Journal of the American Society for Mass Spectrometry最新文献

Algae typically respond to environmental changes by regulating the production and release of metabolites that affect water quality and cause various environmental issues. In this study, we investigated the role of algal organic matter (AOM) in copper [Cu(II)] using high-resolution mass spectrometry and a molecular-network-based nontargeted screening. The abundance and activity of algae were inhibited after the addition of Cu(II). Lipids, proteins, lignins, condensed aromatic structures, CHO-only classes, and nitrogenous organic matter are the primary components of AOM. The addition of extracellular organic matter (EOM) and intracellular organic matter (IOM) promoted the generation of carbohydrates that bonded to Cu(II), thus weakening Cu(II) toxicity. Furthermore, 1006 and 589 unique formulas were observed in the Cu(II)-EOM and Cu(II)-IOM groups, respectively, illustrating that EOM and IOM can induce algae to produce different metabolites to resist Cu(II) stress. Six novel phosphatidylethanolamines (PEs) and three novel phosphatidylglycerols (PGs) were identified in the EOM of the Cu(II)-EOM group. Therefore, AOM addition enhanced the synthesis of novel low-unsaturation and palmitoylated PEs, thereby regulating the immune response of algal cells under Cu(II) stress. Overall, these results demonstrated that Cu(II) can perturb lipid utilization and storage, whereas algae can alleviate Cu(II) toxicity by synthesizing and secreting different lipids.

The common method of mass analysis in a linear ion trap mass spectrometer is to eject ions with different mass-to-charge ratios (m/z) at different times by resonance excitation. Ions are ejected onto an electron multiplier (EM), generating pulsed signals called the ejection signal. Typically, the ejection signal consisting of multiple pulses is smoothed into a wider Gaussian-like pulse due to the limited bandwidth of the electronic system. For different scan rates and different samples, this bandwidth-limited electronic system and signal smoothing process will differentially increase the peak width and thus decrease the mass resolution. In this study, experiments at different scan rates were performed on a "Brick" miniature linear ion trap mass spectrometer. We propose a multistage linear frequency scanning mode that achieves improved mass resolution and extended mass range, in which fractional Fourier transform is applied to process the ejection signals. The proposed signal processing method improves the mass resolution and mitigates the impact of ghost peaks attributed to scattered ion ejection. A peak width of 0.84 Da at a m/z value of 242 and a scan rate of 8000 Da/s was achieved in experiments.

Native mass spectrometry (nMS) is rapidly emerging as a pivotal technique for exploring protein conformations and protein-ligand interactions. Pioneering research has demonstrated that the charge state distribution (CSD) of proteins in native mass spectra can be indicative of their solvent accessible surface area (SASA). Moreover, beyond SASA, it is postulated that the abundance of acidic and basic amino acids on the protein surface may also impact the CSD. Specifically, basic amino acids tend to acquire positive charges during electrospray ionization (ESI), whereas acidic amino acids are prone to adopting negative charges. Consequently, this study investigates the CSDs of globular proteins in both positive and negative ion modes to provide a comprehensive characterization of protein SASA. Experiments were conducted under both native ESI and native nano-ESI conditions. By harnessing the average charges observed across dual polarity nMS data, we achieved significantly enhanced log linear correlations between protein SASA and its CSDs. The coefficient of determination (R²) improved from 0.9866 to 0.9888 under ESI conditions and from 0.9677 to 0.9902 under nano-ESI conditions when compared to models utilizing only positive ion mode data. These findings suggest that the SASA of globular proteins can be effectively characterized through the CSDs derived from dual polarity nMS analysis.

Therapeutic antibodies are a class of glycoproteins that commonly carry conserved N-glycans at the Fc domain, and the attached N-glycans play a pivotal role in their biological function and efficacy. In this study, we conducted a detailed N-glycan profiling of an afucosylated monoclonal antibody using the Waters GlycoWorks RapiFluor-MS kit and hydrophilic interaction liquid chromatography coupled with fluorescence detection and mass spectrometry (HILIC-FLD-MS). We discovered and reported for the first time novel G0 glycan isomers on a monoclonal antibody. The G0 glycan has a composition of two additional N-acetylglucosamine (GlcNAc) units in addition to the core pentasaccharide structure of N-glycans. The MS/MS profile revealed few fragmentation differences for RapiFluor-MS-labeled glycan isomers. To enhance structural elucidation, a reduction and permethylation assay was performed. Reversed-phase liquid chromatography (RP-LC) separated permethylated glycans due to their altered hydrophobic properties and revealed the presence of additional isomers. The fragmentation of sodium adducts of the permethylated glycans provided distinct patterns among isomers, indicating a bisecting structure for the novel G0 glycan isomers previously identified. Since the bisecting glycans possess one GlcNAc on mannose with 1-4 linkage (bisecting GlcNAc), the other GlcNAc could occupy either branching antenna, resulting in the additional subtle positional isomers, which agrees with our observation. This study underscores the utility of permethylation coupled with advanced chromatography and mass spectrometry techniques to resolve glycan isomers and contributes to a deeper understanding of glycan structural diversity in biologic therapeutic development.

