Journal of the American Society for Mass Spectrometry最新文献

{"title":"Capturing Current Practices and Characterizing Measurement Reproducibility for Seized Drug Analysis Using Ambient Ionization Mass Spectrometry: An Interlaboratory Study","authors":"Edward Sisco*,&nbsp;, ,&nbsp;Dennis D. Leber,&nbsp;, and ,&nbsp;Arun S. Moorthy,&nbsp;","doi":"10.1021/jasms.5c00213","DOIUrl":"10.1021/jasms.5c00213","url":null,"abstract":"<p >The use of ambient ionization mass spectrometry (AI-MS) to aid in the preliminary screening of seized drug evidence has steadily increased over the past two decades. Unlike gas chromatography–mass spectrometry (GC-MS), where electron ionization using a single quadrupole analyzer is commonplace, a wide range of ionization sources and mass spectrometers can be used in AI-MS. Differences in instrument configuration can lead to substantial variability in the mass spectral data obtained. An interlaboratory study, consisting of 35 participants from 17 laboratories, was conducted to begin to understand the landscape and the differences in the data that are produced. Laboratories analyzed a series of 21 solutions across multiple days using their own instrumental methods. Mass spectra were extracted and compared to understand operator, within-lab, and between-lab reproducibility for common compounds and mixtures observed in seized drug analysis. In addition, five participants analyzed the 21 solutions using prescribed method parameters to measure reproducibility improvements when using identical instrumental conditions. Mass spectral reproducibility, measured through pairwise cosine similarity, was found to be generally quite high, regardless of sample type, instrument type, method, or operator. Low-fragmentation spectra showed the lowest variability, as they were dominated by intact protonated molecule peaks. Several potential issues that increased variability were identified, including carryover from mass calibrants, poor sample introduction, and mass spectrometer inlets that required cleaning. The use of uniform method parameters was shown to increase the reproducibility of mass spectra across laboratories, most notably at higher in-source collision-induced dissociation energies. This study provides initial insights into the current landscape of AI-MS in seized drug analysis and lays the foundation for future studies that can provide needed data for the development of documentary standards, standard methods, and possibly the establishment of error rates.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2239–2252"},"PeriodicalIF":2.7,"publicationDate":"2025-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/jasms.5c00213","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144938196","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}

{"title":"Optimization of q Value for Sensitive Detection of Uridine and Thymidine Nucleosides by MS3","authors":"Zheng Duan,&nbsp;, ,&nbsp;Chen Wang,&nbsp;, and ,&nbsp;Yinsheng Wang*,&nbsp;","doi":"10.1021/jasms.5c00245","DOIUrl":"10.1021/jasms.5c00245","url":null,"abstract":"<p >Liquid chromatography-tandem mass spectrometry has been widely used to quantify modified nucleosides enzymatically released from RNA and DNA. Compared to MS/MS, MS³ offers higher specificity for analytes in complex sample matrices because coeluting chemical interferences can be more effectively filtered out. To date, the detection of uracil- and thymine-containing nucleosides and their modified derivatives still suffers from poor sensitivity, owing, in part, to their relatively low proton affinities. In this study, we explored various settings of <i>q</i> values in the MS³ analysis of unmodified and chemically modified uridine and thymidine nucleosides on a linear ion trap mass spectrometer. Our results showed that increasing the <i>q</i> value led to markedly improved sensitivities for detecting some of these nucleosides using MS³. Our work suggests that optimizing the <i>q</i> value constitutes a useful approach for the highly sensitive detection of modified nucleosides and other types of analytes by MS³.