Use of Polyetheretherketone as a Material for Solid Phase Extraction of Hydroxylated Metabolites of Polycyclic Aromatic Hydrocarbons in Human Urine

In this study, a novel application of polyetheretherketone (PEEK) tubing for solid phase extraction (SPE) of urinary hydroxylated metabolites of polycyclic aromatic hydrocarbons (OH-PAHs) is presented. The use of PEEK tubing for extracting nine OH-PAHs (2–5 rings) from different matrixes (e.g., urine, acid/enzymatic hydrolysis solution) was demonstrated; a facile method for fast (<2 min) quantification of urinary 1-hydroxypyrene (1-OHPyr) was also developed by the use of PEEK tubing extraction coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS). Although no optimization was performed for the extraction process, a limit of detection (LOD) as low as 0.01 μg L^–1 was obtained, and the ratio of signal intensity of 1-OHPyr to that of 1-OHPyr-d9 (internal standard) was linearly related with the 1-OHPyr concentration over the range of 0.05–5.00 μg L^–1 (<i>R</i>² = 0.9995). Satisfactory recoveries (87–91%) were achieved, and less than 2 min was required to carry out the whole analytical procedure including sample pretreatment and mass spectrometric detection. In a biomonitoring study, the PEEK tubing extraction based method was successfully applied to the quantification of 1-OHPyr in eight human urine samples, further confirming the potential of PEEK tubing for SPE of organic compounds

Influence of Dimehylsulfoxide on Protein–Ligand Binding Affinities

Because of its favorable physicochemical properties, DMSO is the standard solvent for sample storage and handling of compounds in drug discovery. To date, little attention was given to how DMSO influences protein–ligand binding strengths. In this study we investigated the effects of DMSO on different noncovalent protein–ligand complexes, in particular in terms of the binding affinities, which we determined using nanoESI-MS. For the investigation, three different protein–ligand complexes were chosen: trypsin–Pefabloc, lysozyme–tri-<i>N</i>-acetylchitotriose (NAG₃), and carbonic anhydrase–chlorothiazide. The DMSO content in the nanoESI buffer was increased systematically from 0.5 to 8%. For all three model systems, it was shown that the binding affinity decreases upon addition of DMSO. Even 0.5–1% DMSO alters the <i>K</i>_D values, in particular for the tight binding system carbonic anhydrase–chlorothiazide. The determined dissociation constant (<i>K</i>_D) is up to 10 times higher than for a DMSO-free sample in the case of carbonic anhydrase–chlorothiazide binding. For the trypsin–Pefabloc and lysozyme–NAG₃ complexes, the dissociation constants are 7 and 3 times larger, respectively, in the presence of DMSO. This work emphasizes the importance of effects of DMSO as a co-solvent for quantification of protein–ligand binding strengths in the early stages of drug discovery

Solid-Phase Microextraction Coupled to Capillary Atmospheric Pressure Photoionization-Mass Spectrometry for Direct Analysis of Polar and Nonpolar Compounds

A novel capillary ionization source based on atmospheric pressure photoionization (cAPPI) was developed and used for the direct interfacing between solid-phase microextraction (SPME) and mass spectrometry (MS). The efficiency of the source was evaluated for direct and dopant-assisted photoionization, analyzing both polar (e.g., triazines and organophosphorus pesticides) and nonpolar (polycyclic aromatic hydrocarbons, PAHs) compounds. The results show that the range of compound polarity, which can be addressed by direct SPME-MS can be substantially extended by using cAPPI, compared to other sensitive techniques like direct analysis in real time (DART) and dielectric barrier discharge ionization (DBDI). The new source delivers a very high sensitivity, down to sub parts-per-trillion (ppt), making it a viable alternative when compared to previously reported and less comprehensive direct approaches

