Analytical Chemistry

BackgroundCerebrospinal fluid (CSF) leak is typically diagnosed by detecting a protein marker {beta}2-transferrin ({beta}2-Tf) in secretion samples. {beta}2-Tf and {beta}1-transferrin ({beta}1-Tf) are glycoforms of human transferrin (Tf). A novel affinity capture technique for sample preparation, called microprobe-capture in-emitter elution (MPIE), was incorporated with high-resolution mass spectrometry (HR-MS) to analyze the Tf glycoforms and elucidate the structures of {beta}1-Tf and {beta}2-Tf. MethodsTo implement MPIE, an analyte is first captured on the surface of a microprobe, and subsequently eluted from the microprobe inside an electrospray emitter. The capture process is monitored in real-time via next-generation biolayer interferometry (BLI). When electrospray is established from the emitter to a mass spectrometer, the analyte is immediately ionized via electrospray ionization (ESI) for HR-MS analysis. Serum, CSF, and secretion samples were analyzed using MPIE-ESI-MS. ResultsBased on the MPIE-ESI-MS results, the structures of {beta}1-Tf and {beta}2-Tf were solved. As Tf glycoforms, {beta}1-Tf and {beta}2-Tf share the amino acid sequence but have varying N-glycans. {beta}1-Tf, the major serum-type Tf, has two G2S2 N-glycans on Asn413 and Asn611. {beta}2-Tf, the major brain-type Tf, has an M5 N-glycan on Asn413 and a G0FB N-glycan on Asn611. ConclusionsThe structures of {beta}1-Tf and {beta}2-Tf were successfully elucidated by MPIE-ESI-MS analysis. The resolving power of the novel MPIE-ESI-MS method was demonstrated in this study. On the other hand, knowing the N-glycan structures on {beta}2-Tf allows for the design of other novel test methods for {beta}2-Tf in the future.

Sphingolipids are diverse lipids with sphingobases and N-acyl fatty acids as the hydrophobic moieties. While the importance of the in-depth elucidation of hydrophobic structures is widely recognized in lipid biology, mass spectrometry-based annotation of ceramides in the commonly used protonated form is often hindered by in-source dehydration during electrospray ionization in the heated state and variable water losses in the product ion spectrum. In this study, we investigated the sodium ion form and its product ions in ceramides with the use of electron-activated dissociation tandem mass spectrometry (EAD MS/MS) in addition to collision-induced dissociation to facilitate indepth structural elucidation. While dehydrated ions from the protonated form were frequently observed, the sodium adduct ions remained stable because of their higher activation energy compared with the protonated form, which was validated using quantum chemical calculations. Using the three adduct forms under optimized conditions increased confidence in annotating the ceramide peaks through retention-time matching. Furthermore, EAD MS/MS of the sodium adduct ions facilitated the positional determination of double bonds and hydroxyl groups in the ceramide hydrophobic moiety. Our approach is showcased by the annotation of phytoceramides with N-acyl 2- and 3-hydroxyl groups in mouse feces and ceramides with N-acyl n-6 very long-chain polyunsaturated 2-hydroxy fatty acids in mouse testis.