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2017–2021"},"PeriodicalIF":2.7,"publicationDate":"2025-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144938332","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":"Absolute Quantitation of Coeluting Impurities in Peptide Drugs Using High Resolution Mass Spectrometry: Glucagon a Case Study in Pharmaceutical Development","authors":"Anusha Kodidasu,&nbsp;, ,&nbsp;Koushik Acharyya*,&nbsp;, and ,&nbsp;Vasanthakumar Ganga Ramu*,&nbsp;","doi":"10.1021/jasms.5c00105","DOIUrl":"10.1021/jasms.5c00105","url":null,"abstract":"<p >Synthetic peptide-based drugs provide customized therapeutic solutions, but developing a peptide medicine presents various challenges, especially in terms of impurity management. This holds true when traditional techniques like RP-HPLC fail to separate low-abundance coeluting impurities. In this regard, liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS) has proven to be effective for identifying and characterizing peptide impurities, although its application for accurate quantitation is still limited. This study developed and validated two quantitation strategies using UPLC-HRMS, employing full scan and pseudo-MRM (<i>p</i>-MRM) modes, to identify and accurately quantify two structurally similar coeluting peptide impurities (des-Gly⁴ glucagon and des-Thr⁵ glucagon) observed in synthetic glucagon. The methods exhibited high specificity and demonstrated good linearity (<i>R</i>² &gt; 0.99) across a concentration range of 0.25–25 μg/mL. The limits of detection (LODs) for des-Gly⁴-glucagon and des-Thr⁵-glucagon were determined to be as low as 0.01% and 0.02%, with limits of quantitation (LOQs) at 0.02% and 0.04%, respectively. Precision (RSD%) was recorded at less than 10%, and recovery ranged from 100% to 120%. A comparative analysis of the results indicated that both quantifitation methods performed similarly in terms of accuracy, precision, and recovery, producing comparable impurity estimates in three glucagon API samples. However, <i>p</i>-MRM showed slightly better linearity and sensitivity compared to the full scan EIC-based quantitation method.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2072–2078"},"PeriodicalIF":2.7,"publicationDate":"2025-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144938193","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":"Side Chain Driven Mass Spectral Fragmentation of Lys Containing Peptides: Cα-CO Bond Cleavage in Xxx-Lys-Xxx Sequences","authors":"Sanjeev Kumar,&nbsp;, ,&nbsp;Marimuthu Vijayasarathy,&nbsp;, ,&nbsp;Venkatesha M. Achanna,&nbsp;, and ,&nbsp;Padmanabhan Balaram*,&nbsp;","doi":"10.1021/jasms.5c00154","DOIUrl":"10.1021/jasms.5c00154","url":null,"abstract":"<p >Mass spectral fragmentation of the tripeptide amide Ala-Lys-Ala-amide (AKA*) yields a product ion at <i>m</i>/<i>z</i> 228.1, which corresponds to a neutral loss of 43 Da from the <i>b</i>₃ ion (<i>m</i>/<i>z</i> 271.1). Similar losses of 43 Da are observed from <i>b</i>_n ions in the hexapeptide AKAAKA* and tetrapeptide AKAA*. The role of the Lys2 residue, in mediating the neutral loss, is supported by the absence of the corresponding product ion in the MS² spectrum of AVA* and attenuation of the intensity in analogs containing ornithine/diaminobutyric acid residues at position 2. The role of the N-terminus amino group is suggested by the absence of this neutral loss when the residue Ala1 amino group is acetylated or dimethylated. In XKA* sequences, where X = Gly, Ala, Leu, Aib and Pro, the observed neutral losses from <i>b</i>₃ are 29, 43, 85, 57, and 69 Da, respectively, indicative of C^α-CO bond cleavage releasing the R-CH = NH fragment. Isotope labeling and comparison with mass spectral fragments generated from synthetic N^α-formyl Lys-Ala-amide (fKA*) establish the identity of the ion at <i>m</i>/<i>z</i> 228 in the MS² spectrum of AKA* as the <i>b</i> ion [fKA]. A charge remote fragmentation pathway, involving proton abstraction from the terminal amino group, by the Lys2 ε amino group, followed by C^α-CO bond cleavage, is proposed as a plausible mechanism for the observed noncanonical cleavage process.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 10","pages":"2134–2141"},"PeriodicalIF":2.7,"publicationDate":"2025-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144938325","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":"Interdomain Interactions Modulate Refolding Kinetics and Aggregation in a Monoclonal Antibody.","authors":"Philipp Trolese, Andrea Pierangelini, Benedetta Fongaro, Patrizia Polverino de Laureto","doi":"10.1021/jasms.5c00166","DOIUrl":"https://doi.org/10.1021/jasms.5c00166","url":null,"abstract":"<p><p>Understanding the structural determinants of antibody stability and aggregation is essential for therapeutic development. In this study, we investigated the unfolding and refolding behavior of bevacizumab under denaturing conditions using dynamic light scattering (DLS), circular dichroism (CD), and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Unfolding was induced by incubating the antibody in 4 M guanidine hydrochloride (Gnd-HCl), followed by refolding through dilution with 1 M Gnd-HCl. Each domain exhibited distinct unfolding kinetics: the C_H2 and V_H domains