Journal of the American Society for Mass Spectrometry最新文献

Oxazolochlorins are synthetic porphyrinoids that are distinguished from regular tetrapyrrolic pigments by the presence of an oxazoline moiety in place of a pyrrole. They are amenable to electrospray ionization mass spectrometric analysis. Ionization can take place via protonation, the loss of an alkoxy side chain forming an α-oxacarbocation, or in the case of oxazolochlorinato metal complexes by (formal) oxidation of the metal center. Structural aspects of the ions were studied by using cryogenic ion vibrational spectroscopy. Comparison of the vibrational band patterns generated using density-functional theory calculations with the experimental spectra indicates that it is possible to distinguish specific protonation sites as well as oxazolochlorin constitutional macrocycle isomers. Information is also obtained regarding the nature of the alkoxy side chains on the protonated oxazolochlorins. Lastly, we explore how centrally coordinated metal ions (Ni²⁺ and Ag²⁺, both square planar coordinated divalent metals) influence the vibrational band patterns displayed by the macrocycles. This study provides a proof of concept that cryogenic infrared ion spectroscopy is suitable to expand our structural understanding of the ions formed by porphyrinoids under electrospray conditions.

Complex carbohydrates, such as N-linked glycans, are highly important biomolecules with roles ranging from signaling to recognition and immune response. Ion mobility spectrometry–mass spectrometry (IMS-MS) has emerged as a rapid and orthogonal analytical technique to condensed-phase separations for studying carbohydrates, but many challenges exist in their analyses with IMS-MS due to their isomeric and conformational heterogeneity. Specifically, glycan IMS-MS separations often display more peaks than what can be predicted based on structure and/or much broader than expected peaks presumably from their metal-adducted conformers. This has precluded IMS-MS from being routinely used to analyze complex glycans largely because of the reduction in overall peak capacity and thus difficulty in deconvolving mixtures. In this work, we present a traveling wave-based ion heating strategy that uses activating traveling wave conditions. We demonstrated that this ion heating approach can improve peak capacity for individual glycan species as well as for those in mixtures. Importantly, we did not observe any significant loss in sensitivity and comparable resolution to glycans analyzed at gentle traveling wave conditions. Additionally, we demonstrated that isomeric glycans could be repeatedly cycled resulting in scalable resolution without significant ion losses. Overall, our approach can be broadly implemented on any traveling wave-based IMS-MS platform, and we envision utility toward other molecular classes desiring improved IMS-MS peak capacities.

Investigations of protein function and interactions with native mass spectrometry (MS) have yielded significant insights into protein dynamics, transient reaction intermediates, and pharmacokinetic targets. The pursuit of these studies and their outcomes depend on the preparation of protein samples in a manner able to support native conformation, active site chemistry, and protein–ligand interactions. Although ammonium acetate is a commonly utilized volatile buffer in MS-based analyses, the gap in buffer capacity near physiological pH calls into question whether this or other volatile buffer solutions are able to facilitate native conformation and protein–ligand interactions in the gas phase. We report enzymatic activity of the cysteine desulfurase IscS in four volatile buffer solutions comparable to that observed in traditionally utilized buffers such as Tris and HEPES, which is heavily influenced by buffer contributions to protein conformation and stability. We present a dual analysis of MS charge state and enzyme kinetics in the context of protein and solution physical properties, providing a chemical justification for the positive and negative effects of specific buffers. Ultimately, these results demonstrate how native MS technology can be used to identify protein conformational and dynamic interactions modulated by buffer systems to guide mechanistic studies.