O-Acetylation, a common modification in rhamnogalacturonan I (RG-I), is critical for various biological processes, including plant growth, stress responses, and pathogen defense. Precise determination of the degree and specific positions of acetylation is therefore essential. To date, nuclear magnetic resonance (NMR) and tandem mass spectrometry have been employed to identify acetyl positions in pectin oligosaccharides. Although NMR is effective, it requires pure, high-concentration samples. Tandem mass spectrometry (MS), which uses lower sample amounts, faces challenges due to acetyl migration between monosaccharide positions. The multiple steps in pectin sample analysis can further promote O-acetyl migration, especially near free hydroxyl groups. Moreover, during tandem MS, acetyl groups may detach, complicating accurate tracking. This study presents an approach to lock O-acetyl groups by introducing trideuteroacetyl and propionyl substituents onto free hydroxyls of RG-I or partially acetylated RG-I. By combining matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS and electrospray ionization (ESI) MS with MS/MS or tandem mass spectrometry (MSn), we devised a way to determine the monosaccharide sequence in the oligomer and precise positions of acetyl groups in partially acetylated RG-I. This method enables the study of the regiospecificity of recombinant pectin O-acetyltransferases and can be applied to other oligosaccharides to determine acyl positions. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/694011v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1384347org.highwire.dtl.DTLVardef@c8f21forg.highwire.dtl.DTLVardef@8fed52org.highwire.dtl.DTLVardef@125c4a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Currently approved therapeutic monoclonal antibodies (mAbs) are based on immunoglobulin G (IgG) 1, 2 or 4 isotypes, which differ in their specific inter-chains disulfide bridge connectivities. Different analytical techniques have been reported for mAb isotyping, among which native ion mobility mass spectrometry (IM-MS) and collision induced unfolding (CIU) experiments. However, mAb isotyping by these approaches is based on detection of subtle differences and thus remains challenging at the intactlevel. We report here on middle-level (after IdeS digestion) IM-MS and CIU approaches to afford better differentiation of mAb isotypes. Our method provides simultaneously CIU patterns of F(ab)2 and Fc domains within a single run. Middle-level CIU patterns of F(ab)2 domains enable more reliable classification of mAb isotypes compared to intact level CIU, while CIU fingerprints of Fc domains are overall less informative for mAb isotyping. F(ab)2 regions can thus be considered as diagnostic domains providing specific CIU signatures for mAb isotyping. Benefits of middle-level IM-MS and CIU approaches are further illustrated on the hybrid IgG2/IgG4 eculizumab. While classical analytical techniques led to controversial results, middle-level CIU uniquely allowed to face the challenge of eculizumab << hybridicity >>, highlighting that its F(ab)2 and Fc CIU patterns corresponds to an IgG2 and an IgG4, respectively. Altogether, the middle-level CIU approach is more clear-cut, accurate and straightforward for canonical but also more complex, engineered next generation mAb formats isotyping. Middle-level CIU thus constitutes a real breakthrough for therapeutic protein analysis, paving the way for its implementation in R&D laboratories.

AO_SCPLOWBSTRACTC_SCPLOWMass spectrometry is the premier tool for identifying and quantifying site-specific protein glycosylation globally. Analysis of intact glycopeptides often requires an enrichment step, after which the samples remain highly complex and exhibit a broad dynamic range of abundance. Here, we evaluated the analytical benefits of high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to nano-liquid chromatography mass spectrometry (nLC-MS) for analyses of intact glycopeptide devoid of any enrichment step. We compared the effects of compensation voltage on the transmission of N- and O-glycopeptides derived from heterogeneous protein mixtures using two FAIMS devices. We comprehensively demonstrate the performance characteristics of the FAIMS device for glycopeptide analysis and recommend optimal electrode temperature and compensation voltage (CV) settings for N- and O-glycopeptide analysis. Under optimal CV settings, FAIMS-assisted gas-phase fractionation in conjunction with chromatographic reverse phase separation resulted in a 31% increase in the detection of both N- and O-glycopeptide compared to control experiments without FAIMS. Overall, our results demonstrate that FAIMS provides an alternative means to access glycopeptides without any enrichment providing an unbiased global glycoproteome landscape. In addition, our work provides the framework to verify difficult-to-identify glycopeptide features.

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of healthy and diseasedriven biological functions. Previously, we developed a mucin-selective enrichment strategy by employing a catalytically inactive mucinase (StcE) conjugated to solid support. While this method was effective, it suffered from low throughput and high sample requirements. Further, the elution step required boiling in SDS, thus necessitating an in-gel digest with trypsin. Here, we optimized our previous enrichment method to include elution conditions amenable to mucinase digestion and downstream analysis with mass spectrometry. This increased throughput and lowered sample input while maintaining mucin selectivity and enhancing glycopeptide signal. We then benchmarked this technique against different O-glycan binding moieties for their ability to enrich mucins from various cell lines and human serum. Overall, the new method outperformed our previous procedure and all other enrichment techniques tested. This allowed for effective isolation of more mucin-domain glycoproteins, resulting in a high number of O-glycopeptides, thus enhancing our ability to analyze the mucinome.

