Journal of the American Society for Mass Spectrometry最新文献_第4页

High-Specificity and Sensitivity Imaging of Neutral Lipids Using Salt-Enhanced MALDI TIMS 盐增强MALDI TIMS对中性脂的高特异性和灵敏度成像。

IF 2.7 2区化学

Journal of the American Society for Mass Spectrometry Pub Date : 2025-09-04 DOI: 10.1021/jasms.5c00202

Kameron R. Molloy, , , Martin Dufresne, , , Madeline E. Colley, , , Lukasz G. Migas, , , Raf Van de Plas, , and , Jeffrey M. Spraggins*,

{"title":"High-Specificity and Sensitivity Imaging of Neutral Lipids Using Salt-Enhanced MALDI TIMS","authors":"Kameron R. Molloy, , , Martin Dufresne, , , Madeline E. Colley, , , Lukasz G. Migas, , , Raf Van de Plas, , and , Jeffrey M. Spraggins*, ","doi":"10.1021/jasms.5c00202","DOIUrl":"10.1021/jasms.5c00202","url":null,"abstract":"Neutral lipids are vital to various cellular processes and disease pathologies. However, their characterization by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) remains challenging due to poor ionization efficiency and difficulties distinguishing subtle structural differences among numerous isomeric and isobaric species. In this study, we enhanced neutral lipid detection by incorporating isotonic metal–cation washes into our MALDI IMS sample preparation workflow. Resulting salt adducts improved neutral lipid isobar and isomer separation by using trapped ion mobility spectrometry (TIMS). This approach increased both sensitivity and specificity for neutral lipid IMS experiments across multiple organ types, including murine brain, rabbit adrenal gland, human colon, and human kidney. Comparative analyses revealed that the most effective salt wash was tissue-dependent. However, the Na+ carbonate buffer solution (CBS) wash showed the greatest overall increase in neutral lipid detection. These findings provide a robust framework for mapping neutral lipids across multiple tissues and disease states and allow for the detailed characterization of neutral lipid isomers and isobars in complex biological tissues.","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2213–2221"},"PeriodicalIF":2.7,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/jasms.5c00202","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144999299","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Evaluation of the QCxMS2 Method for the Calculation of Collision-Induced Dissociation Spectra via Automated Reaction Network Exploration QCxMS2方法在自动反应网络探索中计算碰撞诱导解离谱的评价

IF 2.7 2区化学

Journal of the American Society for Mass Spectrometry Pub Date : 2025-09-04 DOI: 10.1021/jasms.5c00234

Johannes Gorges, , , Marianne Engeser*, , and , Stefan Grimme*,

{"title":"Evaluation of the QCxMS2 Method for the Calculation of Collision-Induced Dissociation Spectra via Automated Reaction Network Exploration","authors":"Johannes Gorges, , , Marianne Engeser*, , and , Stefan Grimme*, ","doi":"10.1021/jasms.5c00234","DOIUrl":"10.1021/jasms.5c00234","url":null,"abstract":"Collision-induced dissociation mass spectrometry (CID-MS) is an important tool in analytical chemistry for the structural elucidation of unknown compounds. The theoretical prediction of the CID spectra plays a critical role in supporting and accelerating this process. To this end, we adapt the recently developed QCxMS2 program originally designed for the calculation of electron ionization (EI) spectra to enable the computation of CID-MS. To account for the fragmentation conditions characteristic of CID within the automated reaction network discovery approach of QCxMS2 we adapted the internal energy distribution to match the experimental conditions. This distribution can be adjusted via a single parameter to approximate various activation settings, thereby eliminating the need for explicit simulations of the collisional process. We evaluate our approach on a test set of 13 organic molecules with diverse functional groups, compiled specifically for this study. All reference spectra were recorded consistently under the same measurement conditions, including both CID and higher-energy collisional dissociation (HCD) modes. Overall, QCxMS2 achieves a good average entropy similarity score (ESS) of 0.687 for the HCD spectra and 0.773 for the CID spectra. The direct comparison to experimental data demonstrates that the QCxMS2 approach, even without explicit modeling of collisions, is generally capable of computing both CID and HCD spectra with reasonable accuracy and robustness. This highlights its potential as a valuable tool for integration into structure elucidation workflows in analytical mass spectrometry.","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2276–2289"},"PeriodicalIF":2.7,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144991149","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}

