Journal of the American Society for Mass Spectrometry最新文献

To understand the mechanism of action of a drug and assess its clinical usefulness and viability, it is imperative that its affinity for its putative targets is determined. When coupled to mass spectrometry (MS), energetics-based protein separation (EBPS) techniques, such as a thermal shift assay, have shown great potential to identify the targets of a drug on a proteome scale. Nevertheless, the computational analyses assessing the confidence of drug-target predictions made by these methods have remained tightly tied to the protocol under which the data were produced. To identify drug targets in data sets produced using different EBPS-MS techniques, we have developed a novel flexible Bayesian inference approach named TargetSeeker-MS. We showed that TargetSeeker-MS identifies known and novel drug targets in Caenorhabditis elegans and HEK 293 samples treated with the fungicide benomyl. We also demonstrated that TargetSeeker-MS' drug-target identifications are reproducible in C. elegans samples that were processed using two different EBPS techniques (thermal shift assay and a differential precipitation of proteins, named DiffPOP). In addition, we validated a novel benomyl target by measuring its altered enzymatic activity upon drug treatment in vitro. TargetSeeker-MS, which is available as a web server (https://targetseeker.scripps.edu/), allows for the rapid, versatile, and confident identification of targets of a drug on a proteome scale, thereby providing a better understanding of its mechanisms and facilitating the evaluation of its clinical viability.

The interplay between the anionic phospholipid cardiolipin (CL) and cytochrome c (cyt c) holds significance in the early stages of apoptosis. Despite identification of up to four potential sites of interaction between cytochrome c and cardiolipin bearing membranes, the exact mode of interaction remains unexplained, especially given that some of the putative binding surfaces are mutually exclusive. In this study, we utilize millisecond time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry (TRESI-HDX-MS) to investigate conformational and dynamic changes in cytochrome c in the presence of various phospholipids (DMPC, POPG, and CL) incorporated into nanodiscs. We observe that, among the proposed binding sites, the adjacent "L"- and "A"-sites exhibited a decrease in deuterium exchange, while the "N" site remained unperturbed, suggesting a specific orientation of cytochrome c with respect to cell membranes upon binding. We also demonstrate that negatively charged phospholipids with physical differences (i.e., POPG and CL) exhibit essentially the same interaction with cytochrome c, supporting the utility of POPG nanodiscs as a model for cytochrome c-membrane interactions.

Sodium dodecyl sulfate (SDS) plays a pivotal role in protein denaturation, tissue extraction, and protein mass-based electrophoretic separations. However, even modest concentrations of SDS can cause column overpressure, retention time shifts, and ionization signal suppression during liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Thus, SDS removal is a critical step for LC-MS/MS analysis of protein digests containing SDS. This study describes an inexpensive and high-throughput method to remove SDS from protein digests using weak-anion exchange (WAX) resins in 96-well filter plates. Requiring less than 3 min, this method can reduce SDS concentrations from 0.1-0.4% to less than 5 ppm and from 0.6-1% to less than 100 ppm. After SDS removal, the recoveries of unmodified tryptic peptides and phosphorylated peptides (at 94.3 nM) were ∼90% and ∼70%, respectively. Additionally, when using aqueous 1% SDS to solubilize trastuzumab-spiked mouse serum and subsequently removing the SDS using the WAX resin, quantitation of trastuzumab exhibited excellent linearity (R² = 0.9996) together with a low coefficient of variation (<10%). Calculated concentrations were within 20% of the expected value for spiked standard samples (0.5, 1, and 2 μg/mL trastuzumab in mouse serum). The method is about 20× more cost-effective versus commercialized SDS removal kits and both the resin and filter plate are readily available, so the method should easily transfer to other laboratories.