Carboxylic acids are central metabolites in bioenergetics, signal transduction and post-translation protein regulation. Unlike its genomic and transcriptomic counterparts, the quest for metabolomic profiling in trace amounts of biomedical samples is prohibitively challenging largely due to the lack of sensitive and robust quantification schemes for carboxylic acids. Based on diazo-carboxyl click chemistry, here we demonstrate DQmB-HA method as a rapid derivatization strategy for the sensitive analysis of hydrophilic, low-molecular-weight carboxylic acids. To the investigated metabolites, DQmB-HA derivatization method renders 5 to 2,000-fold higher response on mass spectrometry along with improved chromatographic separation on commercial UHPLC-MS machines. Using this method, we present the near-single-cell analysis of carboxylic acid metabolites in mouse egg cells before and after fertilization. Malate, fumarate and {beta}-hydroxybutyrate were found to decrease in mouse zygotes. We also showcase the kinetic profiling of TCA-cycle intermediates inside adherent cells cultured in one well of 96-well plates during drug treatment. FCCP and AZD3965 were shown to have overlapped but different effects on the isotope labeling of carboxylic acids. Finally, we apply DQmB-HA method to plasma or serum samples (down to 5 L) from mice and humans collected on pathological and physiological conditions. The measured changes of succinate, {beta}-hydroxybutyrate, and lactate in blood corroborate previous literatures in ischemia-reperfusion injury mouse model, acute fasting-refeeding mouse model, and human individuals diagnosed with mitochondrial dysfunction diseases, respectively. Overall, DQmB-HA method offers a sensitive, rapid and user-friendly quantification scheme for carboxylic acid metabolites, paving the road toward the ultimate goals of single-cell metabolomic analysis and bedside monitoring of biofluid samples.

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 micrograms of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g. H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 micrograms of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamlines the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/586403v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@16d595aorg.highwire.dtl.DTLVardef@55acforg.highwire.dtl.DTLVardef@18cf92eorg.highwire.dtl.DTLVardef@3b5fb4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Metals are essential for the molecular machineries of life, and microbes have evolved a variety of small molecules to acquire, compete for, and utilize metals. Systematic methods for the discovery of metal-small molecule complexes from biological samples are limited. Here we describe a two-step native electrospray ionization mass spectrometry method, in which double-barrel post-column metal-infusion and pH adjustment is combined with ion identity molecular networking, a rule-based informatics workflow. This method can be used to identify metal-binding compounds in complex samples based on defined mass (m/z) offsets of ion features with the same chromatographic profiles. As this native metal metabolomics approach can be easily implemented on any liquid chromatography-based mass spectrometry system, this method has the potential to become a key strategy for elucidating and understanding the role of metal-binding molecules in biology.

We present a biophysical imaging strategy based on linear unmixing Forster resonance energy transfer (lux-FRET) for investigating protein-protein interactions and receptor-mediated signaling in live cells. This method utilizes spectral unmixing of FRET signals acquired via confocal laser scanning microscopy (LSM), enabling high-resolution quantification of molecular interactions with both spatial and temporal precision. Applying lux-FRET, we examined receptor-receptor interactions and downstream signaling events, including agonist specificity for 5-HT receptors. Ratiometric FRET measurements with a genetically encoded cAMP biosensor allowed us to assess biosensor sensitivity to cyclic nucleotides and receptor efficacy. Additionally, we explored physiological interactions between CD44 and 5-HT receptors and characterized the oligomerization state of the 5-HT1A receptor through apparent FRET efficiency analysis. Our findings demonstrate the utility of lux-FRET combined with quantitative molecular microscopy as a powerful tool for dissecting dynamic signaling mechanisms in live cells. This approach offers broad applicability for researchers studying receptor pharmacology, cellular signaling, and protein interaction dynamics. RESEARCH HIGHLIGHTWe present a real-time imaging strategy combining lux-FRET with quantitative molecular microscopy to study protein interactions and receptor signaling in living cells. Using spectral and ratiometric FRET analysis, this method enables high-resolution visualization of dynamic molecular processes under physiological conditions. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/673709v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@42beecorg.highwire.dtl.DTLVardef@4a746org.highwire.dtl.DTLVardef@181f61corg.highwire.dtl.DTLVardef@144d940_HPS_FORMAT_FIGEXP M_FIG C_FIG

