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Rapid and Non-Targeted Qualitative and Quantitative Detection of miRNA in Complex Biological Samples Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry with a 3-Aminoquinoline and 2′,4′,6′-Trihydroxyacetophenone Ionic Liquid Matrix
IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-30 DOI: 10.1021/jasms.4c0036910.1021/jasms.4c00369
Shiwen Zhou, Jiancong Liao, Kailin Jiang, Huiwen Wang, Yaqin Liu, Hangming Xiong, Ping Wang, Yuanjiang Pan* and Hongru Feng*, 

A novel ionic liquid MALDI matrix, 3-aminoquinoline/2′,4′,6′-trihydroxyacetophenone monohydrate (3-AQ/THAP), was developed for the rapid qualitative and quantitative detection of miRNA from biological samples. Compared to the traditional matrix 2,5-dihydroxybenzoic acid (DHB) and previously reported oligonucleotide-specific matrices, such as 3-aminopicolinic acid (3-APA), 3-hydroxypicolinic acid (3-HPA), and 6-aza-2-thiothymine (ATT), the 3-AQ/THAP matrix offers several advantages. It produces fewer alkali metal adduct peaks, exhibits higher sensitivity, and ensures better spot-to-spot repeatability. The 3-AQ/THAP matrix provides broader mass coverage and can effectively detect oligonucleotides ranging from 3-mer to 50-mer while delivering single-base resolution and sequence information. Additionally, it significantly reduces the “sweet spot” effect with an RSD of less than 7% over 36 single-spot analyses. For oligonucleotides ranging from 16-mer to 26-mer, the linear range extends from 0.4 μM to 40 μM per spot, with an R2 greater than 0.988. Finally, miRNA in human plasma, fetal equine serum, and fetal bovine serum was successfully identified both qualitatively and quantitatively using the 3-AQ/THAP matrix. This matrix demonstrated excellent practicability for the detection of multiple miRNAs in complex biological samples.

{"title":"Rapid and Non-Targeted Qualitative and Quantitative Detection of miRNA in Complex Biological Samples Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry with a 3-Aminoquinoline and 2′,4′,6′-Trihydroxyacetophenone Ionic Liquid Matrix","authors":"Shiwen Zhou,&nbsp;Jiancong Liao,&nbsp;Kailin Jiang,&nbsp;Huiwen Wang,&nbsp;Yaqin Liu,&nbsp;Hangming Xiong,&nbsp;Ping Wang,&nbsp;Yuanjiang Pan* and Hongru Feng*,&nbsp;","doi":"10.1021/jasms.4c0036910.1021/jasms.4c00369","DOIUrl":"https://doi.org/10.1021/jasms.4c00369https://doi.org/10.1021/jasms.4c00369","url":null,"abstract":"<p >A novel ionic liquid MALDI matrix, 3-aminoquinoline/2′,4′,6′-trihydroxyacetophenone monohydrate (3-AQ/THAP), was developed for the rapid qualitative and quantitative detection of miRNA from biological samples. Compared to the traditional matrix 2,5-dihydroxybenzoic acid (DHB) and previously reported oligonucleotide-specific matrices, such as 3-aminopicolinic acid (3-APA), 3-hydroxypicolinic acid (3-HPA), and 6-aza-2-thiothymine (ATT), the 3-AQ/THAP matrix offers several advantages. It produces fewer alkali metal adduct peaks, exhibits higher sensitivity, and ensures better spot-to-spot repeatability. The 3-AQ/THAP matrix provides broader mass coverage and can effectively detect oligonucleotides ranging from 3-mer to 50-mer while delivering single-base resolution and sequence information. Additionally, it significantly reduces the “sweet spot” effect with an RSD of less than 7% over 36 single-spot analyses. For oligonucleotides ranging from 16-mer to 26-mer, the linear range extends from 0.4 μM to 40 μM per spot, with an R<sup>2</sup> greater than 0.988. Finally, miRNA in human plasma, fetal equine serum, and fetal bovine serum was successfully identified both qualitatively and quantitatively using the 3-AQ/THAP matrix. This matrix demonstrated excellent practicability for the detection of multiple miRNAs in complex biological samples.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 3","pages":"495–503 495–503"},"PeriodicalIF":3.1,"publicationDate":"2025-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143547799","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
Remembrance: Dr. Jean H. Futrell
IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-30 DOI: 10.1021/jasms.4c0047910.1021/jasms.4c00479
Richard D. Smith*,  and , David W. Koppenaal*, 
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引用次数: 0
Quantitative MALDI-TOF Mass Spectrometry of Star-Shaped Polylactides Based on Chromatographic Hyphenation
IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-30 DOI: 10.1021/jasms.4c0049110.1021/jasms.4c00491
Jana Falkenhagen, Mete-Sungur Dalgic and Steffen M. Weidner*, 

