Orbitrap-based charge detection mass spectrometry (CDMS) enables the mass analysis of biomolecules at the individual-ion level. Recording longer transients boosts resolution and sensitivity for CDMS measurements. However, singly charged analytes (∼1-2 kDa) remained elusive to Orbitrap-based CDMS due to their low signal-to-noise ratios (S/N). To overcome this limitation, we implemented CDMS on a high-field Orbitrap mass analyzer (HF-OT, Exploris 480 mass spectrometer) coupled to an external data acquisition system. The HF-OT analyzer should theoretically improve the resolution by 1.7-fold and the S/N by 1.4-fold compared to a standard Orbitrap analyzer (S-OT, UHMR mass spectrometer). We first adapted an Exploris 480 mass spectrometer to allow robust detection across an extended m/z range (∼1 kDa to 500 kDa) and enable transient recording up to 20 s. Experimentally, the HF-OT outperformed the S-OT in charge and mass accuracy for a variety of systems, such as insulin, BSA, and a monoclonal antibody, and allowed us to achieve an unprecedented mass resolution far above 3 million even at m/z ∼ 4250. On the lower side of the m/z range, the modified HF-OT allowed the recording of individual molecules carrying just a single charge. For the first time, the peptides angiotensin I and bradykinin could be unambiguously detected and mass analyzed above the noise threshold (S/N = 2), albeit only at extended transient times, creating a new lower mass and charge limit for Orbitrap-based CDMS.

The use of peptides labeled with radioactive iodine isotopes enables rapid and highly sensitive assessment of their dynamic distribution in the body, as well as effective visualization and quantification of their metabolites in biological fluids and tissues down to femtomolar concentrations. In this work, we present the laboratory protocol for radioiodination of peptides containing oxidation-labile amino acids. Products of iodination of the histidine- and tyrosine-containing peptides were analyzed by NMR and high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) spectra. The proposed protocol describes a simple laboratory method for monitoring the extent of iodine isotope incorporation and a technique for isolating labeled peptides (3-10 amino acids) of high chemical and radiochemical purity without using HPLC. Using HPLC with fluorescence detection, we demonstrate the absence of significant oxidation of labile amino acids (Met, Trp, Tyr) during the radiolabeling process. The peptides labeled according to the protocol are obtained as lyophilizates with minimal inorganic salt content, allowing their direct use in cellular and animal model studies.

Single-cell proteomics by mass spectrometry (scp-MS) holds the potential to provide unprecedented insights into molecular features directly linked to the cellular phenotype while deconvoluting complex organisms into their basic building blocks. Tailored sample preparation that maximizes the extracted amount of material that is introduced into the mass spectrometer has rapidly propelled the field forward. However, the measured signal is still at the lower edge of detection, approaching the sensitivity boundary of current instrumentation. Here, we investigate the capacity of the enhanced sensitivity of the Orbitrap Astral mass spectrometer to facilitate deeper proteome profiles from low-input to single-cell samples. We carry out a comprehensive data acquisition method survey to pinpoint which parameters provide the most sensitivity. Furthermore, we explore the quantitative accuracy of the obtained measurements to ensure that the obtained abundances are in line with expected ground truth values. We culminate our technical exploration by generating small datasets from two cultured cell lines and a primary bone marrow sample, to showcase obtainable proteome coverage differences from different source materials. Finally, as a proof of concept, we explore protein covariation to showcase how information on known protein complexes is captured inherently in our scp-MS data.

Alzheimer's disease (AD) is a multifactorial systemic disease that is triggered, at least in part, by the accumulation of β-amyloid (Aβ) peptides in the brain, but it also depends on immune system-mediated regulation. Recent studies suggest that B cells may play a role in AD development and point to the accumulation of clonally expanded B cells in AD patients. However, the specificity of the clonally expanded B cells is unknown, and the contribution of Aβ-specific B cells to AD pathology development is unclear. In this study, we have developed a novel method to identify Aβ-specific B cells by flow cytometry using fluorescent tetramers. The suggested method also enables the identification of B-cell clones specific to a more pathology-provoking form of Aβ with an isomerized Asp7 residue (Iso-D7-Aβ) that accumulates in elderly people and in AD patients. The method has been verified using mice immunized with antigens containing the isomerized or non-isomerized Aβ N-terminus peptides. In addition, we describe a new method for the detection of Iso-D7-Aβ-specific antibodies, which was tested on mouse serum. These methods are of potential importance in research aimed at studying AD and may be also utilized for diagnostic and therapeutic purposes.

