsin. As a result, an integrated approach was made possible, where both digestion (pH 8) and (18)O/(16)O-labeling of the resulting peptides (pH 6) were done using immobilized trypsin beads. This simplified the sample handling and reduced the overall reaction time significantly: the setup enabled tryptic digestion and (18)O/(16)O-labeling without sample transfer steps within 3.5 h with average (18)O/(16)O-ratios of 0.96±0.13 in aqueous buffer. The initial results were confirmed with a more complex matrix, by spiking urine with the model proteins, yielding results comparable with the ratios obtained in buffer. Satisfying ratios were also achieved regarding urinary proteins identified in a full scale bottom-up experiment. Average (18)O/(16)O-peptide ratios of 0.83±0.13 and 0.91±0.27 indicated good performance in a highly relevant matrix for biomarker discovery.     

ON THE FORMATION AND REACTIVITY OF DIOXIRANE INTERMEDIATES IN THE REACTION OF PEROXOANIONS WITH ORGANIC SUBSTRATES*

Abstract— Kinetics and ¹⁸O-labeling studies have provided evidence for the involvement of dioxirane intermediates (V) in the ketone-catalysed decomposition of peroxomonosulfate (Caroate) HSO;. Reaction rates depend on ketone structure. In competition with catalysis of peroxide decomposition, the dioxirane intermediate is capable of oxidizing several organic and inorganic substrates. Thus, cis- and rrans-cinnammic acids could be converted stereospecifically into the corresponding epoxides. Also, oxidation of phenylpropiolic acid, a substrate which is representative of weakly nucleophilic alkynes, could be carried out using the Caroatehetone oxidizing system. Under the conditions adopted, no oxidation of the substrates examined was found to occur in the absence of ketone. The possibility that formation of dioxathiirane intermediates (XXII) occurs following a side pathway in the reaction of Caroate with sulfoxides (which produces sulfones in high yield) has been explored. Preliminary experiments using ¹⁸O-labeling of p-tolyl phenylsulfoxide, however. failed to support this hypothesis. pointing out the need for further detailed studies.     

Electron spray ionization mass spectrometry and 2D 31P NMR for monitoring 18O/16O isotope exchange and turnover rates of metabolic oligophosphates

A new method was here developed for the determination of ¹⁸O-labeling ratios in metabolic oligophosphates, such as ATP, at different phosphoryl moieties (α-, β-, and γ-ATP) using sensitive and rapid electrospray ionization mass spectrometry (ESI-MS). The ESI-MS-based method for monitoring of ¹⁸O/¹⁶O exchange was validated with gas chromatography–mass spectrometry and 2D ³¹P NMR correlation spectroscopy, the current standard methods in labeling studies. Significant correlation was found between isotopomer selective 2D ³¹P NMR spectroscopy and isotopomer less selective ESI-MS method. Results demonstrate that ESI-MS provides a robust analytical platform for simultaneous determination of levels, ¹⁸O-labeling kinetics and turnover rates of α-, β-, and γ-phosphoryls in ATP molecule. Such method is advantageous for large scale dynamic phosphometabolomic profiling of metabolic networks and acquiring information on the status of probed cellular energetic system.     

[F]Fluoromethyl iodide ([F]FCH2I): preparation and reactions with phenol, thiophenol, amide and amine functional groups

In this study, we report the synthesis and reactivity of [¹⁸F]fluoromethyl iodide ([¹⁸F]FCH₂I) with various nucleophilic substrates and the stabilities of [¹⁸F]fluoromethylated compounds. [¹⁸F]FCH₂I was prepared by reacting diiodomethane (CH₂I₂) with [¹⁸F]KF, and purified by distillation in radiochemical yields of 14-31% (n = 25). [¹⁸F]FCH₂I was stable in organic solvents commonly used for labeling and aqueous solution with pH 1-7, but was unstable in basic solutions. [¹⁸F]FCH₂I displayed a high reactivity with various nucleophilic substrates such as phenol, thiophenol, amide and amine. The [¹⁸F]fluoromethylated compounds synthesized by the reactions of phenol, thiophenol and tertiary amine with [¹⁸F]FCH₂I were stable for purification, formulation and storage. In contrast, the [¹⁸F]fluoromethylated compounds synthesized by the reactions of primary or secondary amines, and amide with [¹⁸F]FCH₂I were too unstable to be detected or purified from the reaction mixtures. Defluorination of these [¹⁸F]fluoromethyl compounds was a main decomposition route.     