The end groups of three- and four-arm star-shaped polylactides (PLA) with trimethylolpropane and pentaerythritol core structures were functionalized with acetic acid. Reaction products with different degrees of functionalization were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Additional gradient elution liquid adsorption chromatography (GELAC) measurements were performed to determine the degree of functionalization. This technique enabled clear separation and sufficient quantification of the formed species. These chromatographic data could be used inversely to quantify mass spectrometric results, which are usually biased by the unknown ionization probabilities of different polymer end group structures. Our results showed that, in this particular case, the peak intensity in the MALDI-TOF mass spectra can be used to semiquantitatively determine the degree of functionalization in incompletely functionalized multiarm PLA.

{"title":"Quantitative MALDI-TOF Mass Spectrometry of Star-Shaped Polylactides Based on Chromatographic Hyphenation","authors":"Jana Falkenhagen,&nbsp;Mete-Sungur Dalgic and Steffen M. Weidner*,&nbsp;","doi":"10.1021/jasms.4c0049110.1021/jasms.4c00491","DOIUrl":"https://doi.org/10.1021/jasms.4c00491https://doi.org/10.1021/jasms.4c00491","url":null,"abstract":"<p >The end groups of three- and four-arm star-shaped polylactides (PLA) with trimethylolpropane and pentaerythritol core structures were functionalized with acetic acid. Reaction products with different degrees of functionalization were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Additional gradient elution liquid adsorption chromatography (GELAC) measurements were performed to determine the degree of functionalization. This technique enabled clear separation and sufficient quantification of the formed species. These chromatographic data could be used inversely to quantify mass spectrometric results, which are usually biased by the unknown ionization probabilities of different polymer end group structures. Our results showed that, in this particular case, the peak intensity in the MALDI-TOF mass spectra can be used to semiquantitatively determine the degree of functionalization in incompletely functionalized multiarm PLA.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 3","pages":"613–621 613–621"},"PeriodicalIF":3.1,"publicationDate":"2025-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/jasms.4c00491","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143547798","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
Correction to “Advancing the Prediction of MS/MS Spectra Using Machine Learning”
IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-28 DOI: 10.1021/jasms.5c0000110.1021/jasms.5c00001
Julia Nguyen, Richard Overstreet, Ethan King and Danielle Ciesielski*, 
{"title":"Correction to “Advancing the Prediction of MS/MS Spectra Using Machine Learning”","authors":"Julia Nguyen,&nbsp;Richard Overstreet,&nbsp;Ethan King and Danielle Ciesielski*,&nbsp;","doi":"10.1021/jasms.5c0000110.1021/jasms.5c00001","DOIUrl":"https://doi.org/10.1021/jasms.5c00001https://doi.org/10.1021/jasms.5c00001","url":null,"abstract":"","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 3","pages":"627 627"},"PeriodicalIF":3.1,"publicationDate":"2025-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143547795","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
A Fast-Pass, Desorption Electrospray Ionization Mass Spectrometry Strategy for Untargeted Metabolic Phenotyping
IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-27 DOI: 10.1021/jasms.4c0045910.1021/jasms.4c00459
Hawkins S. Shepard, Jody C. May, Baltazar E. Zuniga, Joshua P. Abraham, Brian F. Pfleger, Jamey D. Young and John A. McLean*, 

Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) provides direct analytical readouts of small molecules that can be used to characterize the metabolic phenotypes of genetically engineered bacteria. In an effort to accelerate the time frame associated with the screening of mutant libraries, we have developed a high-throughput DESI-MSI analytical workflow implementing a single raster line-scan strategy that facilitates the collection of location-resolved molecular information from engineered strains on a subminute time scale. Evaluation of this “Fast-Pass” DESI-MSI phenotyping workflow on analytical standards demonstrated the capability of acquiring full metabolic profiling information with a throughput of ∼40 s per sample. This Fast-Pass strategy was implemented in the analysis of genetically edited Escherichia coli strains that have been engineered to produce various free-fatty acids (FFAs) for applications relevant to biofuels. Due to the untargeted nature of DESI-MSI, the investigation of these strains yielded molecular information for both global metabolites and targeted detection of accumulated bioproducts, allowing simultaneous readouts of strain-specific chemical profiles and comparative measurements of FFA production levels.

{"title":"A Fast-Pass, Desorption Electrospray Ionization Mass Spectrometry Strategy for Untargeted Metabolic Phenotyping","authors":"Hawkins S. Shepard,&nbsp;Jody C. May,&nbsp;Baltazar E. Zuniga,&nbsp;Joshua P. Abraham,&nbsp;Brian F. Pfleger,&nbsp;Jamey D. Young and John A. McLean*,&nbsp;","doi":"10.1021/jasms.4c0045910.1021/jasms.4c00459","DOIUrl":"https://doi.org/10.1021/jasms.4c00459https://doi.org/10.1021/jasms.4c00459","url":null,"abstract":"<p >Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) provides direct analytical readouts of small molecules that can be used to characterize the metabolic phenotypes of genetically engineered bacteria. In an effort to accelerate the time frame associated with the screening of mutant libraries, we have developed a high-throughput DESI-MSI analytical workflow implementing a single raster line-scan strategy that facilitates the collection of location-resolved molecular information from engineered strains on a subminute time scale. Evaluation of this “Fast-Pass” DESI-MSI phenotyping workflow on analytical standards demonstrated the capability of acquiring full metabolic profiling information with a throughput of ∼40 s per sample. This Fast-Pass strategy was implemented in the analysis of genetically edited <i>Escherichia coli</i> strains that have been engineered to produce various free-fatty acids (FFAs) for applications relevant to biofuels. Due to the untargeted nature of DESI-MSI, the investigation of these strains yielded molecular information for both global metabolites and targeted detection of accumulated bioproducts, allowing simultaneous readouts of strain-specific chemical profiles and comparative measurements of FFA production levels.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"400–408 400–408"},"PeriodicalIF":3.1,"publicationDate":"2025-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/jasms.4c00459","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143127216","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
Faces of Mass Spectrometry/Ljiljana Paša-Tolić
IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-24 DOI: 10.1021/jasms.4c0051310.1021/jasms.4c00513
Anne Brenner,  and , J. D. Brookbank, 
{"title":"Faces of Mass Spectrometry/Ljiljana Paša-Tolić","authors":"Anne Brenner,&nbsp; and ,&nbsp;J. D. Brookbank,&nbsp;","doi":"10.1021/jasms.4c0051310.1021/jasms.4c00513","DOIUrl":"https://doi.org/10.1021/jasms.4c00513https://doi.org/10.1021/jasms.4c00513","url":null,"abstract":"","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"232–235 232–235"},"PeriodicalIF":3.1,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143127438","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
Characterization of Sugammadex-Related Isomeric Cyclodextrin Impurities Using Cyclic Ion Mobility High-Resolution Mass Spectrometry
IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-24 DOI: 10.1021/jasms.4c0024310.1021/jasms.4c00243
Péter S. Szakály, Dávid Papp, Arnold Steckel, Erzsébet Varga and Gitta Schlosser*, 