The FXR1 protein regulates the stability and translation of a number of RNA molecules and plays an important role in the regulation of cellular processes under normal conditions and stress. In particular, this protein is known to be a negative regulator of the key proinflammatory cytokine TNF alpha. We had previously shown that FXR1 functioned in the amyloid form in neurons of the brain of jawed vertebrates. Under stress conditions, FXR1 is incorporated into stress granules in some cell lines, but such studies have not been conducted for neuronal cells. Here, we showed the ability of the FXR1 protein to form cytoplasmic granules in a neuroblastoma cell line under various types of stress. This protein colocalizes with core proteins of neuronal stress granules upon heat shock and sodium arsenite treatment. We also showed that FXR1 colocalizes with anti-amyloid antibodies OC under both normal and stress conditions. Given that stress granules are dynamic structures, we propose that amyloid FXR1-containing RNP particles interact with other stress granule proteins through weak intermolecular hydrogen bonds. Using a yeast model system, we found that FXR1 colocalizes and physically interacts with stress granule proteins such as TIA-1, FMRP, FXR2, and SFPQ. Overall, our results provide new insights into the role of the RNA-binding protein FXR1 in neuronal stress response. We believe that FXR1 inactivation in neuronal stress granules can contribute to an increase in the level of the proinflammatory cytokine TNF alpha in neurodegenerative diseases.

NADPH oxidase enzymes (NOXs) are a family of enzymes generating superoxide, which form reactive oxygen species. NOX2 activity is a causative agent for the progression of many diseases: neurodegenerative, cardiovascular, immune dysregulations, and even hereditary diseases and cancer. Administering antioxidants helps in inhibiting NOX2 activity; however, the development of selective inhibitors may provide greater improvement in the therapy of diseases. Here, an optimized synthesis of two most promising NOX2 inhibitors based on the 3-(indolin-6-yl)-4-(N-pyrazole-sulfonamide)-1H-pyrrolo [2,3-b]pyridine structure, namely, GSK2795039 and NCATS-SM7270, and an isomeric derivative of the same class, IMBIOC-1, is reported. The new modified procedures simplify the isolation, reduce byproduct formation, and improve the yields in 0.1–1 g scale preparations. Molecular modeling of the structures of NOX2 complexes with inhibitors validated their binding at the same site as NADPH, with IMBIOC-1 forming the largest number of intermolecular interactions with the NOX2 active site. Testing the effects of the compounds on amyloid beta-induced oxidative stress and toxicity in HMC3 microglial cells showed that all three inhibitors completely prevented the pathological amyloid-beta effect. At the same time, NCATS-SM7270 and IMBIOC-1 provided a stronger protective effect on microglial cell survival than GSK2795039, which allowed us to assert the potential of those compounds as neuroprotective agents.

Recently, a conceptually new mass analyzer was introduced by pairing a quadrupole Orbitrap mass spectrometer with an asymmetric track lossless (Astral™) analyzer. This system provides >200 Hz MS/MS scanning speed, high resolving power, sensitivity, and mass accuracy. Due to its speed, the instrument allows for a narrow-window data-independent acquisition (nDIA) strategy, representing a new technical milestone in peptide-centric proteomics. However, this new system may also be applied to other complex and clinically important proteomes, such as the human plasma N -glycoproteome. Here, we evaluate the Orbitrap Astral mass spectrometer for the in-depth analysis of the plasma N -glycoproteome and pioneer a dedicated nDIA workflow, termed “nGlycoDIA”, on glycopeptide enriched and crude plasma. This strategy leads to the cumulative identification of over 3000 unique glycoPSMs derived from 181 glycoproteins in just 40 minutes and covers a dynamic range of 7 orders of magnitude for a glycopeptide enriched plasma sample. Notably, we detect several glycosylated cytokines that have reported plasma concentrations in the ng/L range. Furthermore, shortening the gradient to 10 min still allows for the detection of almost 1850 (95% CI [1840-1860]) unique glycoPSMs, indicating that high-throughput in-depth clinical plasma glycoproteomics may be within reach.