unfolded rapidly, while the C_H3 domain retained its structure until 45 min, consistent with its known thermodynamic stability. Aggregation, detected by DLS, was prevalent only after 120 min and overnight unfolding, coinciding with C_H3 destabilization. Notably, aggregation-prone regions were identified in both the Fc and Fab portions of the antibody. Specifically, interactions at the C_H2-C_H3 and C_H3-C_H3 interfaces appear disrupted upon C_H3 unfolding, leading to misfolded and aggregation-prone states in both domains. In parallel, the V_H CDR H1 region exhibited aberrant protection after refolding, suggesting its involvement in aggregation. These findings highlight the cooperative nature of C_H2-C_H3 refolding and underscore the critical role of the C_H3 stability in preventing aggregation. The involvement of both constant and variable domains emphasizes the complex, interdependent nature of monoclonal antibody aggregation. This work provides mechanistic insights into domain-specific contributions to folding and aggregation, offering guidance for the design of more stable therapeutic antibodies.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":2.7,"publicationDate":"2025-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144938349","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":"Detection and Characterization of Cyanometabolite Complexes between Microcystin-RR and a Microguanidine by UHPLC-ESI-MS","authors":"Sanduni H. Premathilaka,&nbsp; and ,&nbsp;Dragan Isailovic*,&nbsp;","doi":"10.1021/jasms.5c00165","DOIUrl":"https://doi.org/10.1021/jasms.5c00165","url":null,"abstract":"<p >Microcystins (MCs) are hepatotoxic cyclic peptides produced by cyanobacteria, with MC-RR being one of the most polar and commonly detected MC congeners in water collected during cyanobacterial harmful algal blooms (cHABs). Microguanidines (MGDs) are sulfated metabolites produced by <i>Microcystis</i> sp. that have not been reported during Lake Erie cHABs. Herein, both MGD AL772 (also abbreviated here as MGD) and previously unreported complex ions containing MC-RR and MGD AL772 were discovered after UHPLC-ESI-HRMS, MS/MS, and MS/MS/MS analyses of the Lake Erie cHAB samples in positive and negative modes. Initially, a triply charged [(MC-RR)₂-MGD+3H]³⁺ ion was detected at the same retention time as coeluting [MC-RR+2H]²⁺ and [MGD-H]⁻ ions. Characterization of this complex ion by HCD, CID, ETD and thiol derivatization indicates that two [MC-RR+2H]²⁺ ions are bonded to one [MGD-H]⁻, probably through interactions of positively charged guanidinium groups of arginine (R) residues and negatively charged MGD’s sulfate groups, to form the triply charged complex during ESI. Four other complexes, including [MC-RR-MGD] and its in-source fragment [(MC-RR-MGD)-SO₃], were also detected as positive and negative ions at the same time due to coelution of MC-RR and MGD. These findings not only underscore the importance of an efficient separation for quantification of cyanobacterial metabolites but also provide novel insights into their interactions during ESI, which enabled the present report on the complexes between MC-RR and a sulfated cyanobacterial metabolite, MGD AL772.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 9","pages":"1950–1958"},"PeriodicalIF":2.7,"publicationDate":"2025-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144929034","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":"Active Humidity Control Chamber for Desorption Electrospray Ionization-Mass Spectrometry Imaging Applications","authors":"Hawkins S. Shepard,&nbsp;Robert L. G. Gottschalk,&nbsp;Jody C. May and John A. McLean*,&nbsp;","doi":"10.1021/jasms.5c00111","DOIUrl":"https://doi.org/10.1021/jasms.5c00111","url":null,"abstract":"<p >Ambient ionization techniques enable mass spectrometry (MS) to expand into broader experimental contexts, although it is increasingly clear that results are influenced by the environmental conditions at the site of sampling. Desorption electrospray ionization (DESI), in particular, is affected by variations in relative humidity (RH) levels. Here we describe the design, development, and construction of an environmental control chassis that can actively modulate RH within ± 3% of user-defined set points across a broad humidity range (15%–70% RH). Preliminary characterization demonstrated differential analyte responses across a range of set points, with observed enhancement of leucine-enkephalin, sulfadimethoxine (negative mode), and maltose (positive mode) in response to increased humidity. The measurable differences in analyte signals across discrete humidity set points underscore the importance of environmental control in ambient ionization strategies. The humidity control system outlined here can be translated to other DESI platforms, with construction information provided herein.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 9","pages":"1995–1999"},"PeriodicalIF":2.7,"publicationDate":"2025-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/jasms.5c00111","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144929054","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}

