{"title":"Discovery and Characterization of Novel G0 Glycan Isomers in an Afucosylated Therapeutic Antibody Using Liquid Chromatography-Mass Spectrometry.","authors":"Miyang Li, Sean Shen, Simon Letarte, Tawnya Flick","doi":"10.1021/jasms.4c00474","DOIUrl":"https://doi.org/10.1021/jasms.4c00474","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143750545","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Masashi Serizawa, Pieter van Delft, Peter J Schoenmakers, Ron A H Peters, Andrea F G Gargano
{"title":"Size-Exclusion Chromatography-Electrospray-Ionization Mass Spectrometry To Characterize End Group and Chemical Distribution of Poly(lactide-<i>co</i>-glycolide) Copolymers.","authors":"Masashi Serizawa, Pieter van Delft, Peter J Schoenmakers, Ron A H Peters, Andrea F G Gargano","doi":"10.1021/jasms.4c00447","DOIUrl":"https://doi.org/10.1021/jasms.4c00447","url":null,"abstract":"<p><p>The characterization of the microstructure of <i>in vivo</i> 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.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143750605","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Seth W Croslow, Chen H Sirois, Jonathan V Sweedler
{"title":"Factorial-Design-Based Optimization of a Commercial MALDI-2 timsTOF Mass Spectrometer for Lipid Analysis.","authors":"Seth W Croslow, Chen H Sirois, Jonathan V Sweedler","doi":"10.1021/jasms.4c00424","DOIUrl":"https://doi.org/10.1021/jasms.4c00424","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143741744","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jaskiran Kaur, Kawthar Z Alzarieni, Judy Kuan Yu Liu, Wanru Li, Ruth O Anyaeche, Hilkka I Kenttämaa
{"title":"Methane Elimination upon Collision-Activated Dissociation of Selected Product Ions Formed Upon Gas-Phase Reactions of (Isopropenyloxy)trimethylsilane Facilitates the Mass Spectrometric Classification of Specific Oxygen- and/or Nitrogen-Containing Compounds.","authors":"Jaskiran Kaur, Kawthar Z Alzarieni, Judy Kuan Yu Liu, Wanru Li, Ruth O Anyaeche, Hilkka I Kenttämaa","doi":"10.1021/jasms.4c00504","DOIUrl":"https://doi.org/10.1021/jasms.4c00504","url":null,"abstract":"<p><p>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 <i>methane</i> 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.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143708002","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Functional Testing and Localization of Tyrosine Sulfation in a Trispecific Antibody.","authors":"Armelle Martelet, Valerie Garrigue, Hélène Le Borgne, Claire Borel, Sylvie Alexandre, Ronan Crépin, Bruno Genet, Haichuan Liu, Yuzhuo Zhang, Séverine Clavier","doi":"10.1021/jasms.4c00432","DOIUrl":"https://doi.org/10.1021/jasms.4c00432","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143708000","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Mapping Binding Domains of Viral and Allergenic Proteins with Dual-Cleavable Cross-Linking Technology.","authors":"Akash Talukder, Saiful M Chowdhury","doi":"10.1021/jasms.4c00398","DOIUrl":"https://doi.org/10.1021/jasms.4c00398","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-03-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143690766","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Mapping Binding Domains of Viral and Allergenic Proteins with Dual-Cleavable Cross-Linking Technology","authors":"Akash Talukder, and , Saiful M. Chowdhury*, ","doi":"10.1021/jasms.4c0039810.1021/jasms.4c00398","DOIUrl":"https://doi.org/10.1021/jasms.4c00398https://doi.org/10.1021/jasms.4c00398","url":null,"abstract":"<p >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.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 4","pages":"721–731 721–731"},"PeriodicalIF":3.1,"publicationDate":"2025-03-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143746241","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Michelle Rajkovic, Thorsten Benter, Walter Wißdorf
{"title":"Investigation of Surface-Induced Dissociation Processes via Molecular Dynamics Simulations of Wall Collisions of Large Droplets Produced by Electrospray Ionization.","authors":"Michelle Rajkovic, Thorsten Benter, Walter Wißdorf","doi":"10.1021/jasms.4c00449","DOIUrl":"https://doi.org/10.1021/jasms.4c00449","url":null,"abstract":"<p><p>Electrospray ionization is one of the most utilized ionization techniques in atmospheric pressure mass spectrometry. Recent experimentally reported results are in disagreement with fundamentals revolving around ESI droplet sizes and their lifetimes. Specifically, much larger droplet sizes and longer lifetimes have been experimentally observed to exist in typical ESI ion sources. Experiments involving a custom scan mode on a triple quadrupole system have shown that high-mass fragments of large ESI droplets can be observed in mass spectra. Initial hypotheses rationalizing these results were focused on the creation of droplet fragments by collision-induced dissociation (CID). The collision energy accumulated by CID is most likely too small to lead to the observed mass spectra. In response, surface-induced dissociation (SID) was proposed as an additional mechanism to provide large amounts of collision energy to the droplets. The present work thus investigates the possible fragmentation pathways and dynamics of droplet fragments resulting from aspirated ESI droplets upon surface collisions through classical molecular dynamics simulations. Different types of collisions are simulated, where the impact of the simulated droplet fragments is either frontal or angled. The resulting fragmentation dynamics are thoroughly analyzed, showing the possibility for charged fragments to be liberated through SID events. A second, much larger droplet fragment is employed to illustrate the altered collision dynamics found for such larger aggregates, where no charged clusters are released through the surface collision. Since approximated force fields have to be used to model the interactions between the particles observed in the simulation, a sensitivity study is carried out regarding the critical parameters governing such processes. Further modifications of the MD system have to be carried out, including more realistic walls and much larger ESI droplets, to clarify the possibility of charged fragment releases from larger droplet fragments through SID.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143673151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Pneumatically Assisted Microfluidic Probe for Enhanced Mass Spectrometry Imaging Performance.","authors":"Li-Xue Jiang, Julia Laskin","doi":"10.1021/jasms.5c00011","DOIUrl":"https://doi.org/10.1021/jasms.5c00011","url":null,"abstract":"<p><p>A pneumatically assisted microfluidic probe (MFP) with two microfluidic channels has been developed for nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI) of biological samples. This design simplifies the experimental setup, making it independent of the vacuum suction at the mass spectrometer inlet. The implementation of pneumatically assisted solvent flow through the probe enables stable, high solvent flow rates required to maintain a consistent liquid bridge during high-throughput MSI experiments. This approach addresses challenges associated with using MFP nano-DESI probes on mass spectrometers that have limited vacuum suction and the operation of MFPs with small microfluidic channels. We demonstrate the robustness of the pneumatically assisted MFP with 30 μm channels, which cannot be used for high-throughput MSI experiments without pneumatic assistance, by successfully imaging five mouse brain tissue sections without interruptions.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143668541","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chris Heintz, Hendrik Kersten, Thorsten Benter and Walter Wißdorf*,
{"title":"Signatures of Charged Droplets from ESI: A Statistical Analysis of Non-summed Mass Spectra Compared to APCI","authors":"Chris Heintz, Hendrik Kersten, Thorsten Benter and Walter Wißdorf*, ","doi":"10.1021/jasms.4c0050810.1021/jasms.4c00508","DOIUrl":"https://doi.org/10.1021/jasms.4c00508https://doi.org/10.1021/jasms.4c00508","url":null,"abstract":"<p >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.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 4","pages":"839–849 839–849"},"PeriodicalIF":3.1,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143746098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}