Cyclic ion mobility–mass spectrometry (cIM-MS) is a powerful technique for separating and identifying isomeric mixtures of compounds. When coupled with chromatography, cIM-MS creates a multidimensional separation system, with high resolving power and peak capacity. In this study, we report the cyclic ion mobility separation and high-resolution mass spectrometry identification of four regioisomers of a Sugammadex-related impurity, abbreviated as Di-OH-SGM. Separation using multipass cyclic ion mobility was achieved by selecting the [M + 2Na]2+ ion, while other adducts, such as [M + Na]+, [M + 2H]2+, [M + H + Na]2+, and [M – 2H]2– did not yield isomer separation. Two methods were developed for ion mobility separation of the isomers: a conventional multipass method and a slicing method. Isomer assignment was based on the characteristic fragment ions. The collision cross section values (cTWCCSN2) of the resolved cyclodextrin isomers were also determined. Ion mobility separation of structurally different fragment ions was demonstrated. Additionally, by coupling cIM-MS with reversed-phase liquid chromatography (HPLC-cIM-MS), two-dimensional separation of the isomers was achieved. The isomers, separated using HPLC-cIM-MS, were identified with the same approach as with cIM-MS alone, and their elution order provided insights into their relative hydrophobicity.

{"title":"Characterization of Sugammadex-Related Isomeric Cyclodextrin Impurities Using Cyclic Ion Mobility High-Resolution Mass Spectrometry","authors":"Péter S. Szakály,&nbsp;Dávid Papp,&nbsp;Arnold Steckel,&nbsp;Erzsébet Varga and Gitta Schlosser*,&nbsp;","doi":"10.1021/jasms.4c0024310.1021/jasms.4c00243","DOIUrl":"https://doi.org/10.1021/jasms.4c00243https://doi.org/10.1021/jasms.4c00243","url":null,"abstract":"<p >Cyclic ion mobility–mass spectrometry (cIM-MS) is a powerful technique for separating and identifying isomeric mixtures of compounds. When coupled with chromatography, cIM-MS creates a multidimensional separation system, with high resolving power and peak capacity. In this study, we report the cyclic ion mobility separation and high-resolution mass spectrometry identification of four regioisomers of a Sugammadex-related impurity, abbreviated as Di-OH-SGM. Separation using multipass cyclic ion mobility was achieved by selecting the [M + 2Na]<sup>2+</sup> ion, while other adducts, such as [M + Na]<sup>+</sup>, [M + 2H]<sup>2+</sup>, [M + H + Na]<sup>2+</sup>, and [M – 2H]<sup>2–</sup> did not yield isomer separation. Two methods were developed for ion mobility separation of the isomers: a conventional multipass method and a slicing method. Isomer assignment was based on the characteristic fragment ions. The collision cross section values (<sup>cTW</sup>CCS<sub>N2</sub>) of the resolved cyclodextrin isomers were also determined. Ion mobility separation of structurally different fragment ions was demonstrated. Additionally, by coupling cIM-MS with reversed-phase liquid chromatography (HPLC-cIM-MS), two-dimensional separation of the isomers was achieved. The isomers, separated using HPLC-cIM-MS, were identified with the same approach as with cIM-MS alone, and their elution order provided insights into their relative hydrophobicity.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"258–264 258–264"},"PeriodicalIF":3.1,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/jasms.4c00243","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143127202","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
Locating Polyubiquitin Receptors on the 19S Regulatory Proteasome of S. cerevisiae by Cross-Linking Mass Spectrometry
IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-22 DOI: 10.1021/jasms.4c0038110.1021/jasms.4c00381
Yiran Ma, Bingqing Zhao, Amit K. S. Gautam, Caroline Davis, Jonathan C. Trinidad, James P. Reilly, David E. Clemmer* and Andreas Matouschek*, 

The effectiveness of state-of-the-art cross-linking strategies and mass spectrometry (MS) detection was explored in an important biological context, namely, the ubiquitin-proteasome system, which is responsible for most of the regulated protein degradation in eukaryotic cells. The locations of possible binding sites on the S. cerevisiae 19S proteasome regulatory particle for Lys48 linked polyubiquitin chains were examined using cross-linking strategies and MS based detection by comparing two types of cross-linkers: a (bis)-sulfosuccinimidyl suberate (BS3) and diethyl suberothioimidate (DEST). The well-established BS3-based strategy produced 328 cross-linked peptides; however, no ubiquitin-19S cross-links were observed. The recently developed DEST-based approach produced fewer (146) linkages overall, but these included six ubiquitin-19S cross-links. Some of these cross-links are predicted by the canonical view of ubiquitin recognition, but others suggest novel insights into how the proteasome recognizes its substrates. A discussion of these strategies and structural implications for polyubiquitin-proteasome binding is provided.