{"title":"Combined Trapped Ion Mobility and Infrared Ion Spectroscopy Study of Protonation Sites in Aromatic Amines","authors":"Laura Finazzi,&nbsp;Lara van Tetering,&nbsp;Jelle L. Schuurman,&nbsp;Jonathan Martens,&nbsp;Giel Berden and Jos Oomens*,&nbsp;","doi":"10.1021/jasms.5c00164","DOIUrl":"https://doi.org/10.1021/jasms.5c00164","url":null,"abstract":"<p >The protonation site of aromatic amines in the gas phase has been under substantial debate, as it involves a subtle competition between the higher electronegativity of the amine nitrogen and the better charge delocalization ability of the fused aromatic rings. Previous studies have unambiguously shown, especially by ion mobility measurements, that higher-energy tautomers are easily observed depending on the experimental conditions in the ion source, including voltage settings and the type of solvent used in spray sources. Here, we use a combination of ion mobility and ion spectroscopy and focus on the tautomeric structure <i>after</i> ion mobility separation, in particular for protonated 1-aminonaphthalene and 1-aminoanthracene. We employ an atmospheric pressure chemical ionization (APCI) source, with a direct insertion probe to avoid any solvent influence, mounted on an FTICR mass spectrometer with a trapped ion mobility (TIMS) unit and optical access to the ions to perform infrared (IR) multiple-photon dissociation spectroscopy using the Free-Electron Laser for Infrared eXperiments (FELIX). TIMS analysis indeed reveals the presence of both N- and C-protonated species, but the IR spectra recorded in the ICR cell also suggest that mobilization and scrambling of the proton occur after TIMS separation. We computationally investigate the energetics of tautomerization and experimentally explore ion activation after TIMS separation.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 9","pages":"1940–1949"},"PeriodicalIF":2.7,"publicationDate":"2025-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/jasms.5c00164","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144929063","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}

{"title":"Faces of Mass Spectrometry/Pratik Jagtap.","authors":"Anne Brenner, J D Brookbank","doi":"10.1021/jasms.5c00264","DOIUrl":"https://doi.org/10.1021/jasms.5c00264","url":null,"abstract":"","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":2.7,"publicationDate":"2025-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144938356","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":"Collision-Induced Unfolding of High-m/z Native-like Protein Ions within a Trapped Ion Mobility Spectrometer","authors":"Olakunle O. Akinola,&nbsp; and ,&nbsp;Nicholas B. Borotto*,&nbsp;","doi":"10.1021/jasms.5c00246","DOIUrl":"https://doi.org/10.1021/jasms.5c00246","url":null,"abstract":"<p >Native mass spectrometry (nMS) is a powerful tool for the rapid characterization of protein ions and protein–ligand complexes. By coupling nMS with ion mobility spectrometry (IMS), and collisional activation, we can rapidly obtain insights into protein conformation, and stability can be rapidly obtained. Originally incapable of this workflow, recent work enabled this collision-induced unfolding (CIU) process on commercially available Bruker timsTOF instruments. This early work, however, faced challenges in transmitting larger proteins and sought to unfold only small proteins up to 29 kDa. In this study, we continue the development of this technique and optimized instrument settings to enable the transmission of proteins up to 8,000 Th. The technique also demonstrates the capability to sufficiently energize ions to unfold native-like dimers of superoxide dismutase and β-lactoglobulin and the 45 kDa monomeric ovalbumin. When this TIMS activation technique is applied to large protein ions, however, limited unfolding was observed for bovine serum albumin, and no unfolding was observed for immunoglobulin G likely reflecting the limit of activation for this workflow.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 9","pages":"1988–1994"},"PeriodicalIF":2.7,"publicationDate":"2025-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144929138","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}