INTRODUCTION:Identifying early risks of developing Alzheimer's disease (AD) is a major challenge as the number of patients with AD steadily increases and requires innovative solutions. Current molecular diagnostic modalities, such as cerebrospinal fluid (CSF) testing and positron emission tomography (PET) imaging, exhibit limitations in their applicability for large-scale screening. In recent years, there has been a marked shift toward the development of blood plasma-based diagnostic tests, which offer a more accessible and clinically viable alternative for widespread use. Furthermore, advances in large-scale proteomics technologies have boosted an interest in identifying novel biomarkers and developing panels of AD-associated proteins. AREAS COVERED:This review mainly examines the results of recent searches for proteomic markers of AD in blood plasma (from 2022-2024 PubMed), focuses on some aspects for special attention in further studies, and discusses the prospects for their further application. EXPERT OPINION:Recent advances in AD plasma/serum proteomic studies are largely driven using novel Olink/PEA and SomaScan/aptamer technologies, which complement the 'gold standard' of MS-based quantitative proteomics (MRM/SRM), and particularly expand the capabilities for studying low-abundant proteins.

Beta-amyloid (Aβ) is an important factor in the development of pathology in Alzheimer’s disease. Level of beta-amyloid precursor protein (APP) is increased in neurites with age and in Alzheimer’s disease model mice. However, it is unclear whether Aβ can affect APP levels in cells. The aim of this study was to evaluate the effect of Aβ on the level and trafficking of APP in human neuroblastoma cells and to identify the role of cardiotonic steroid (CTS) ouabain in this process. Western blot analysis revealed that 30-min incubation of the cells with 100 nM Aβ increased APP levels by 75%. Confocal microscopy showed that Aβ alters APP trafficking, promoting its movement into neurites. This effect establishes a positive feedback loop that accelerates Aβ formation in neurites. The rise in APP was associated with Src kinase activation triggered by Aβ binding to Na,K-ATPase. Notably, Src kinase inhibition completely blocked the Aβ-induced increase in APP, indicating that beta-amyloid effect on APP is mediated by Src kinase activation. Furthermore, 100 nM CTS ouabain, a specific Na,K-ATPase ligand, significantly decreased Aβ′s impact on APP and Src kinase activation. Given that CTS are naturally present in the human body, these findings are important for developing therapeutic strategies to counteract Aβ-driven APP accumulation and for understanding the role of endogenous CTS in regulating Aβ formation.

Alzheimer's disease (AD) is a worldwide problem due to the lack of effective therapy and accurate methods for timely diagnosis. The complexity of AD's pathophysiology complicates the development of effective therapeutic agents, as most drugs act on only one therapeutic target, bypassing others. The design and development of multifunctional agents capable of altering metal ion-induced abnormalities, oxidative stress, and toxic beta amyloid (Aβ) aggregates is of interest. Herein, we report the first boron dipyrromethene (BODIPY) based bifunctional copper chelator with clioquinol, BDP-CLQ, capable of both optical detection of Aβ fibrils and copper chelation, with multiple anti-AD properties. Foremost, BDP-CLQ demonstrated a 3-fold and 5-fold fluorescence increase at 650 nm and 565 nm in the presence of Aβ and effective copper chelation (pKd = 16.6 ± 0.3). In addition, BDP-CLQ demonstrated a potent inhibition of Aβ aggregation, reduction in Aβ-induced stiffness of neuronal cells, and antioxidant activity. BDP-CLQ is the first BODIPY-based fluorescent probe with multiple anti-AD activities, as well as the first clioquinol-based probe capable of Aβ optical visualization. This study demonstrates the prospects of the development of clioquinol-based theranostic probes since this allows combining several promising anti-AD actions in a single molecule and developing multi-targeted drugs.

