Journal of the American Society for Mass Spectrometry最新文献

The Omnitrap-Orbitrap-Booster (OOB) mass spectrometry (MS) platform was developed to advance the top-down (TD) MS analysis of proteins. It integrates a multimodal tandem mass spectrometry (MS/MS) ion trap system (Omnitrap), a high-resolution Orbitrap Fourier transform mass spectrometer (FTMS), and a high-performance data acquisition system (FTMS Booster) to improve fragmentation efficiency and spectral quality by increasing the signal-to-noise (S/N) ratio of product ions. In this study, we evaluate the OOB platform for the electron capture dissociation (ECD)-based TD MS analysis of a P15 multiple myeloma antibody light chain. Single precursor charge state analysis of P15 23+ yielded relatively high sequence coverage of 68%, albeit indicating a limitation caused by the overlap of certain product ions with the charge reduced precursors. The corresponding method development, leveraging consecutive analysis of multiple precursor charge states (15+ to 19+) across triplicate LC-MS/MS runs on the OOB platform, enhanced P15 sequence coverage to 93%, demonstrating its capacity for comprehensive protein characterization. In addition, we demonstrate that the obtained ECD-based TD MS performance on the OOB platform for P15 light chain is comparable to the “gold-standard” electron transfer/higher-energy collision dissociation (EThcD)-based TD MS on an Orbitrap Eclipse. Serendipitously, ECD exhibits a lower spectral peak density (i.e., reduced spectral congestion) due to reduced redundancy of product ions. These results establish the OOB platform as a powerful and efficient tool for TD MS of proteins.

Glycosylation is the most common protein post-translational modification, affecting protein properties and functions. Abnormal variations in glycosylation are associated with diseases, e.g., coronavirus disease COVID-19. Matrix-assisted laser desorption/ionization mass spectrometry has been widely utilized for studying protein glycosylation, after proper purification of glycopeptides or glycans using hydrophilic interaction liquid chromatography (HILIC) or laboratory-synthesized hydrophilic materials. Here, glass wool tips were developed to enrich immunoglobulin G glycopeptides and applied in the analysis of COVID-19 patient samples as a proof-of-concept. A significant decrease in galactosylation was detected in the COVID-19 patient plasma sample compared to the reference sample. The tips developed in this work provided a cheap and simple enrichment alternative to commercial HILIC tips for studying protein glycosylation.

Post-translational modifications (PTMs) within the Complementarity-Determining Regions (CDRs) of monoclonal antibodies (mAbs) can impact their target-binding capabilities, making them potential critical quality attributes (CQAs) during therapeutic mAb development. Conventional methods for assessing PTM criticality often involve variant enrichment followed by target-binding testing, which face limitations in throughput and complexity. To address these challenges, affinity enrichment-based strategies have emerged, offering a valuable alternative for PTM assessment. Notably, the combination of competitive binding, size exclusion chromatography (SEC) separation, and MS detection, has proven highly effective in assessing the criticality of PTMs in a multiplexed fashion. Recently, we introduced a new technique termed affinity-resolved SEC-MS, which employs SEC to separate free and target-bound mAbs, followed by postcolumn denaturation (PCD)-assisted intact mass measurements. While highly effective in interrogating PTMs associated with significant mass shifts, this technique is less suitable for studying PTMs with subtle mass changes, such as asparagine (Asn) deamidation and aspartic acid (Asp) isomerization - two prevalent and often critical PTMs found in therapeutic mAbs. To overcome this limitation, we combined affinity-resolved SEC separation with online strong cation exchange chromatography-MS (SCX-MS) analysis. This 2D-LC-MS approach leverages the excellent selectivity of SCX separation for mAb CDR modifications at the Fab fragment level, enabling effective evaluation of Asn deamidation and Asp isomerization with site-specific resolution. The utility of this new approach was demonstrated through two case studies: examining a single Asn deamidation in an in-house mAb and assessing multiple site-specific Asn deamidation and Asp isomerization in trastuzumab, all occurring within the CDRs. Additionally, we detailed a quantitative approach to estimate the relative fold change in dissociation constant (K_D) for antigen–antibody interactions resulting from each individual CDR modification.