Antibody–drug conjugates (ADCs) represent a rapidly evolving class of potent biopharmaceuticals, combining the selective targeting of monoclonal antibodies with the cytotoxic power of small-molecule drugs. These targeted cancer therapies are reliant on cleavable linkers, often peptide-based, to connect cytotoxic drugs to monoclonal antibodies. Cathepsin-sensitive dipeptide linkers such as Valine–Citrulline are commonly used in current therapeutic ADCs. These linkers are designed for controlled drug release by tumor-associated enzymes like cathepsin B. Critically, the stereochemical configuration of components, such as citrulline, within these linkers dictates their enzymatic cleavability and biological activity. For instance, L-citrulline can be cleaved by cathepsin B, while D-citrulline is not. High chiral purity ensures that the linker is recognized and cleaved efficiently by specific enzymes, enhancing the effectiveness of the drug-delivery system. Beyond the linker, the stereoisomerism of the drug payload itself (in the case of a chiral drug payload) also profoundly impacts ADC efficacy and safety. Ion mobility–mass spectrometry (IM–MS) is an emerging molecular characterization technique that offers a powerful orthogonal dimension of separation by differentiating ions based on their size, shape, and charge, prior to MS analysis. This technique provides collision cross-section (CCS) measurements, which are invaluable for resolving isobaric and isomeric compounds that cannot be differentiated by mass alone. In this work, traveling wave IM spectrometry (TWIMS) enabled by the structures for lossless ion manipulation (SLIM) architecture was evaluated for the separation of isobaric epimers of a cleavable Val–Cit drug linker (DL). SLIM coupled with quadrupole time-of-flight (qTOF) MS was used to separate not only the epimers around the cleavable citrulline center of the DL (compounds A and B) but also the coeluting epimers from the other chiral centers of the drug payload (compounds C and D). A novel aspect of this approach lies in its ability to selectively exploit different adducts of the same charge state to enable the separation of distinct stereoisomeric or isomeric pairs, which are otherwise difficult to separate. Separation of the epimers was optimized by varying the traveling wave frequency, amplitude, and gas pressure in the SLIM chamber. Using this novel approach, citrulline-containing DL’s, compounds A–D, were all baseline resolved with a single set of TWIMS parameters. This work underscores the critical role of high-resolution SLIM-IM–MS in resolving subtle structural differences in chiral ADC components, providing essential insights into their development and quality control.

Ion–molecule reactions at near-ambient temperatures provide a controlled means to study gas-phase interactions that underpin fundamental chemical processes, such as binding, molecular recognition, and reactivity. Here, we demonstrate that such experiments can be performed on a commercially manufactured quadrupole time-of-flight mass spectrometer (Q-ToF MS) by electronically reconfiguring the collision cell into a stacked-ring ion trap (SRIT), without mechanical modification. Ten different test ions were successfully stored, including singly charged ions with m/z values from 59 to 556, each demonstrating stable confinement for at least 2 s. The 7+ charge state of lysozyme formed from a neutral aqueous solution (m/z 1788) was also stably confined, highlighting the wide mass range over which reliable trapping is achieved. A proton-transfer reaction was also performed in SRIT using isolated cytochrome c 16+ (m/z 773). The resulting product ions, including cytochrome c 15+ (m/z 825) and protonated dimethylacetamide (m/z 88), were simultaneously observed. Unlike when using a linear ion trap mass spectrometer, both the high-m/z and low-m/z products of the ion–molecule reaction can be detected directly from a single isolated precursor ion. To assess collisional heating, the para-methoxybenzylammonium ion, which has a labile C–N bond with a dissociation energy of 105.8 kJ mol^–1, was stored for 2 s, and no detectable fragmentation was observed. Ion temperatures remained close to room temperature and increased only slightly from 292.2  ±  3.2 K to 303.2  ±  3.5 K as the RF peak-to-peak amplitude was raised from 17 to 75 V, corresponding to a typical range required for effective trapping across different m/z values under these conditions. Repurposing the collision cell into a SRIT provides a straightforward approach for studying spontaneous ion–molecule reactions under near-ambient conditions on commercial Q-ToF MS instruments. It is anticipated to be useful for fundamental investigations of gas-phase ion chemistry and for analytical applications, such as selective ion–molecule tagging, structural isomer discrimination, and diagnostic adduct formation.

Characterization of high-molecular-weight species (HMWS) that are formed under forced stress conditions is a key component of degradation pathway studies performed during late-stage development for monoclonal antibody (mAb) therapeutics. Native mass spectrometry (MS) is a powerful technique for probing the structure and composition of HMWS. It offers high mass accuracy (surpassing that of the gold-standard SEC-MALS analysis), good sensitivity, and specificity. However, the routine implementation of this method in biopharmaceutical industry laboratories has been hindered by the special considerations required for data acquisition and interpretation. In this study, we present a systematic evaluation and the development of a platform method for native SEC-MS characterization of large aggregates in stressed mAb samples. Our approach utilizes conventional quadrupole time-of-flight mass spectrometry without hardware modifications to general-purpose instruments, making it widely accessible. We applied this platform to characterize aggregates in a model IgG4 antibody subjected to thermal and low-pH stress conditions. By implementing postcolumn denaturation (PCD), we successfully identified the nature of the formed aggregates, distinguishing between covalent and noncovalent species. Our work provides a practical guideline for incorporating native SEC-MS methods into degradation pathway study workflows, offering a robust and versatile tool for HMWS characterization. This method enhances our understanding of stress-induced antibody aggregation, potentially improving the development of monoclonal antibody therapeutics.