Tubulins are among the most successful targets for cancer chemotherapy. However, the emergence of drug resistance stimulates the continuous search for novel chemotherapeutics. Here, we discover that coumarin-30, a widely available laser dye, binds to the colchicine site of tubulin and inhibits microtubule dynamics and cancer cell division at submicromolar concentrations. By combining coumarin-30 as a fluorescent probe with the microscale thermophoresis approach, we develop a versatile assay for detecting tubulin–ligand interactions and simultaneously sorting ligands into binders of the colchicine site versus other protein pockets. The assay’s performance is demonstrated on a wide panel of compounds. Using this methodology, we identify several potent tubulin polymerization inhibitors and determine their binding sites. The results are verified with studies of microtubule dynamics in vitro and the cell cycle in cancer cell culture. Thus, the coumarin-30-based assay is a fast, accurate, and cost-effective method for characterizing tubulin ligands with diverse binding pockets.

Recently, the use of ultralong transients has enabled exceptional resolution and sensitivity in Orbitrap-based charge detection mass spectrometry (CDMS). Nevertheless, measuring small analytes carrying a few charges remains a challenge. Prolonged trapping should, in theory, allow for the detection of lower charged ions (<10+) due to enhanced signal-to-noise (S/N) ratios. However, in practice, due to ion decay through frequency drifts, or collision-induced fragmentations, low m/z ions deviate from the ideal coherent trajectories in the Orbitrap. Here, by incorporating electron capture charge reduction (ECCR) in the gas phase prior to CDMS, we show that charge reduction significantly improves the stability of ion trajectories when ions are trapped for long periods in the Orbitrap analyzer. Using proteins with molecular weights ranging from 12 to 900 kDa, we demonstrate that ECCR-CDMS enhances ion survival by up to 60-fold, even enabling the detection of doubly charged individual ions from cytochrome c that typically elude conventional Orbitrap-based CDMS.

Recombinant adeno-associated viruses (rAAVs) play an important role in gene therapy, yet the optimal preparation of these biotherapeutics remains challenging, with often incomplete incorporation of genome cargo, negatively affecting therapeutic use. Genome packaging has traditionally been difficult to investigate. Charge detection mass spectrometry (CDMS) has gained prominence, as it provides the ability to mass analyze and resolve empty and filled rAAVs, enabling the quantitative determination of empty-to-filled rAAV ratios. Such measurements require a high mass resolving power and depend on the unbiased detectability of the distinct rAAV particles. The mass resolving power in the Orbitrap mass analyzers scales with transient recording times. Therefore, we extended the capability of recording ions from 1 or 2 to 24 s. When we record these 24 s transients to analyze rAAVs, we not only observe a substantial improvement in accuracy and mass resolution but also find that this can lead to erroneous artifacts in the empty-to-filled ratios. Artifacts originate from the distinct behavior between orbiting ions from either empty or filled particles, mainly due to differences in desolvation and charge losses. Ions from empty rAAVs appear much more susceptible to charge losses, which negatively affect their tracing, artificially decreasing the empty-to-filled ratios. Further elucidating the causes of this undesirable ion behavior, we provide the means to minimize charge losses, thus regaining accurate AAV quantification at an increased charge precision and mass-resolving power. As the Orbitrap-based CDMS has become a method of choice to determine this important quality control attribute, our findings are important to avoid reporting incorrect empty-to-filled ratios.