引用次数: 0

Experiment and Simulation Study of Automatic Temperature Control FAIMS Chip 温度自动控制FAIMS芯片的实验与仿真研究。

IF 2.7 2区化学

Journal of the American Society for Mass Spectrometry Pub Date : 2025-09-04 DOI: 10.1021/jasms.4c00427

Xiaoxia Du, , , Haonan Liang, , , Yao Li, , , Xingxing A Luo, , , Yuqiao Zhang, , and , Hua Li*,

{"title":"Experiment and Simulation Study of Automatic Temperature Control FAIMS Chip","authors":"Xiaoxia Du, , , Haonan Liang, , , Yao Li, , , Xingxing A Luo, , , Yuqiao Zhang, , and , Hua Li*, ","doi":"10.1021/jasms.4c00427","DOIUrl":"10.1021/jasms.4c00427","url":null,"abstract":"The temperature of the carrier gas will affect the performance of high-field asymmetric ion waveform mobility spectrometry (FAIMS), changing the height and position of the peaks of the FAIMS spectrum. In this study, we explored the influence of temperature on the FAIMS spectrum through experiment and simulation. In the experiment, the PCB self-heating temperature control FAIMS system was used to study the effects of temperature changes on the FAIMS spectra of ethanol, acetic acid, acetone, and ethyl acetate, and the coefficient solving methods of mobility a2 and a4 were derived in detail. Based on the experimental data, the mobility coefficients a2 and a4 were solved and replaced by the simulation software SIMION. The results show that as the temperature increases, the peak height of the spectrum of FAIMS decreases, and there will be a certain amount of offset in the position of the peaks in the spectrum. Through simulation, it is found that during the heating process, the number of ions decreases from approximately 60 to 6, and the compensation voltages of acetone, ethanol, acetic acid, and ethyl acetate change by about 1.1, 0.7, 0.3, and 0.1 V, respectively. This study provides a detailed solution process for the calculation of the ion mobility coefficients in the SIMION simulation, and the use of temperature control can effectively improve the resolution and accuracy of FAIMS in volatile organic compounds (VOCs) detection.","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2026–2035"},"PeriodicalIF":2.7,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144999296","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}

引用次数: 0

An Online Buffer Exchange Platform for Charge Detection Mass Spectrometry Analysis of AAVs and AAV-Antibody Complexes aav及其抗体复合物电荷检测质谱分析的在线缓冲交换平台

IF 2.7 2区化学

Journal of the American Society for Mass Spectrometry Pub Date : 2025-09-03 DOI: 10.1021/jasms.5c00206

Chen Du, , , Victoria C. Cotham*, , , Garima Thakur, , , Sheldon Mink, , , Andrew D. Tustian, , , Sven Møller-Tank, , , Shunhai Wang*, , and , Ning Li,

{"title":"An Online Buffer Exchange Platform for Charge Detection Mass Spectrometry Analysis of AAVs and AAV-Antibody Complexes","authors":"Chen Du, , , Victoria C. Cotham*, , , Garima Thakur, , , Sheldon Mink, , , Andrew D. Tustian, , , Sven Møller-Tank, , , Shunhai Wang*, , and , Ning Li, ","doi":"10.1021/jasms.5c00206","DOIUrl":"10.1021/jasms.5c00206","url":null,"abstract":"Adeno-associated viruses (AAVs) are leading vectors in gene therapy that have demonstrated great potential in combating a wide range of human diseases. To enhance specificity and reduce dosing requirements, antibody-retargeted AAVs have emerged as a promising strategy to redirect vectors to novel receptors, thereby achieving improved efficacy and safety. However, effective characterization of AAVs and AAV-antibody complexes is complicated by heterogeneities that arise from variations in capsid protein assembly, genome integrity, and antibody decorations, demanding high-resolution techniques beyond traditional methods. Charge detection mass spectrometry (CDMS) is an emerging technique that effectively characterizes complex AAV samples by directly measuring ion charge. This technique is typically performed using static infusion-based nanoelectrospray ionization with long acquisition times, which is highly manual and subject to unstable spray and sample instability. To address limitations of static infusion in CDMS, we have developed a novel automated platform: size-exclusion chromatography-based flow injection coupled with CDMS (SEC-FI-CDMS). This platform streamlines and automates sample introduction with a dual-flow setup, enabling fast online desalting in high-flow mode and stable, prolonged infusion in low-flow mode for Orbitrap-based CDMS analysis. It accurately determines AAV genome packaging ratios and masses, differentiates capsid assembly and payload variations, and resolves antibody decoration on AAV-antibody complexes. This comprehensive characterization supports AAV manufacturing and development, paving the way for more effective next generation gene therapies.","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2230–2238"},"PeriodicalIF":2.7,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/jasms.5c00206","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144938273","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Characterization of Micrococcus luteus Lipidome Containing Novel Lipid Families by Multiple Stage Linear Ion-Trap with High Resolution Mass Spectrometry 用多级线性离子阱-高分辨质谱法鉴定含有新型脂系的黄体微球菌脂质体。