An Improved Synthesis of N-(4-[18F]Fluorobenzoyl)-Interleukin-2 for the Preclinical PET Imaging of Tumour-Infiltrating T-cells in CT26 and MC38 Colon Cancer Models

Positron emission tomography (PET) imaging of activated T-cells with N-(4-[¹⁸F]fluorobenzoyl)-interleukin-2 ([¹⁸F]FB-IL-2) may be a promising tool for patient management to aid in the assessment of clinical responses to immune therapeutics. Unfortunately, existing radiosynthetic methods are very low yielding due to complex and time-consuming chemical processes. Herein, we report an improved method for the synthesis of [¹⁸F]FB-IL-2, which reduces synthesis time and improves radiochemical yield. With this optimized approach, [¹⁸F]FB-IL-2 was prepared with a non-decay-corrected radiochemical yield of 3.8 ± 0.7% from [¹⁸F]fluoride, 3.8 times higher than previously reported methods. In vitro experiments showed that the radiotracer was stable with good radiochemical purity (>95%), confirmed its identity and showed preferential binding to activated mouse peripheral blood mononuclear cells. Dynamic PET imaging and ex vivo biodistribution studies in naïve Balb/c mice showed organ distribution and kinetics comparable to earlier published data on [¹⁸F]FB-IL-2. Significant improvements in the radiochemical manufacture of [¹⁸F]FB-IL-2 facilitates access to this promising PET imaging radiopharmaceutical, which may, in turn, provide useful insights into different tumour phenotypes and a greater understanding of the cellular nature and differential immune microenvironments that are critical to understand and develop new treatments for cancers.     

Isotopic Traceability (13C and 18O) of Greek Olive Oil

In recent years, isotopic analysis has been proven a valuable tool for the determination of the origin of various materials. In this article, we studied the ¹⁸O and ¹³C isotopic values of 210 olive oil samples that were originated from different regions in Greece in order to verify how these values are affected by the climate regime. We observed that the δ¹⁸O isotopic values range from 19.2 ‰ to 25.2 ‰ and the δ¹³C values range from −32.7 ‰ to −28.3 ‰. These differences between the olive oils’ isotopic values depended on the regional temperature, the meteoric water, and the distance from the sea. Furthermore, we studied the ¹³C isotopic values of biophenolic extracts, and we observed that they have same capability to differentiate the geographic origin. Finally, we compared the isotopic values of Greek olive oils with samples from Italy, and we concluded that there is a great dependence of oxygen isotopes on the climatic characteristics of the different geographical areas.     

Discrepancy in infrared measurement results of carbon monoxide in nitrogen mixtures due to variations of the 13C/12C isotope ratio

Significant errors in the non-dispersive infrared (NDIR) analyses of carbon monoxide (CO) can be made when the ¹³C/¹²C isotope ratio in the sample and the calibrant differ significantly. This paper shows that variations in the ¹³C/¹²C isotope ratio of 5×10⁻² mol/mol CO in nitrogen mixtures on three different NDIR CO analysers may lead to serious deviations in the instrument response, whereas the instrument response using GC-TCD is unaffected. The observed deviations in the assigned amount-of-substance fraction CO for a ¹³C depleted mixture vary from +2 to −5% relative to the gravimetric amount-of-substance fraction for different NDIR analysers. A GC-MS method has been developed to perform a pre-screening of the isotopic composition of CO in nitrogen mixtures. This method proved to be an adequate tool to measure differences in the ¹³C/¹²C ratio. Based on the GC-MS results a suitable measurement technique can be selected, or information about a possible error in NDIR analysis can be given to the producer or user of the calibration gas mixture. Presented at 3. International Gas Analysis Symposium, October 6–8, 2004, in Amsterdam     