Electrospray ionization (ESI) is the most widely used technique for the ionization of liquid samples, for example, from liquid chromatography (LC) coupled to mass spectrometry. Recent experiments demonstrate the penetration of charged droplets or at least large clusters into the high-vacuum region of different ESI mass spectrometers. Using a Bruker micrOTOF equipped with a standard Bruker Apollo ESI source, we demonstrated that time-of-flight (TOF) MS can detect signatures of these droplets by analyzing the statistics of individual TOF spectra, resulting from a single orthogonal acceleration (oa) stage pulse. A custom experimental setup allows additional online monitoring of the ion current in the oa-stage by coupling an oscilloscope to an auxiliary secondary electron multiplier (SEM). The results obtained with ESI are compared to mass spectra recorded under similar conditions using atmospheric pressure chemical ionization (APCI). Our findings reveal that the observation of droplet signatures is unique to the ESI process, with their frequency and intensity strongly determined by the ion source settings. We also report that the majority of the individual spectra obtained do not contain ion signals. The observed intensity in the summed spectra stems from a few very intense spectra, which result from single droplet fragment bursts. In contrast, APCI provides an almost continuous and stable ion current, without intense signal bursts characteristic for ESI. Additional optical monitoring strongly suggests that these signatures are not a result of spray instability, but are common even for undisturbed, continuous spray operation. The variation of ion source parameters shows that specific capillary voltages, nebulizer pressures, and dry gas flows lead to an increase in the frequency of droplet occurrence. Since these parameters are fundamental and frequently altered in analytical measurements, the results reported in this contribution underscore the significance of understanding droplet dynamics in ESI-MS and provide insights regarding droplets affecting the ESI signal intensity recorded in analytical runs.

Mass spectrometry contributed significantly to the creation of proteomics as a science and is now the principal analytical tool of proteomics. Although the bottom-up approach remains the most popular, more and more studies apply the top-down method for sequencing. Novel tandem mass spectrometry methods resolved numerous sequencing problems and allowed for the analysis of intact peptides and even proteins. Natural frog peptides are convenient models to develop mass spectrometry methods for de novo sequencing of intact biomolecules. Here we demonstrate the ability of ExD tandem mass spectrometry to sequence intact peptides from skin secretions of Pelophylax ridibundus from the Rostov (Russia) population. Studying that secretion, we detected an array of mono- and diformylated skin peptides at various sites of the backbone. The main goal of the study dealt with the determination of the formylation sites of these up to 46-mer long peptides. ExD reliably solved the problem without any additional mass spectrometry experiments or preliminary chemical derivatization. We propose a mechanism for the observed formylation and a biochemical and experimental rationale.

To facilitate the assessment of the internal energy distribution of ions activated by collision-induced dissociation with collision energy in the laboratory frame exceeding 20 eV, the screening of fluorine-containing benzylpyridiniums was performed by evaluating their dissociation energies through quantum chemistry calculations at the CCSD(T)/6-311++G(d,p)//M06-2X-D3/6-311++G(d,p) level of theory. The dissociation energy increased with the presence of a trifluoromethyl group at the 4-position and fluorine atoms at positions 2, 3, 5, and 6 of benzylpyridinium. However, the fluorine atom at the 4-position in benzylpyridinium slightly reduced the dissociation energy, suggesting its electron-donating nature. 4-Trifluoromethyl-2,3,5,6-tetrafluorobenzylpyridinium (CF₃,F₄-BnPy⁺) has the highest dissociation energy among the reported benzylpyridiniums and is a suitable thermometer ion for characterizing the internal energy distributions of ions activated by collision-induced dissociation (CID) during tandem mass spectrometry. Using CF₃,F₄-BnPy⁺ allowed the evaluation of the internal energy distribution of CID-activated ions up to a collision energy of 30 eV in the laboratory frame. The survival yield method using fluorine-substituted benzylpyridiniums and 4-methylbenzylpyridiniums facilitates the optimization of tandem mass spectrometry parameters for the analysis of small molecules, such as metabolites.

Profiling of steroid hormones is incredibly valuable in clinical settings for diagnosing endocrine disorders. However, the presence of matrix effects and labor-intensive manual work in LC-MS/MS analysis has hindered its routine application. In the present study, a highly efficient and automated magnetic bead extraction method was developed to address matrix effects and quantitatively profile 15 steroid hormones in human serum. Octadecyl (C18) and N-vinylpyrrolidone divinylbenzene (HLB) modified magnetic beads were compared for enriching steroids from human serum. Following enrichment, the beads were separated using a magnetic field; the matrix was cleaned, and the steroid hormones were eluted from the beads for LC-MS/MS analysis. This entire process of enrichment, cleanup, and elution was conducted automatically, making it simple, fast, and cost-effective. The results indicated that steroid hormones could be selectively enriched from human serum in just 1 min using C18 magnetic beads. The absolute matrix effect, evaluated as the relative response between human serum matrix and methanol solution, ranged from 89.2% to 113.1% for low levels, from 82.3% to 112.0% for medium levels, and from 91.7% to 111.0% for high levels. The intrabatch coefficients of variation (CVs) and interbatch CVs were between 3.1% and 13.4% and between 3.0% and 13.7%, respectively. Recoveries were between 87.6% and 114.3% for low levels, 94.0% and 105.0% for medium levels, and 91.9% and 111.7% for high levels. The clinical application was demonstrated by profiling steroid hormones in 160 pregnant women at various gestational weeks. The results suggested that the automated magnetic bead extraction method for LC-MS/MS could effectively address matrix effects in profiling steroid hormones. To our knowledge, this is the first automated magnetic bead extraction method for LC-MS/MS profiling of steroid hormones in clinical practice.