IF 2.7 2区化学

Journal of the American Society for Mass Spectrometry Pub Date : 2025-09-02 DOI: 10.1021/jasms.5c00133

Brian A. Kleiboeker, and , Fong-Fu Hsu*,

{"title":"Characterization of Micrococcus luteus Lipidome Containing Novel Lipid Families by Multiple Stage Linear Ion-Trap with High Resolution Mass Spectrometry","authors":"Brian A. Kleiboeker,  and , Fong-Fu Hsu*, ","doi":"10.1021/jasms.5c00133","DOIUrl":"10.1021/jasms.5c00133","url":null,"abstract":"Micrococcus luteus (M. luteus) is a ubiquitous, long-existing Gram-positive bacterium with a distinctive yellow or golden color. It is a model organism for laboratory studies due to its small genome and ease of cultivation. However, only limited knowledge about its constituent lipid structure is known, and its entire lipid profile remains unclear. Here, we applied linear ion trap (LIT) multiple-stage mass spectrometry (MSn) with high resolution for structural characterization of the native lipid extract, along with GC/MS analysis of the acid hydrolysate to reveal the structural details of the entire lipidome, which includes the major phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylinositol (PI), dimannosyl diacylglycerol (DMDG), and minor diacylglycerol (DAG) lipid families. Importantly, we also found two extra lipid families, the new phosphatidyl 1,3-propanediol and the known polyprenyl 1-phosphosate that was not previously reported for M. luteus. We also revealed the unique lipidome simplified by the dominance of branched 15:0-fatty acid substituents (>90% branched 15:0-FA with anteiso-15:0 to iso-15:0 abundance ratio of 4/1), which is in line with the small genome of M. luteus. In addition, the applied LIT MSn mass spectrometry revealed a fragmentation pathway that undergoes internal loss of a glycerol residue specific to DMDG, leading to its structural characterization.","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2117–2125"},"PeriodicalIF":2.7,"publicationDate":"2025-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144938329","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}

引用次数: 0

Profiling Multiple Steroid Subclasses Enabled by Rounded Turn SLIM Ion Mobility. 通过圆转弯SLIM离子迁移率分析多个类固醇子类。

IF 2.7 2区化学

Journal of the American Society for Mass Spectrometry Pub Date : 2025-09-01 DOI: 10.1021/jasms.5c00218

Cole L Frank, Breland M Jones, Emmaleigh Efird, Makenna Hoover, Selena Kingsley, Heidi M Sabatini, Christopher D Chouinard

{"title":"Profiling Multiple Steroid Subclasses Enabled by Rounded Turn SLIM Ion Mobility.","authors":"Cole L Frank, Breland M Jones, Emmaleigh Efird, Makenna Hoover, Selena Kingsley, Heidi M Sabatini, Christopher D Chouinard","doi":"10.1021/jasms.5c00218","DOIUrl":"https://doi.org/10.1021/jasms.5c00218","url":null,"abstract":"Ion mobility-mass spectrometry has shown tremendous potential for improving the analysis of various subclasses of steroids. Its speed and ability to separate isobaric and isomeric species makes it ideal for biomedical, clinical, food, environmental, and antidoping analyses. But while other high-resolution ion mobility (HRIM) techniques have begun to see increased use in steroidomics, Structures for Lossless Ion Manipulations (SLIM) is a relative newcomer to the field. HRIM methods are generally challenged with low molecular weight (MW < 400) analysis, but a recently developed SLIM platform has incorporated rounded turns, helium buffer gas, and increased SLIM RF frequency to enable improved low mass transmission. In the present work, we investigate this low mass-tailored SLIM system for the analysis of anabolic androgens, bile acids, endocrine hormones, and ketosteroids. Linear regression-corrected SLIMCCSHe (CCS = collision cross section) values show excellent agreement with values obtained via direct measurement with a drift tube instrument but also underlie the importance of using class- and adduct-specific correction factors. Next, separation performance was evaluated for a range of challenging isomers and demonstrated good resolution for species with ΔCCS of <1%. Finally, the new rounded turn system was compared quantitatively with the traditional 90° (square turn) system in both nitrogen and helium buffer gases and revealed significantly improved signal across the board for rounded turn analyses with helium. Overall, this low mass-tailored system has remarkable promise for development of rapid, targeted steroid analysis in a range of applications.","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144938354","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}