Membrane-bound Na+,K+-ATPase as the cardiac glycoside receptor

DSC studies are carried out to characterize Na⁺,K⁺-ATPase isolated from pig kidney in its natural membrane environment as well as in its purified state upon detergent treatment. The transition temperatures of the investigated thermal protein unfolding process, observed between 43 and 64.5° C, depend on the local membrane environment as well as onpH. In addition, the transition temperature is significantly increased upon binding of different cations and ligands which are known to interact with the enzyme. Evidence for a lipid phase transition around 23 °C in the original biological membrane is reported.The application of a calorimeter equipped with removable cells appears to be more suitable for the investigation of this type of membrane sample than an instrument with fixed capillary cells. Filling the sample capillary cell with an usual syringe, consisting of a long and thin needle, can influence the experimental results.Na⁺, K⁺ -ATPase acts as the receptor for cardiac glycoside binding. The thermodynamic parameters of this binding process are determined by titration calorimetry. The binding of ouabain, as a typical example, is unusually slow and is enthalpy driven. The enthalpy change upon binding enthalpy is –75 kJ mol^–1 at 25 °C. The surprisingly low stoichiometric coefficient, resulting from an evaluation based on a simple one step binding model, is interpreted in terms of a dimeric receptor unit.The authors are grateful to E. Lewitzki, H. Ruf, E. Schick and F. Thommen for stimulating discussions, to A. Schacht for skilfull enzyme preparations and to the German Research Foundation for financial support (SFB 169).     

Review on Applications of 17O in Hydrological Cycle

The triple oxygen isotopes (¹⁶O, ¹⁷O, and ¹⁸O) are very useful in hydrological and climatological studies because of their sensitivity to environmental conditions. This review presents an overview of the published literature on the potential applications of ¹⁷O in hydrological studies. Dual-inlet isotope ratio mass spectrometry and laser absorption spectroscopy have been used to measure ¹⁷O, which provides information on atmospheric conditions at the moisture source and isotopic fractionations during transport and deposition processes. The variations of δ¹⁷O from the developed global meteoric water line, with a slope of 0.528, indicate the importance of regional or local effects on the ¹⁷O distribution. In polar regions, factors such as the supersaturation effect, intrusion of stratospheric vapor, post-depositional processes (local moisture recycling through sublimation), regional circulation patterns, sea ice concentration and local meteorological conditions determine the distribution of ¹⁷O-excess. Numerous studies have used these isotopes to detect the changes in the moisture source, mixing of different water vapor, evaporative loss in dry regions, re-evaporation of rain drops during warm precipitation and convective storms in low and mid-latitude waters. Owing to the large variation of the spatial scale of hydrological processes with their extent (i.e., whether the processes are local or regional), more studies based on isotopic composition of surface and subsurface water, convective precipitation, and water vapor, are required. In particular, in situ measurements are important for accurate simulations of atmospheric hydrological cycles by isotope-enabled general circulation models.     

A Bayesian Markov-Chain-Based Heteroscedastic Regression Model for the Analysis of <Superscript>18</Superscript>O-Labeled Mass Spectra

To reduce the influence of the between-spectra variability on the results of peptide quantification, one can consider the ¹⁸O-labeling approach. Ideally, with such labeling technique, a mass shift of 4 Da of the isotopic distributions of peptides from the labeled sample is induced, which allows one to distinguish the two samples and to quantify the relative abundance of the peptides. It is worth noting, however, that the presence of small quantities of ¹⁶O and ¹⁷O atoms during the labeling step can cause incomplete labeling. In practice, ignoring incomplete labeling may result in the biased estimation of the relative abundance of the peptide in the compared samples. A Markov model was developed to address this issue (Zhu, Valkenborg, Burzykowski. J. Proteome Res. 9, 2669–2677, 2010). The model assumed that the peak intensities were normally distributed with heteroscedasticity using a power-of-the-mean variance funtion. Such a dependence has been observed in practice. Alternatively, we formulate the model within the Bayesian framework. This opens the possibility to further extend the model by the inclusion of random effects that can be used to capture the biological/technical variability of the peptide abundance. The operational characteristics of the model were investigated by applications to real-life mass-spectrometry data sets and a simulation study.     