The characterization of the microstructure of in vivo degradable polyesters is gaining increased interest thanks to their high-performance applications, such as drug delivery systems. The design of such material requires a high level of understanding of the critical material attributes of the polyesters, such as molecular-weight distribution (MWD), chemical-composition distribution (CCD), and end-groups (functionality-type distribution, FTD). Size-exclusion chromatography (SEC) hyphenated with mass spectrometry (MS) is an effective method for analyzing the microstructure of polymers. While the MWD can be determined by size-exclusion chromatography hyphenated with ultraviolet spectrometry and refractive index, the CCD and FTD can be determined by SEC-MS. However, previous applications of SEC-MS have not assessed if polymer fragmentation can occur during the analysis process. In order to correctly interpret CCD and FTD, it is important to establish whether SEC-MS methods can be applied to biodegradable polymers and to recognize if fragmentation processes occur. In this study, we investigate whether SEC-MS methods can be applied to PLGA biodegradable polyesters. The research demonstrates that the choice of alkali metal salt used during ionization can influence the stability of PLGA during SEC-MS analysis. CsI was found to minimize fragmentations during ESI-MS, simplifying the MS spectra and allowing isomeric PLGA structures to be distinguished. The resulting method facilitates FTD and CCD determination. Additionally, when combined with selective degradation, the described method can provide insights into the "blockiness" of the polymer and support the development of sequence-controlled PLGA synthesis.

Matrix-assisted laser desorption/ionization mass spectrometry with laser postionization (MALDI-2 MS) has become an important technique for the analysis of a wide range of biomolecules. It has traditionally been limited to custom lab-built setups until the recent introduction of a commercial timsTOF fleX MALDI-2 system. A comprehensive optimization of the timsTOF fleX system for lipid analysis was performed using a factorial design of experiments (DOE). By examining 13 instrumental parameters across three full factorial DOEs, we performed over 1500 individual runs to assess the impact and cross interactions of these parameters on the lipid signal intensity. We found optimal values for both ion transmission and MALDI-2 parameters to maximize the signals within the lipid region. These results show that laser shot frequency, collision RF, and pre pulse storage were essential for enhancing lipid ion transmission, resulting in a nearly 5-fold increase in signal intensity compared to default parameters. For MALDI-2 optimization, positive and negative modes showed similar optimized values, with TIMS In pressure and laser power being crucial. Overall, optimization of ion optics and MALDI-2 resulted in signal enhancements of nearly 2 orders of magnitude for certain lipid species.

Unambiguous identification of drug impurities is of the utmost importance for the pharmaceutical sector. Therefore, access to analytical techniques that enable the reliable characterization of drug impurities during and after the drug development process is required. In this study, tandem mass spectrometry combined with gas-phase ion-molecule reactions followed by collision-activated dissociation (CAD) of specific ion-molecule reaction product ions is demonstrated to enable the identification of nucleophilic compounds with at least one H atom on their nucleophilic atom or a H atom on an adjacent heavy atom. Compounds with different oxygen- and/or nitrogen-containing functionalities and their combinations, such as carboxylic acids, phenols, aldehydes, hydroxylamines, amides, anilines, and sulfonamides, were tested. All analytes were protonated via atmospheric pressure chemical ionization (APCI), transferred into a linear quadrupole ion trap, isolated, and allowed to react with (isopropenyloxy)trimethylsilane (ITS). All protonated compounds studied, including the ones mentioned above as well as many others, react with ITS to form an addition product that has eliminated an acetone molecule. Subjecting these product ions to CAD generated diagnostic fragment ions via the loss of methane for most of the compounds with at least one H atom on their nucleophilic atom or a H atom on an adjacent heavy atom. Only three exceptions were identified. Quantum chemical calculations were employed to delineate the likely mechanisms for the formation of the relevant product ions upon ion-molecule reactions and their fragmentation via elimination of methane.

Mass spectrometry (MS) is a tool of choice for the in-depth characterization of new biotherapeutic molecules such as a complex naturally derived trispecific antibody (tsAb) that presents a tyrosine sulfation within the variable domain. Although tyrosine sulfation is an important post-translational modification responsible for strengthening protein-protein interactions, its localization is challenging, as the sulfate group is very labile using conventional positive ion mode fragmentation techniques. In this work, we describe the combination of functional testing and MS-based methods to study the impact of tyrosine sulfation in the tsAb. The presence of sulfation was confirmed by intact mass and peptide mapping analyses. For unambiguous localization of the sulfate group, electron activated dissociation (EAD) MS/MS experiments were employed to generate diagnostic fragments carrying an intact sulfate group. We also demonstrated that a significant decrease in binding of the tsAb to the target antigen was observed following the sulfatase treatment. Taken together, the results from this study support the notion that tyrosine sulfation plays an important role in protein-protein interactions.

The dual-cleavable nature of the cross-linking technology (DUCCT) enhances the reliable identification of cross-linked peptides via mass spectrometry. The DUCCT approach uses a cross-linking agent that can be selectively cleaved by two different tandem mass spectrometry techniques: collision-induced dissociation (CID) and electron transfer dissociation (ETD). This results in distinct signatures in two independent mass spectra for the same cross-linked precursor, leading to unambiguous identification and the validation of the spectra. In this study, we expanded the application of the DUCCT cross-linker to evaluate the binding domains of a specific cat dander allergen, Fel d 1, which exists as the Fel d 1 A and B protein complex, and a viral spike protein from SARS-CoV-2, which invades host cells. To assess the cross-linked products obtained by DUCCT, we utilized a software tool called Cleave-XL, which effectively identified cross-linked sites using data from CID and ETD. Dual cleavable cross-linking studies identified cross-linked peptides in these complexes, which have been reported in bioinformatics analysis and proposed for immunotherapy using synthetic peptides. A benchmark study was also conducted using a commercial cross-linker disuccinimidyl suberate (DSS). Overall, we expect that DUCCT cross-linking technology will greatly facilitate the rapid screening of binding interfaces, thereby advancing structural biology and cell signaling investigations.

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.