引用次数: 0

Relational Graph Convolutional Network for Robust Mass Spectrum Classification 鲁棒质谱分类的关系图卷积网络。

IF 2.7 2区化学

Journal of the American Society for Mass Spectrometry Pub Date : 2025-09-01 DOI: 10.1021/jasms.5c00055

Raphaël La Rocca, , , Anthony Cioppa, , , Enrico Ferrarini, , , Monica Höfte, , , Marc Van Droogenbroeck, , , Edwin De Pauw, , , Gauthier Eppe, , and , Loïc Quinton*,

{"title":"Relational Graph Convolutional Network for Robust Mass Spectrum Classification","authors":"Raphaël La Rocca, , , Anthony Cioppa, , , Enrico Ferrarini, , , Monica Höfte, , , Marc Van Droogenbroeck, , , Edwin De Pauw, , , Gauthier Eppe, , and , Loïc Quinton*, ","doi":"10.1021/jasms.5c00055","DOIUrl":"10.1021/jasms.5c00055","url":null,"abstract":"Supervised machine learning methods have shown impressive performance in interpreting mass signals and automatically segmenting spatially meaningful regions in Mass Spectrometry Imaging (MSI). Such segmentation generates maps that provide researchers with valuable insights into sample composition and serve as a foundation for downstream statistical analyses. However, these models often require data set-specific preprocessing and do not fully exploit the rich mass features available in high-resolution mass spectrometry (HRMS). Unlike low-resolution mass spectrometry, HRMS reveals additional features such as mass defects and repeated mass differences that carry important chemical information. In this work, we propose a novel deep learning architecture based on a Relational Graph Convolutional Network (R-GCN) that captures and leverages those HRMS mass features. Our model explicitly encodes structural features such as mass defects and known mass differences to represent each spectrum as a graph, enabling the learning of associations between chemically related ion families. To the best of our knowledge, no existing deep learning models for MSI classification incorporate this level of chemically informed mass structure. Most existing methods treat spectra as flat vectors or image-like inputs, thereby ignoring the underlying mass relationships. We evaluate our R-GCN approach against several conventional machine learning and deep learning baselines across diverse MSI data sets, demonstrating its robustness to common signal variations (e.g., mass shift, ion loss). Finally, we integrate Class Activation Mapping (CAM) to enhance model interpretability, enabling the identification of ion families that are relevant to specific biological or spatial regions.","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2036–2047"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144938374","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}

引用次数: 0

Effect of pH on the Kinetics of Cysteine-to-Cystine Conversion in Thin Films Formed by ESI Microdroplet Deposition pH对ESI微滴沉积薄膜中半胱氨酸到胱氨酸转化动力学的影响。

IF 2.7 2区化学

Journal of the American Society for Mass Spectrometry Pub Date : 2025-08-29 DOI: 10.1021/jasms.5c00195

Marta Managò, , , Chiara Salvitti, , , Anna Troiani, , , Alessia Di Noi, , , Andreina Ricci, , and , Federico Pepi*,

引用次数: 0

Simulating Collision-Induced Dissociation Tandem Mass Spectrometry (CID-MS/MS) for the Blood Exposome Database Using Quantum Chemistry Methods - A Pilot Study 使用量子化学方法模拟碰撞诱导解离串联质谱（CID-MS/MS）用于血液暴露体数据库-一项试点研究。

IF 2.7 2区化学

Journal of the American Society for Mass Spectrometry Pub Date : 2025-08-28 DOI: 10.1021/jasms.5c00179