Pd(0) or Pd(II)-catalyzed ring-opening reactions of benzylidene- and alkylidenecyclopropyl ketones and aldehydes

Pd(0) catalyzed reactions of methylenecyclopropyl carbonyl compounds afforded a convenient method for the synthesis of conjugate (E,E)-1,3-diene derivatives 2 in good to excellent yields. Moreover, we also found that Pd(II)-catalyzed reactions of methylenecyclopropyl carbonyl compounds with water gave 1,5-diketones in good to high yields via a carbene-palladium intermediate. The plausible reaction mechanisms have also been provided on the basis of control and ¹⁸O-labeling experiments.     

Alpha selective epoxide opening with F: synthesis of 4-(3-[F]fluoro-2-hydroxypropoxy)benzaldehyde ([F]FPB) for peptide labeling

Strained tricyclic ring systems such as epoxides are rarely used as precursors for the introduction of anionic fluorine-18 into organic compounds intended for positron emission tomography (PET). Here we report the alpha selective ring opening of epoxides for the introduction of fluorine-18 into small as well as larger biomolecules via 1- and 2-step protocols. [¹⁸F]fluoromisonidazole ([¹⁸F]MISO), a tracer for hypoxia imaging, and the tumor targeting peptide Tyr³-octreotate (TATE) were radiolabeled using epoxide opening reactions. In the latter case, the new prosthetic labeling synthon 4-(3-[¹⁸F]fluoro-2-hydroxypropoxy)benzaldehyde ([¹⁸F]FPB) has been used for ¹⁸F-introduction.     

New accessory for studies of isotopic 1H/2H exchange and biomolecular interactions using transmission infrared spectroscopy

We present here a new accessory for IR transmission measurements of ¹H/²H exchange, as an ancillary tool for monitoring structural features of biomolecules in aqueous solution. This new accessory results from the combination of two dialysis membranes and a conventional liquid cell having two cylinders containing ²H₂O buffer. When compared with conventional transmission measurements, carried out either after dissolving lyophilized biomolecules in ²H₂O or after dialyzing the aqueous solution considered against ²H₂O buffer, this accessory shows the following advantages: (1) controlled measurements over the initial steps of this isotopic exchange and absence of molecular aggregation, and (2) smaller sample amounts. This new Fourier transform IR cell can also be used to analyze ligand–biomolecule and drug–cell interactions.      

A Simplified Method for the Determination of Labeled Alkane Hydrocarbons in Mammalian Tissue and Blood After Exposure to Radiolabeled Cigarette Smoke

Abstract

A simplified procedure for the determination of ¹⁴C- labeled alkanes in mammalian tissue and blood is proposed. A portion of the ethanolic-KOH digest of the sample is counted in a standard toluene scintillator solution; consequently no secondary scintillator solvent or special tissue solubilizer is required. A wide variety of sample types and sizes, including whole organs, can be handled without homogenization or drying. Accuracy and precision are good. Sensitivity is generally inversely related to sample size.     

Differentiation of the Stereochemistry and Anomeric Configuration for 1-3 Linked Disaccharides Via Tandem Mass Spectrometry and <Superscript>18</Superscript>O-labeling