Computational approaches are increasingly used to predict monoclonal antibody (mAb) candidates from BCR-seq datasets. However, the reliable identification of B cells encoding antibodies against rare antigenic epitopes remains challenging. We employed a repertoire-guided workflow combining antigen-tetramer sorting of B cells from immunized mice, followed by low-input bulk BCR-seq yielding informative clonal repertoires. Clustering and supporting somatic hypermutation (SHM) lineage analysis allowed us to identify IGH and IGK clonotypes potentially targeting β–amyloid and its isoAsp7 variant (isoD7–Aβ1-16), implicated in Alzheimer′s disease. We observed recurrent IGHV8-12 and IGKV1-117 usage, consistent with canonical mouse anti-Aβ responses. Focusing on isoD7–Aβ binders, we selected ten candidate IGH—IGK pairs for recombinant expression. One recombinant mAb demonstrated preferential binding to isoD7–Aβ by microscale thermophoresis, supporting the feasibility of the approach but underscoring the challenge of accurate chain pairing. This highlights the potential of bioinformatic workflows to identify mAbs even under low-input conditions. ### Competing Interest Statement The authors have declared no competing interest. the Ministry of Science and Higher Education of the Russian Federation, 075-15-2024-530

Orbitrap mass spectrometry is widely used in the life-sciences. However, like all mass spectrometers, non-uniform (heteroscedastic) noise introduces bias in multivariate analysis complicating data interpretation. Here, we study the noise structure of an Orbitrap mass analyser integrated into a secondary ion mass spectrometer (OrbiSIMS). Using a stable primary ion beam to provide a well-controlled source of ions from a silver sample, we find that noise has three characteristic regimes: at low signals the Orbitrap detector noise and a censoring algorithm dominates; at intermediate signals counting noise specific to the ion emission process is most significant; and at high signals additional sources of measurement variation become important. Using this understanding, we developed a generative model for Orbitrap data that accounts for the noise distribution and introduce a scaling method, termed WSoR, to reduce the effects of noise bias in multivariate analysis. We compare WSoR performance with no-scaling and existing scaling methods for three biological imaging data sets including drosophila central nervous system, mouse testis and a desorption electrospray ionisation (DESI) image of a rat liver. WSoR consistently performed best at discriminating chemical information from noise. The performance of the other methods varied on a case-by-case basis, complicating the analysis.

Great progress has been made in the detection of large biomolecular analytes by native mass spectrometry; however, characterizing highly heterogeneous samples remains challenging due to the presence of many overlapping signals from complex ion distributions. Electron-capture charge reduction (ECCR), in which a protein cation captures free electrons without apparent dissociation, can separate overlapping signals by shifting the ions to lower charge states. The concomitant shift to higher m/z also facilitates the exploration of instrument upper m/z limits if large complexes are used. Here we perform native ECCR on the bacterial chaperonin GroEL and megadalton scale adeno-associated virus (AAV) capsid assemblies on a Q Exactive UHMR mass spectrometer. Charge reduction of AAV8 capsids by up to 90% pushes signals well above 100,000 m/z and enables charge state resolution and mean mass determination of these highly heterogeneous samples, even for capsids loaded with genetic cargo. With minor instrument modifications, the UHMR instrument can detect charge-reduced ion signals beyond 200,000 m/z. This work demonstrates the utility of ECCR for deconvolving heterogeneous signals in native mass spectrometry and presents the highest m/z signals ever recorded on an Orbitrap instrument, opening up the use of Orbitrap native mass spectrometry for heavier analytes than ever before.

Along with mass spectrometry (MS), ion mobility separations (IMS) are advancing to ever larger biomolecules. The emergence of electrospray ionization (ESI) and native MS enabled the IMS/MS analyses of proteins up to ∼100 kDa in the 1990s and whole protein complexes and viruses up to ∼10 MDa since the 2000s. Differential IMS (FAIMS) is substantially orthogonal to linear IMS based on absolute mobility K and offers exceptional resolution, unique selectivity, and steady filtering readily compatible with slower analytical methods such as electron capture or transfer dissociation (ECD/ETD). However, the associated MS stages had limited FAIMS to ions with m/z < 8000 and masses under ∼300 kDa. Here, we integrate high-definition FAIMS with the Q-Exactive Orbitrap UHMR mass spectrometer that can handle m/z up to 80,000 and MDa-size ions in the native ESI regime. In the initial evaluation, the oligomers of monoclonal antibody adalimumab (148 kDa) are size-selected up to at least the nonamers (1.34 MDa) with m/z values up to ∼17,000. This demonstrates the survival and efficient separation of noncovalent MDa assemblies in the FAIMS process, opening the door to novel analyses of the heaviest macromolecules.