Proteins can be modified by lipids in various ways, for example by myristoylation, palmitoylation, farnesylation, and geranylgeranylation--these processes are collectively referred to as lipidation. Current chemical proteomics using alkyne lipids has enabled the identification of lipidated protein candidates but does not identify endogenous lipidation sites and is not readily applicable to in vivo systems. Here, we introduce a proteomic methodology for global analyses of endogenous lipidation sites that combines liquid-liquid extraction of hydrophobic lipidated peptides with liquid chromatography-tandem mass spectrometry using a gradient program of acetonitrile in the high concentration range. We applied this method to explore lipidation sites in HeLa cells, and identified a total of 90 lipidation sites, including 75 protein N-terminal myristoylation sites, which is more than the number of high-confidence lipidated proteins identified by myristic acid analog-based chemical proteomics. Isolation of lipidated peptides from digests prepared with different proteases enabled the identification of different lipidated sites, extending the coverage. Moreover, our peptide-centric approach successfully identified dually modified peptides having myristoylation and palmitoylation. Finally, we analyzed in vivo myristoylation sites in mouse tissues and found that the lipidation profile is tissue-specific. This simple method (not requiring chemical labeling or affinity purification) should be a promising tool for global profiling of various protein lipidations.

Proteome Integral Solubility Alteration (PISA) assay is widely used for identifying drug targets, but it is labour-intensive and time-consuming, and requires a substantial amount of biological sample. Aiming at enabling automation and greatly reducing the sample amount, we developed One-Pot Time-Induced (OPTI)-PISA. Here we demonstrate OPTI-PISA performance on identifying targets of multiple drugs in cell lysate and scaling down the sample amount to sub-microgram levels, making PISA method suitable for NanoProteomics.

Highly multiplexed protein and RNA in situ concurrent detection on a single tissue section is highly desirable for both basic and applied biomedical research. CODEX is a new and powerful platform to visualize up to 60 protein biomarkers in situ and RNAscope in situ hybridization (RNAscope) is a novel RNA detection system with single-copy sensitivity and unprecedent specificity at a single cell level. Nevertheless, to our knowledge, the combination CODEX and RNAscope remained unreported until this study. Here we report a simple and reproducible combination of CODEX and RNAscope (Comb-CODEX-RNAscope). We also determined the cross-reactivities of CODEX anti-human antibodies to rhesus macaques, a widely used animal model of human disease.

Protein post-translational modifications (PTMs) are crucial and dynamic players in a large variety of cellular processes and signaling, and proteomic technologies have emerged as the method of choice to profile PTMs. However, these analyses remain challenging due to potential low PTM stoichiometry, the presence of multiple PTMs per proteolytic peptide, PTM site localization of isobaric peptides, and labile PTM groups that lead to neutral losses. Collision-induced dissociation (CID) is commonly used for to characterize PTMs, but the application of collision energy can lead to neutral losses and incomplete peptide sequencing for labile PTM groups. In this study, we compared CID to an alternative fragmentation, electron activated dissociation (EAD), operated on a recently introduced fast-acquisition quadrupole-time-of-flight (QqTOF) mass spectrometer. We analyzed a series of synthetic modified peptides, featuring phosphorylated, succinylated, malonylated, and acetylated peptides. We performed targeted, quantitative parallel reaction monitoring (PRM or MRMHR) assays to assess the performances of EAD to characterize, site-localize and quantify peptides with labile modifications. The tunable EAD kinetic energy allowed the preservation of labile modifications and provided better peptide sequence coverage with strong PTM-site localization fragment ions. Zeno trap activation provided significant MS/MS sensitivity gains by an average of 6-11-fold for EAD analyses, regardless of modification type. Evaluation of the quantitative EAD PRM workflows revealed high reproducibility with coefficients of variation of typically [~]2%, as well as very good linearity and quantification accuracy. This novel workflow, combining EAD and Zeno trap, offers confident, accurate, and robust characterization and quantification of PTMs.