Gas-Phase Protonation Thermochemistry of Adenosine

The goal of this work was to obtain a detailed insight on the gas-phase protonation energetic of adenosine using both mass spectrometric experiments and quantum chemical calculations. The experimental approach used the extended kinetic method with nanoelectrospray ionization and collision-induced dissociation tandem mass spectrometry. This method provides experimental values for proton affinity, PA(adenosine) = 979 ± 1 kJ·mol−1, and for the “protonation entropy”, ΔpS°(adenosine) = S°(adenosineH+) − S°(adenosine) = −5 ± 5 J·mol−1·K−1. The corresponding gas-phase basicity is consequently equal to: GB(adenosine) = 945 ± 2 kJ·mol−1 at 298K. Theoretical calculations conducted at the B3LYP/6-311+G(3df,2p)//B3LYP/6-31+G(d,p) level, including 298 K enthalpy correction, predict a proton affinity value of 974 kJ·mol−1 after consideration of isodesmic proton transfer reactions with pyridine as the reference base. Moreover, computations clearly showed that N3 is the most favorable protonation site for adenosine, due to a strong internal hydrogen bond involving the hydroxyl group at the 2′ position of the ribose sugar moiety, unlike observations for adenine and 2′-deoxyadenosine, where protonation occurs on N1. The existence of negligible protonation entropy is confirmed by calculations (theoretical ΔpS°(adenosine) ∼ −2/−3 J·mol−1·K−1) including conformational analysis and entropy of hindered rotations. Thus, the calculated protonation thermochemical properties are in good agreement with our experimental measurements. It may be noted that the new PA value is ∼10 kJ·mol−1 lower than the one reported in the National Institute of Standards and Technology (NIST) database, thus pointing to a correction of the tabulated protonation thermochemistry of adenosine

Rapid Profiling of the Glycosylation Effects on the Binding of SARS-CoV‑2 Spike Protein to Angiotensin-Converting Enzyme 2 Using MALDI-MS with High Mass Detection

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 binds directly to angiotensin-converting enzyme 2 (ACE2), mediating the host cell entry of SARS-CoV-2. Both spike protein and ACE2 are highly glycosylated, which can regulate the binding. Here, we utilized high-mass MALDI-MS with chemical cross-linking for profiling the glycosylation effects on the binding between RBD and ACE2. Overall, it was found that ACE2 glycosylation affects the binding more strongly than does RBD glycosylation. The binding affinity was improved after desialylation or partial deglycosylation (N690) of ACE2, while it decreased after degalactosylation. ACE2 can form dimers in solution, which bind more tightly to the RBD than the ACE2 monomers. The ACE2 dimerization and the binding of RBD to dimeric ACE2 can also be improved by the desialylation or deglycosylation of ACE2. Partial deglycosylation of ACE2 increased the dimerization of ACE2 and the binding affinity of RBD and ACE2 by more than a factor of 2, suggesting its high potential for neutralizing SARS-CoV-2. The method described in the work provided a simple way to analyze the protein–protein interaction without sample purification. It can be widely used for rapid profiling of glycosylation effects on protein–protein interaction for glycosylation-related diseases and the study of multiple interactions between protein and protein aggregates in a single system

Gas-Phase Protonation Thermochemistry of Adenosine

The goal of this work was to obtain a detailed insight on the gas-phase protonation energetic of adenosine using both mass spectrometric experiments and quantum chemical calculations. The experimental approach used the extended kinetic method with nanoelectrospray ionization and collision-induced dissociation tandem mass spectrometry. This method provides experimental values for proton affinity, PA(adenosine) = 979 ± 1 kJ·mol−1, and for the “protonation entropy”, ΔpS°(adenosine) = S°(adenosineH+) − S°(adenosine) = −5 ± 5 J·mol−1·K−1. The corresponding gas-phase basicity is consequently equal to: GB(adenosine) = 945 ± 2 kJ·mol−1 at 298K. Theoretical calculations conducted at the B3LYP/6-311+G(3df,2p)//B3LYP/6-31+G(d,p) level, including 298 K enthalpy correction, predict a proton affinity value of 974 kJ·mol−1 after consideration of isodesmic proton transfer reactions with pyridine as the reference base. Moreover, computations clearly showed that N3 is the most favorable protonation site for adenosine, due to a strong internal hydrogen bond involving the hydroxyl group at the 2′ position of the ribose sugar moiety, unlike observations for adenine and 2′-deoxyadenosine, where protonation occurs on N1. The existence of negligible protonation entropy is confirmed by calculations (theoretical ΔpS°(adenosine) ∼ −2/−3 J·mol−1·K−1) including conformational analysis and entropy of hindered rotations. Thus, the calculated protonation thermochemical properties are in good agreement with our experimental measurements. It may be noted that the new PA value is ∼10 kJ·mol−1 lower than the one reported in the National Institute of Standards and Technology (NIST) database, thus pointing to a correction of the tabulated protonation thermochemistry of adenosine