Neurotransmitters are critical for the proper function, signal transmission, and physiological balance of the brain, with γ-aminobutyric acid (GABA) being the main inhibitory neurotransmitter in the central nervous system. GABA is present at relatively low concentrations compared to other neurotransmitters, therefore requiring sensitive analytical methods for accurate identification and quantitation. Described herein is a rapid and facile liquid chromatography mass spectrometry (LC-MS)-based chemical derivatization method to enhance the detection of GABA, demonstrated in both saline culture media and Carassius auratus (goldfish) retina samples. We have expanded the use of trimethylation enhancement using diazomethane (TrEnDi) to permethylate GABA ([GABA^Tr]⁺) at 98–100% yields across all matrix types. Quantitative methylation of the carboxylic acid and amino moieties nullifies any zwitterionic character and fixes a permanent positive charge on [GABA^Tr]⁺, leading to MS sensitivity enhancement. In biological triplicates of goldfish retina samples, [GABA^Tr]⁺ boasted 6.3–27.9-fold increases in MS sensitivity compared to its unmodified counterpart, enabling quantitation with concentrations ranging between 78.6 and 806.5 nM. Calibration curve linearity for [GABA^Tr]⁺ and unmodified GABA was R² = 0.9996 and R² = 0.9923, respectively. Limits of detection and quantitation (LOD/LOQ) for [GABA^Tr]⁺ were 0.053 nM (1.1 fmol)/0.18 nM (3.6 fmol), compared to 2.5 nM (50 fmol)/8.3 nM (167 fmol) for unmodified GABA. This work demonstrates that TrEnDi has the ability to rapidly enhance LC-MS detection of GABA in a relatively facile manner, reducing the probability of reporting false negatives in the analysis of complex biological samples.

Methimazole (MMI), which is primarily known as an antithyroid drug, has received considerable attention for its potent antioxidant properties in a copper(I)-mediated Fenton reaction. However, the antioxidant mechanistic details of MMI, particularly its structural analogy to the natural antioxidant ergothioneine (both of which feature an imidazole-2-thione moiety), remain largely unexplored due to the difficulty of in situ characterizing unstable reaction intermediates. In this study, we combined an in situ reactor-integrated paper-in-tip spray ionization source with mass spectrometry (PTSI-MS) to probe the protection mechanisms of MMI against the Cu^I-catalyzed Fenton reaction online. We present direct MS evidence of the antioxidant mechanisms of MMI (M) in its reduced form (MSH) and oxidized disulfide form (MSSM). The former can directly sequester H₂O₂ (i.e., a H₂O₂ scavenging mechanism). The latter tightly coordinates with Cu^I to form a Cu^I-MSSM complex that stabilizes Cu^I and prevents its oxidation by H₂O₂ and thus the formation of ^•OH (i.e., a metal ion coordination mechanism). The proposed reaction pathway of the Cu^I complexes from MMI disulfide (Cu^I-MSSM, m/z = 289 and 291) to MMI thiosulfonate (Cu^I-MSO₂SM, m/z = 321 and 323) to MMI monosulfide (Cu^I-MSM, m/z 257 and 259) was verified by S–S oxidation, SO₂–S bond cleavage, and extruded sulfur elimination, which confirmed the stable N,N′-bidentate binding of Cu^I to these intermediates, even when attacked by H₂O₂. These findings challenge the traditional radical scavenging mechanism and contribute to our understanding of the relationship between the structure and antioxidant activity of imidazole-2-thiones. Furthermore, the established in situ reactor-integrated PTSI-MS approach provides unique insights into reaction dynamics, offering the advantages of simplicity, speed, and low reaction volume (down to 20 μL).

Ion mobility separates ion in the gas phase based on rotationally averaged cross section, a parameter often correlated with size, providing a versatile measurement strategy when integrated with mass spectrometry. The rapid growth in the field of ion mobility mass spectrometry has been catalyzed by numerous innovative advances in instrumentation that have improved resolution, sensitivity, and the ability to measure collision cross sections. These advances in ion mobility instrumentation and methods have been translated into many applications in the fields of metabolomics, lipidomics, proteomics, and structural biology. This Perspective focuses on developments in ion mobility instrumentation, spanning the impressive capabilities of commercial platforms to customized designs and modifications that establish new benchmarks at the frontiers of ion mobility mass spectrometry.