Since the introduction of the first multidimensional liquid chromatography mass spectrometry (mD-LC-MS) approach for antibody peak characterization, multiple developments have been reported including integration of various chromatographic approaches and precise fractionation, providing high quality and reliable peptide data in a drastically decreased total analysis time. Most of these platforms rely on the use of immobilized enzyme reactors (IMERs) for digestion, limiting the applicability to few enzymes such as trypsin. Recently, the introduction of in-solution enzymatic digestion in mD-LC-MS systems has been proposed as an alternative to IMERs. Here, we make use of current innovations in 2D-LC commercial systems such as active solvent modulation valves, which permits direct mixing of the fractionated peaks with the endoprotease and reducing agent in an online manner, to integrate in-solution digestion in a straightforward manner in mD-LC-MS peak characterization platforms. Following antibody digestion, the generated peptides are automatically trapped, separated and detected by mass spectrometry. Efficient reduction and tryptic digestion was obtained using short incubation times (15 min). The approach was further expanded to alternative endoproteases such as chymotrypsin and thermolysine with other digestion specificities and allow an easy exchange between enzymes with similar buffer and digestion conditions. As a proof-of-principle that strategy was applied to achieve peptide maps from ion exchanged separated mAb peaks showing good digestion efficiency and high sequence coverages.

Chemoproteomics is a powerful method capable of detecting interactions between small molecules and the proteome, however its use as a high-throughput screening method for chemical libraries has so far been limited. To address this need, we have further developed a chemoproteomics workflow to screen cysteine reactive covalent fragments in cell lysates against the deubiquitinating (DUB) enzymes using activity-based protein profiling. By using targeted ubiquitin probes, we have addressed sensitivity and affinity limitations, enabling target identification and covalent fragment library profiling in a 96-well plate format. The use of data independent acquisition (DIA) methods for MS analysis combined with automated Evosep liquid chromatography (LC) reduced instrument runtimes to 21 minutes per sample and simplified the workflow. In this proof-of-concept study, we have profiled 138 covalent fragments against 57 DUB proteins and validated four hit fragments against OTUD7B and UCHL3 through site identification experiments and orthogonal biochemical activity assays. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/526632v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@2e036borg.highwire.dtl.DTLVardef@e27962org.highwire.dtl.DTLVardef@8f1caaorg.highwire.dtl.DTLVardef@1a3159a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Compound identification is an essential task in the workflow of untargeted metabolomics since the interpretation of the data in a biological context depends on the correct assignment of chemical identities to the features it contains. Current techniques fall short of identifying all or even most observable features in untargeted metabolomics data, even after rigorous data cleaning approaches to remove degenerate features are applied. Hence, new strategies are required to annotate the metabolome more deeply and accurately. The human fecal metabolome, which is the focus of substantial biomedical interest, is a more complex, more variable, yet lesser-investigated sample matrix compared to widely studied sample types like human plasma. This manuscript describes a novel experimental strategy using multidimensional chromatography to facilitate compound identification in untargeted metabolomics. Pooled fecal metabolite extract samples were fractionated using offline semi-preparative liquid chromatography. The resulting fractions were analyzed by an orthogonal LC-MS/MS method, and the data were searched against commercial, public, and local spectral libraries. Multidimensional chromatography yielded more than a 3-fold improvement in identified compounds compared to the typical single-dimensional LC-MS/MS approach and successfully identified several rare and novel compounds, including atypical conjugated bile acid species. Most features identified by the new approach could be matched to features that were detectable but not identifiable in the original single-dimension LC-MS data. Overall, our approach represents a powerful strategy for deeper annotation of the metabolome that can be implemented with commercially-available instrumentation, and should apply to any dataset requiring deeper annotation of the metabolome.

Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful analytical technique for analyzing biological macromolecules. A long-standing challenge has been applying LC-MS at physiological pH under native conditions using volatile buffers. The predominant "buffer" used, ammonium acetate (AmAc, pKa 4.75 for acetic acid and 9.25 for ammonium), does not offer sufficient buffering capacity in the physiological pH range of 7.0-7.4. To address this, we evaluated a set of fluorinated ethylamines, 2-fluoroethylamine (MFEA, pKa 8.9), 2,2-difluoroethylamine (DFEA, pKa 7.2), and 2,2,2-trifluoroethylamine (TFEA, pKa 5.5), that together provide buffering across the 4.5-9.8 pH range. We show that protein separations on strong cation- and anion-exchange resins in these volatile mobile phases perform comparably to traditional non-volatile buffers, with similar elution profiles and analyte elution ranking, albeit with slightly broader peaks. Using fully volatile gradients of pH or ionic strength, we chromatographically resolved charge variants of protein analytes such as mAbs and bovine serum albumin. For many of the eluting LC peaks, we obtained high-resolution mass spectra capable of resolving glycoforms of antibodies. Hydrophobic interaction chromatography (HIC) in volatile mobile phases preserved native separation order and further resolved drug-to-antibody ratio (DAR) species of the antibody-drug conjugate brentuximab-vedotin. For each chromatography modality we further compare innovator and biosimilar antibodies, demonstrating the reproducibility of results in the proposed volatile compounds. Together, our results establish fluorinated ethylamines, in combination with ammonium acetate, as a universal volatile buffer system for native LC-MS, broadly applicable across major chromatographic modalities while maintaining compatibility with mass spectrometry.

To date, collision-induced dissociation and methods based on electron transfer dissociation are considered the standard approaches for the mass spectrometry analysis of N- and O-glycopeptides, respectively, allowing for identification of both peptide and glycan compositions. In recent years, alternative fragmentation methods such as ultraviolet photodissociation (UVPD) and more energetic versions of electron-based techniques (such as electron ionisation dissociation, EID) have been shown to be useful for the analysis of glycopeptides, producing rich information on the glycopeptide structure, including types of glycosidic linkages. We evaluated ultraviolet photodissociation (UVPD), electron ionization dissociation (EID), electron capture dissociation (ECD), and activated-ion ECD (AI-ECD) using an Orbitrap-Omnitrap hybrid for LC-MS analysis of complex N-glycopeptides. Both UVPD and EID generated extensive peptide, glycosidic, and cross-ring fragments, enabling detailed structural characterization. While ECD alone produced few glycopeptide identifications, AI-ECD significantly improved yields through supplemental vibrational activation. UVPD and EID achieved comparable identification efficiencies to stepped collisional dissociation and provided additional linkage information. These results establish the Omnitrap as a powerful platform for comprehensive glycoproteomic analysis and highlight the need for enhanced computational tools to interpret complex UVPD and EID spectra.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is widely used for epitope analysis of the antibodies. However, epitope analysis of glycoproteins is challenging because of the heterogeneity of attached N-linked glycans. Recent studies have reported methods where N-linked glycans were removed at low pH following hydrogen deuterium exchange reactions, but the methods for glycoproteins remain controversial. Here, we demonstrate the utility of using antigens with uniformed N-linked glycans in glycoprotein epitope analysis by HDX-MS. By treating HEK293 cells with kifunensine, we were able to prepare antigens mainly with N-linked high-mannose-type glycans. Analysis of epitopes of a monoclonal antibody S309 using antigens prepared with this method allowed us to identify an epitope that included the previously reported N-linked glycan attachment sites for this anti-body. We propose that using antigens with uniformed glycans should be effective for epitope analysis of glycoproteins, such as virus spike proteins covered by glycan shields. Moreover, we believe that this approach accelerates research on vaccines and neutralizing antibodies against viruses that escape host immunity through glycan shields or mutations.