Jesi Lee, and , Dinesh Barupal*,

{"title":"Simulating Collision-Induced Dissociation Tandem Mass Spectrometry (CID-MS/MS) for the Blood Exposome Database Using Quantum Chemistry Methods - A Pilot Study","authors":"Jesi Lee,  and , Dinesh Barupal*, ","doi":"10.1021/jasms.5c00179","DOIUrl":"10.1021/jasms.5c00179","url":null,"abstract":"A significant number of compounds in exposome databases and chemical inventories lack mass spectral data due to the nonavailability of reference standards. To address this limitation, computational chemistry methods can be utilized to extend mass spectral libraries for a set of chemicals. In this pilot study, we employed quantum-chemistry-based software QCxMS to generate collision-induced dissociation mass spectra for 121 compounds from the Blood Exposome Database. We developed a scalable computational framework that integrates QCxMS and additional tools, utilizing a grid-based parameter selection strategy and defined coverage criteria. Our approach systematically explored protomeric isomers and applied predefined parameter combination sets sequentially based on molecular structures. This workflow produced high-quality in silico spectra for 81 compounds that achieved entropy similarity scores ≥700 and at least two matching fragment ions against the NIST23 library, yielding 71% spectral coverage. These results highlight the importance of optimizing simulation parameters and accounting for protomeric diversity to enhance the spectral quality and computational efficiency. This workflow provides a practical strategy to add mass spectral data for most compounds in the Blood Exposome Database at reasonable computational cost, supporting the spectral library expansion for improved compound annotation in exposomics.","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2171–2180"},"PeriodicalIF":2.7,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144938326","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}

引用次数: 0

Electron Capture Dissociation for Discovery Top-Down Proteomics of Peptides and Small Proteins on Chromatographic Time Scales 电子捕获解离在色谱时间尺度上发现多肽和小蛋白自上而下的蛋白质组学。

IF 2.7 2区化学

Journal of the American Society for Mass Spectrometry Pub Date : 2025-08-28 DOI: 10.1021/jasms.5c00116

Lester S. Manly, , , Anne M. Roberts, , , Joseph S. Beckman, , and , Blaine R. Roberts*,

{"title":"Electron Capture Dissociation for Discovery Top-Down Proteomics of Peptides and Small Proteins on Chromatographic Time Scales","authors":"Lester S. Manly, , , Anne M. Roberts, , , Joseph S. Beckman, , and , Blaine R. Roberts*, ","doi":"10.1021/jasms.5c00116","DOIUrl":"10.1021/jasms.5c00116","url":null,"abstract":"Bottom-up proteomics introduces proteoform ambiguity due to the loss of connectivity between peptides and their original proteoforms. Top-down proteomics (TDP) removes the ambiguity through the direct identification and characterization of intact proteoforms and their respective post-translational modifications (PTM). Electron capture dissociation (ECD) is an efficient and gentle peptide and protein fragmentation strategy that can be used for both bottom-up and top-down approaches. Here, we used an Agilent 6550 Q-TOF mass spectrometer retrofitted with an e-MSion ECD cell. Top-down sequencing capabilities of the cell were evaluated by sequencing of intact peptides and proteins on high-performance liquid chromatography (HPLC) time scales. Amyloid beta 1-40 (Aβ40) was first tested due to its pathophysiological role in Alzheimer’s disease and served as our large peptide standard. We sequenced Aβ40 via reverse-phase HPLC-MS and achieved 95% sequence coverage on chromatographic time scales utilizing a data-dependent acquisition (DDA)-based method. Acetone-precipitated protein extracts from human brain were then separated by HPLC and analyzed with a DDA method, which identified 16 proteoforms between 2 and 17 kDa with sequence coverage ranging from 7 to 90% based on proteoform size and composition. In addition to proteoform identification, ECD fragmentation distinguished multiple isoaspartate modifications from aspartate, as well as accurately differentiating leucine from isoleucine residues directly from the human brain sample. Here, we observed isoaspartate within a thymosin beta-4 proteoform. Additionally, we demonstrated the differentiation of leucine and isoleucine within a subunit of ubiquitin. This study advances the application of LC-Q-TOF instrumentation for discovery-based top-down proteomics utilizing ECD as enabled by the e-MSion ECD cell.","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2079–2093"},"PeriodicalIF":2.7,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/jasms.5c00116","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144938376","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0