A common issue in pulsed ambient ionization sources is the presence of background ions, which can cause ionization suppression and negatively influence downstream statistical analysis. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is an ionization technique commonly used for mass spectrometry imaging and high throughput screening, both of which highly benefit from reduced background signals. An electrospray diverter was developed to act as an ion shutter, preventing ions from entering the mass spectrometer when a sample related signal was not present. After timing optimization for both liquid based screening and tissue imaging, reductions in background signal up to 4-fold were observed, as well as increases in sample related signal up to 11-fold. In all cases, a relative increase in the sample related signal was observed, and no negative impacts on sample related data were detected.

Fourier Transform is a low-cost method for improving duty cycle, resolving power, and signal-to-noise ratio in the Ion Mobility Spectrometry (IMS) experiment in a 2-gate IMS cell. By simultaneously pulsing both gates through a frequency sweep, the resulting data may be deconvoluted into a time-based mobility spectrum through a Fast Fourier Transform (FT). However, inconsistencies common in low-cost function generators result in spectral artifacts. In this work, an asynchronous stepped frequency FTIMS method is demonstrated, which uses a simple, software timed pulse generator compatible with any modern Analog-Digital Converter (ADC) system. By unlinking the frequency initiation and data collection using a long rise-time amplifier circuit, a stand-alone FTIMS with Faraday Plate detection was characterized in both single gate and FT modes of operation using the same IMS cell. Asynchronous stepped FTIMS parameters were investigated for system optimization with respect to resolving power, signal-to-noise ratio, and experimental time. Once optimized, asynchronous FTIMS demonstrated significant improvements in resolving power and signal-to-noise ratios without a significant increase in experimental time. By unlinking the frequency generation and data analysis, a simple Python script was demonstrated using a variety of commercially available ADC systems ranging in cost from several thousand to several hundred dollars (USD) without sacrificing spectral fidelity. A custom circuit was developed to allow a Raspberry Pi 4 Single Board Computer (SBC) to function as the data acquisition and control (DAC) interface for a low-cost stand-alone FTIMS solution.

Engineered monoclonal antibodies have proven themselves as invaluable biotherapeutics used in clinics worldwide. In discovery, development, and production, it is essential that they are accurately validated to ensure their homogeneity, consistency, safety, and effectiveness, which are commonly conducted via mass spectrometric analysis. However, validation processes can be manual, time-consuming, and costly. One hindrance in the analytical workflow is the relatively long time required to deglycosylate native mAbs with PNGase F on the benchtop, which is usually done to reduce the spectral complexity, increase the ionization efficiency, and facilitate the identification of glycosylation sites. To circumvent this obstacle, a workflow on the SampleStream Platform was developed and optimized to automate in-channel PNGase F-mediated deglycosylation of native monoclonal antibody at unprecedented speed, accomplishing 86% deglycosylation in 3 min. The presented workflow offers a promising strategy to reduce discovery and development costs and streamline the characterization of antibody-based biopharmaceuticals.

The protonation site of a molecule can significantly influence its gas-phase behavior and fragmentation, especially when multiple protonation sites are accessible. Here, we characterize two gas-phase protonation site isomers (hereafter referred to as protomers) of quizartinib using cyclic ion mobility–mass spectrometry (cIMS-MS), tandem MS, and molecular modeling. Despite density functional theory (DFT) calculations indicating a gas-phase preference for protonation at the central imidazole nitrogen (hereafter N21), two mobility-separated species were observed, suggesting kinetic trapping of a solution-phase protomer. To probe this hypothesis, solvent-phase molecular modeling using implicit water and acetonitrile models was performed, revealing that the morpholine nitrogen (hereafter N15) is the most favorable protonation site in solution. This supports a dual-phase model: one protomer arises from the liquid-phase favored protonation site, and the other from the gas-phase protonation site. Post-IMS fragmentation of the protomers revealed a common m/z 421 product ion, along with other shared fragments at m/z 395 and m/z 114. Product ion m/z 308 was unique to protomer 1, while m/z 334, m/z 307, m/z 281,276 and m/z 87 were unique to protomer 2. To investigate the origins of m/z 421 and m/z 395, these ions were generated by pre-IMS activation, isolated, and subjected to cIMS separation. Both ions exhibited two distinct arrival time peaks, indicating that they retain the protonation-site memory of their precursors. The two mobility-separated m/z 421 ions further yielded unique as well as some common fragments upon dissociation. We propose a charge remote hydrogen transfer mechanism for formation of m/z 421 and m/z 395, initiated from either the morpholine (N15) or imidazole (N21) protonation. Structural assignments were proposed for the major common and unique product ions of each protomer. These findings highlight a mechanistic link between solution- and gas-phase protonation and demonstrate the utility of cIMS-MS for probing structure-specific fragmentation and isobaric dissociation product ion resolution in small molecules with multiple heteroatoms.