Gas-Phase Protonation Thermochemistry of Adenosine

The goal of this work was to obtain a detailed insight on the gas-phase protonation energetic of adenosine using both mass spectrometric experiments and quantum chemical calculations. The experimental approach used the extended kinetic method with nanoelectrospray ionization and collision-induced dissociation tandem mass spectrometry. This method provides experimental values for proton affinity, PA(adenosine) = 979 ± 1 kJ·mol−1, and for the “protonation entropy”, ΔpS°(adenosine) = S°(adenosineH+) − S°(adenosine) = −5 ± 5 J·mol−1·K−1. The corresponding gas-phase basicity is consequently equal to: GB(adenosine) = 945 ± 2 kJ·mol−1 at 298K. Theoretical calculations conducted at the B3LYP/6-311+G(3df,2p)//B3LYP/6-31+G(d,p) level, including 298 K enthalpy correction, predict a proton affinity value of 974 kJ·mol−1 after consideration of isodesmic proton transfer reactions with pyridine as the reference base. Moreover, computations clearly showed that N3 is the most favorable protonation site for adenosine, due to a strong internal hydrogen bond involving the hydroxyl group at the 2′ position of the ribose sugar moiety, unlike observations for adenine and 2′-deoxyadenosine, where protonation occurs on N1. The existence of negligible protonation entropy is confirmed by calculations (theoretical ΔpS°(adenosine) ∼ −2/−3 J·mol−1·K−1) including conformational analysis and entropy of hindered rotations. Thus, the calculated protonation thermochemical properties are in good agreement with our experimental measurements. It may be noted that the new PA value is ∼10 kJ·mol−1 lower than the one reported in the National Institute of Standards and Technology (NIST) database, thus pointing to a correction of the tabulated protonation thermochemistry of adenosine

Elucidating the Role of Ion Suppression in Secondary Electrospray Ionization

Ion suppression is a known matrix effect in electrospray ionization (ESI), ambient pressure chemical ionization (APCI), and desorption electrospray ionization (DESI), but its characterization in secondary electrospray ionization (SESI) is lacking. A thorough understanding of this effect is crucial for quantitative applications of SESI, such as breath analysis. In this study, gas standards were generated by using an evaporation-based system to assess the susceptibility and suppression potential of acetone, deuterated acetone, deuterated acetic acid, and pyridine. Gas-phase effects were found to dominate ion suppression, with pyridine exhibiting the most significant suppressive effect, which is potentially linked to its gas-phase basicity. The impact of increased acetone levels on the volatiles from exhaled breath condensate was also examined. In humid conditions, a noticeable decrease in intensity of approximately 30% was observed for several features at an acetone concentration of 1 ppm. Considering that this concentration is expected for breath analysis, it becomes crucial to account for this effect when SESI is utilized to quantitatively determine specific compounds

Rapid and Precise Measurements of Gas-Phase Basicity of Peptides and Proteins at Atmospheric Pressure by Electrosonic Spray Ionization-Mass Spectrometry

Deprotonation reactions of peptide and protein ions have been studied by introducing volatile reference bases at atmospheric pressure between an electrosonic spray ionization (ESSI) source and the inlet of a hybrid quadrupole time-of-flight mass spectrometer. This new setup offers the unique possibility to measure the apparent gas-phase basicity GBapp of multiply charged ions by a bracketing approach. A very good agreement has been obtained with reference values obtained by Fourier transform-ion cyclotronic resonance (FT-ICR), validating our approach. The measurements were then extended to larger biomolecules such as insulin and myoglobin in native and denaturing buffers. The main advantages of this methodology are measurements at atmospheric pressure with good sensitivity (for concentrations less than 10 μM in denaturing or nondenaturing buffer), very good precision (less than 2%), and in a short time (less than 30 min to screen up to 23 volatile reference bases)