Reflectron-based time-of-flight analyzers rely on sub-nanosecond detector time response to achieve acceptable resolving power for low-mid mass, multiple ion peaks. With the adoption of multi-reflection analyzers, order of magnitude longer folded ion paths relax restrictions on detector response time, allowing implementation of new technologies that greatly improve dynamic range, detector lifetime, and ion detection efficiency. A detection system is presented, integrated into the Astral analyzer, that combines 10 keV post-acceleration and focal plane correction with a unique BxE focusing, optically coupled detector, pre-amplification and dual channel digitization. Calibration and peak handling methods are also described. The instrument demonstrated >1x104 dynamic range in a single shot, >100k resolving power, and a relative immunity to detector ageing.

Post-translational modifications of beta-amyloid (Aβ) play an important role in the pathogenesis of Alzheimer’s disease (AD). Aβ modifications such as Ser8 phosphorylation (pS8-Aβ42) and Asp7 isomerization (iso-Aβ42) can significantly alter the properties of Aβ and have been detected in vivo. One of the reasons for the different pathogenicity of Aβ isoforms may be the activation of different signaling cascades leading to changes in the mechanical properties of cells. In this paper, we used correlative scanning ion-conductance microscopy (SICM) and Pt-nanoelectrodes to compare the effects of Aβ isoforms on the Young’s modulus of SH-SY5Y cells and the level of ROS. It was found that unmodified Aβ42 resulted in the largest increase in cell Young’s modulus of all isoforms after 4 h of incubation, while pS8-Aβ42 induced the greatest increase in stiffness and ROS levels after 24 h of incubation. Analysis of signaling proteins involved in the regulation of the actin cytoskeleton showed that Aβ42, pS8-Aβ42 and iso-Aβ42 have different effects on cofilin, GSK3β, LIMK, ERK and p38. This indicates that post-translational modifications of Aβ modulate its effect on neuronal cells through the activation of various signaling cascades, which affects the mechanical properties of cells.

Mass spectrometry has been increasingly explored in intraoperative studies as a potential technology to help guide surgical decision making. Yet, intraoperative experiments using high-performance mass spectrometry instrumentation present a unique set of operational challenges. For example, standard operating rooms are often not equipped with the electrical requirements to power a commercial mass spectrometer and are not designed to accommodate their permanent installation. These obstacles can impact progress and patient enrollment in intraoperative clinical studies because implementation of MS instrumentation becomes limited to specific operating rooms that have the required electrical connections and space. To expand our intraoperative clinical studies using the MasSpec Pen technology, we explored the feasibility of transporting and acquiring data on Orbitrap mass spectrometers operating on battery power in hospital buildings. We evaluated the effect of instrument movement including acceleration and rotational speeds on signal stability and mass accuracy by acquiring data using direct infusion electrospray ionization. Data were acquired while rolling the systems in/out of operating rooms and while descending/ascending a freight elevator. Despite these movements and operating the instrument on battery power, the relative standard deviation of the total ion current was <5% and the magnitude of the mass error relative to the internal calibrant never exceeded 5.06 ppm. We further evaluated the feasibility of performing intraoperative MasSpec Pen analysis while operating the Orbitrap mass spectrometer on battery power during an ovarian cancer surgery. We observed that the rich and tissue-specific molecular profile commonly detected from ovarian tissues was conserved when running on battery power. Together, these results demonstrate that Orbitrap mass spectrometers can be operated and acquire data on battery power while in motion and in rotation without losses in signal stability or mass accuracy. Furthermore, Orbitrap mass spectrometers can be used in conjunction to the MasSpec Pen while on battery power for intraoperative tissue analysis.