Paper spray mass spectrometry (PS-MS) often employs laser cutting to prepare paper substrates, potentially inducing localized thermal decomposition of the cellulose backbone. This work investigates how cellulose pyrolysis products and inherent background molecules within the paper affect PS-MS signal quality and evaluates paper pretreatment methods to enhance performance. Comparative analyses of laser-cut and razor-cut paper using mass spectrometry and ultraviolet-visible spectroscopy (UV-vis) showed significant differences. Laser-cut paper exhibited elevated MS blank signals and higher absorbance in the 200-400 nm UV region, indicating increased chemical abundance and complexity. Gas chromatography-mass spectrometry (GC-MS) identified over 20 residual compounds on laser-cut paper absent in razor-cut samples, half of which were identified as known cellulose pyrolysis products. Washing the paper substrates with methanol, water, or dilute nitric acid significantly reduced both pyrolysis products and background molecules, with water showing the most improvement. Analyses of morphine, fentanyl, methamphetamine, voriconazole, and fluconazole showed no reduction in the signal after washing, with fentanyl and methamphetamine exhibiting a significantly increased MS signal, regardless of the cutting method. This work reveals that while pyrolysis products from laser cutting contribute to increased chemical noise, inherent background molecules in the paper also play a significant role. A simple water wash mitigates both issues, potentially improving the overall PS-MS performance for a range of analytes.

Investigating and manipulating the ion-molecule reactions within the ionization source of ion mobility spectrometry (IMS) or mass spectrometry can contribute to developing advanced ionization sources and novel analytical techniques. In this study, a pressure-tunable photoionization tandem ion mobility spectrometer (PI-tandem-IMS) was developed to investigate the ionization suppression caused by unusual proton transfer reactions in dual-analyte systems in which high proton affinity (PA) ions are deprotonated by low PA molecules. The proton transfer reaction mechanisms in the toluene-acetone and toluene-ethanol systems were studied. The experimental results showed the linear correlation between the ln(S_X2H⁺·K_0(T⁺₎/S_T⁺·K_0(X2H⁺₎ + 1) and the square of the reactant concentration c_X², as well as the cubic power of the pressure p³. Based on this, the generation of the proton-bound dimers in the toluene-acetone and toluene-ethanol systems was assigned as a termolecular process. The reaction rate coefficients k of the toluene-acetone and toluene-ethanol systems were calculated at different temperatures, and the Arrhenius plot showed that rate coefficients were both negatively correlated with temperature, implying that elevated temperatures suppress the proton transfer reaction. At 313.15 K, the calculated k values for the toluene-acetone and the toluene-ethanol systems were 2.2 × 10^-26 cm⁶/s and 5.2 × 10^-28 cm⁶/s, respectively, suggesting a higher inhibitory effect of acetone on toluene ionization than that of ethanol. Besides, the suppressive effect of reducing the pressure or increasing the reaction region electric field strength on proton transfer reactions was shown, which demonstrated the PI-tandem IMS was a good tool for understanding ion-molecule reactions in the ionization source.

Static limit in secondary ion mass spectrometry (SIMS) is defined as a threshold beam fluence, where secondary ions are desorbed only from the virgin surface. For the common SIMS technique, the static SIMS limit is set to approximately 10¹² ions/cm². Within the present paper, we investigated the applicability of the static limit for a mass spectrometry imaging technique known as MeV-SIMS, where the target surface is bombarded by primary ions within the MeV energy range domain. Here, desorption of secondary ions relies mainly on electronic excitations instead of collision cascades, as is the case for the lower energy primary ion beams. We have measured the disappearance cross sections of several organic targets for three different chlorine primary ion beam energies. Results show how the disappearance cross section depends on the primary ion beam energy. Generally, the static SIMS regime applies for a range of primary ion beam fluences similar to that for the common SIMS technique; however, the dependence of the disappearance cross section within the lower MeV energy domain (up to 10 MeV) exhibits somewhat unexpected characteristics. Further, we thoroughly investigated the dynamics of the secondary ion mass spectra after prolonged primary ion bombardment. Secondary ion yields of various peaks were monitored during analysis, and the corresponding data can be used to identify specific peaks and also to determine fragmentation patterns of analyzed organic molecules.