The effectiveness of collision-induced dissociation (CID) in ion trap mass spectrometry (ITMS) is limited by a low-mass cutoff and weak fragmentation yields. Theoretically, the q value is optimized to balance the fractional product ion mass range with adequate energy deposition to improve fragment ion detection in the CID process; however, many promising technologies still depend on the traditional sinusoidal waveform-driven IT. Additionally, traditional CID-based multistage mass spectrometry (MSⁿ) experiments on ITMS rely on complex and time-consuming “tuning” to optimize CID for a particular ion. The digital ion trap (DIT) has a very promising application field in MSⁿ analysis, because of its many unique features. Herein, we conducted a theoretical and experimental investigation of a developed synchronized reverse scan–CID (SRS-CID) using a digital linear ion trap. Specifically, (1) simulations and experiments demonstrated that in the SRS-CID, ions were sequentially scanned from high to low m/z value via the resonance excitation point (q_excitation), producing multiple fragment ions without the need to know the m/z value or complex radiofrequency (rf) tuning of each product ion. The simulations demonstrated that the heating rate in the SRS-CID could reach 0.022 eV/μs. The experiments demonstrated that the optimal reverse scan speed was −0.053 ns/step. (2) We preliminary increased the period by a fixed value (T_step) to control q_excitation to study the molecule fragmentation approach. Different mass spectra were obtained by controlling t_excitation with a fixed T_step. (3) This paper introduces the phase space method to study the motion trajectories of precursor ions and daughter ions. The calculations used and the entire program were uploaded to GitHub. (4) Changing the duty cycle to advantageously shift q_excitation improved the heating rate (0.033 eV/μs) in SRS-CID. Overall, we demonstrated the effectiveness of the developed SRS-CID technique in fragment ion analysis via theoretical derivation, simulation, and experimentation. Furthermore, DIT mass spectrometry was advantageous in tandem mass spectrometry analysis by facilitating modulation of the driving rf period.

Mass spectrometry imaging (MSI) can be used to survey numerous molecular species from a wide variety of surfaces, including biological tissue sections. Atmospheric-pressure (AP) infrared laser-ablation plasma postionization (IR-PPI) has recently been shown to allow matrix free analysis of small molecules from both fresh frozen and formalin fixed paraffin embedded (FFPE) tissue. Detected ion intensities in IR-PPI as well as other AP inlet modalities such as desorption electrospray ionization (DESI) show a strong dependence on the inlet capillary temperature. In this study, the relationship between detected ion intensity and inlet capillary temperature is evaluated, between room temperature and 650 °C, for analyte pipetted on various substrates, as well as fresh frozen and FFPE tissue, by IR-PPI. Temperature trends for exemplar ions of interest show a variety of dependencies with optimal temperatures observed throughout this temperature range. For example, detection of lactate [M-H]⁻ m/z 89.0244 is optimal at ∼100 °C, glutamine [M-H]⁻ m/z 145.0618 at ∼250 °C, arachidonic acid [M-H]⁻ m/z 303.2324 at ∼150 °C and PI(18:0/20:4) [M-H]⁻ m/z 885.5488 at ∼500 °C. Data reduction and clustering of these data by uniform manifold approximation and projection (UMAP) and k-means provides a summary of all temperature trends within the data and association of different ions with these trends are presented. Finally, the implications of different inlet capillary temperature settings in tissue MSI are demonstrated by comparing detected glucose and lactate ion intensities in response to different inlet temperatures in mouse brain. The choice and control of inlet temperature are shown to be critical variables for the interpretation of biological MSI data in AP modalities.

Metabolites are essential small molecules that are naturally occurring in biological processes as end or intermediate products of various pathways. Matrix-assisted laser desorption/ionization–trapped ion mobility separation–mass spectrometry imaging (MALDI-TIMS-MSI) is an emerging technique that can be used to identify the spatial localization of endogenous compounds on tissue. We evaluated the potential of ammonium fluoride (NH₄F) to enhance the ionization efficiency of metabolites in negative polarity mode when used as a comatrix additive in N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC), 9-aminoacridine (9AA), and 1,5-diaminonaphthalene (DAN) matrices. An extensive list of 234 isotopically labeled metabolites (IROA-IS) was used to establish a quantitative ionization efficiency model with respect to the metabolite chemical structures. In addition, we extended our evaluation to endogenous compounds observed in brain samples collected from male mice. Overall, our study demonstrates that NH₄F improves the sensitivity and ionization efficiency of metabolites and lipids in MALDI-TIMS-MSI. This effect was found to vary depending on the matrix, with the ionization efficiency of the studied metabolites increasing in the order NEDC < 9AA < DAN. The quantitative structure–ionization efficiency relationship model can facilitate the appropriate selection of the matrix in MALDI prior to the analysis of analytes of interest.