Collision-induced dissociation (CID) of deprotonated hexose-containing disaccharides (m/z 341) with 1–2, 1–4, and 1–6 linkages yields product ions at m/z 221, which have been identified as glycosyl-glycolaldehyde anions. From disaccharides with these linkages, CID of m/z 221 ions produces distinct fragmentation patterns that enable the stereochemistries and anomeric configurations of the non-reducing sugar units to be determined. However, only trace quantities of m/z 221 ions can be generated for 1–3 linkages in Paul or linear ion traps, preventing further CID analysis. Here we demonstrate that high intensities of m/z 221 ions can be built up in the linear ion trap (Q3) from beam-type CID of a series of 1–3 linked disaccharides conducted on a triple quadrupole/linear ion trap mass spectrometer. ¹⁸O-labeling at the carbonyl position of the reducing sugar allowed mass-discrimination of the “sidedness” of dissociation events to either side of the glycosidic linkage. Under relatively low energy beam-type CID and ion trap CID, an m/z 223 product ion containing ¹⁸O predominated. It was a structural isomer that fragmented quite differently than the glycosyl-glycolaldehydes and did not provide structural information about the non-reducing sugar. Under higher collision energy beam-type CID conditions, the formation of m/z 221 ions, which have the glycosyl-glycolaldehyde structures, were favored. Characteristic fragmentation patterns were observed for each m/z 221 ion from higher energy beam-type CID of 1–3 linked disaccharides and the stereochemistry of the non-reducing sugar, together with the anomeric configuration, were successfully identified both with and without ¹⁸O-labeling of the reducing sugar carbonyl group.     

The DEPTQ+ Experiment: Leveling the DEPT Signal Intensities and Clean Spectral Editing for Determining CHn Multiplicities

We propose a new ¹³C DEPTQ⁺ NMR experiment, based on the improved DEPTQ experiment, which is designed to unequivocally identify all carbon multiplicities (Cq, CH, CH₂, and CH₃) in two experiments. Compared to this improved DEPTQ experiment, the DEPTQ⁺ is shorter and the different evolution delays are designed as spin echoes, which can be tuned to different ¹J_CH values; this is especially valuable when a large range of ¹J_CH coupling constants is to be expected. These modifications allow (i) a mutual leveling of the DEPT signal intensities, (ii) a reduction in J cross-talk in the Cq/CH spectrum, and (iii) more consistent and cleaner CH₂/CH₃ edited spectra. The new DEPTQ⁺ is expected to be attractive for fast ¹³C analysis of small-to medium sized molecules, especially in high-throughput laboratories. With concentrated samples and/or by exploiting the high sensitivity of cryogenically cooled ¹³C NMR probeheads, the efficacy of such investigations may be improved, as it is possible to unequivocally identify all carbon multiplicities, with only one scan, for each of the two independent DEPTQ⁺ experiments and without loss of quality.     

Optimization of Sample Preparation for Metabolomics Exploration of Urine,Feces, Blood and Saliva in Humans Using Combined NMR and UHPLC-HRMS Platforms

Currently, most clinical studies in metabolomics only consider a single type of sample such as urine, plasma, or feces and use a single analytical platform, either NMR or MS. Although some studies have already investigated metabolomics data from multiple fluids, the information is limited to a unique analytical platform. On the other hand, clinical studies investigating the human metabolome that combine multi-analytical platforms have focused on a single biofluid. Combining data from multiple sample types for one patient using a multimodal analytical approach (NMR and MS) should extend the metabolome coverage. Pre-analytical and analytical phases are time consuming. These steps need to be improved in order to move into clinical studies that deal with a large number of patient samples. Our study describes a standard operating procedure for biological specimens (urine, blood, saliva, and feces) using multiple platforms (¹H-NMR, RP-UHPLC-MS, and HILIC-UHPLC-MS). Each sample type follows a unique sample preparation procedure for analysis on a multi-platform basis. Our method was evaluated for its robustness and was able to generate a representative metabolic map.     

Incorporation of 18Oxygen into Peptide Mixtures and Analysis with Multi‐Dimensional Chromatography and Mass‐Spectroscopy

Abstract

Enzymatic labeling with ¹⁸oxygen has the potential to become a widely applied method of isotope labeling for differential protein expression analysis by mass spectrometry because it is not amino acid specific and the reagents are cost‐effective and readily available. In this work, we investigate experimental parameters that affect efficient ¹⁸O incorporation with a model bovine serum albumin protein system and then use optimized chemistries for labeling the c‐terminus of peptides in a yeast proteome. Additionally, the role of sample handling, including the use of liquid chromatography was examined. An analytical methodology was developed which demonstrates the application of multi‐dimensional chromatography in conjunction with enzymatic labeling.