{"title":"Locating Polyubiquitin Receptors on the 19S Regulatory Proteasome of S. cerevisiae by Cross-Linking Mass Spectrometry","authors":"Yiran Ma,&nbsp;Bingqing Zhao,&nbsp;Amit K. S. Gautam,&nbsp;Caroline Davis,&nbsp;Jonathan C. Trinidad,&nbsp;James P. Reilly,&nbsp;David E. Clemmer* and Andreas Matouschek*,&nbsp;","doi":"10.1021/jasms.4c0038110.1021/jasms.4c00381","DOIUrl":"https://doi.org/10.1021/jasms.4c00381https://doi.org/10.1021/jasms.4c00381","url":null,"abstract":"<p >The effectiveness of state-of-the-art cross-linking strategies and mass spectrometry (MS) detection was explored in an important biological context, namely, the ubiquitin-proteasome system, which is responsible for most of the regulated protein degradation in eukaryotic cells. The locations of possible binding sites on the <i>S. cerevisiae</i> 19S proteasome regulatory particle for Lys<sup>48</sup> linked polyubiquitin chains were examined using cross-linking strategies and MS based detection by comparing two types of cross-linkers: a (bis)-sulfosuccinimidyl suberate (BS<sup>3</sup>) and diethyl suberothioimidate (DEST). The well-established BS<sup>3</sup>-based strategy produced 328 cross-linked peptides; however, no ubiquitin-19S cross-links were observed. The recently developed DEST-based approach produced fewer (146) linkages overall, but these included six ubiquitin-19S cross-links. Some of these cross-links are predicted by the canonical view of ubiquitin recognition, but others suggest novel insights into how the proteasome recognizes its substrates. A discussion of these strategies and structural implications for polyubiquitin-proteasome binding is provided.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"277–285 277–285"},"PeriodicalIF":3.1,"publicationDate":"2025-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143127085","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
Rigorous Analysis of Multimodal HDX-MS Spectra
IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-21 DOI: 10.1021/jasms.4c0047110.1021/jasms.4c00471
Lisa M. Tuttle, Ellie I. James, Florian Georgescauld, Thomas E. Wales, David D. Weis, John R. Engen, Abhinav Nath, Rachel E. Klevit and Miklos Guttman*, 

An inherent strength of hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is its ability to detect the presence of multiple conformational states of a protein, which often manifest as multimodal isotopic envelopes. However, the statistical considerations for accurate analysis of multimodal spectra have yet to be established. Here we outline an unrestrained binomial distribution fitting approach with the corresponding statistical tests to accurately detect and, when possible, deconvolute isotopic distributions that contain multiple subpopulations. The algorithms have been incorporated into an updated version of the freely available software, HX-Express, and validated using known mixtures of peptides deuterated to varying degrees. This approach presents a readily accessible tool to fit and interpret bimodal and trimodal behavior in HDX-MS data for mixed populations, EX1 kinetics, and pulse labeling data.

{"title":"Rigorous Analysis of Multimodal HDX-MS Spectra","authors":"Lisa M. Tuttle,&nbsp;Ellie I. James,&nbsp;Florian Georgescauld,&nbsp;Thomas E. Wales,&nbsp;David D. Weis,&nbsp;John R. Engen,&nbsp;Abhinav Nath,&nbsp;Rachel E. Klevit and Miklos Guttman*,&nbsp;","doi":"10.1021/jasms.4c0047110.1021/jasms.4c00471","DOIUrl":"https://doi.org/10.1021/jasms.4c00471https://doi.org/10.1021/jasms.4c00471","url":null,"abstract":"<p >An inherent strength of hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is its ability to detect the presence of multiple conformational states of a protein, which often manifest as multimodal isotopic envelopes. However, the statistical considerations for accurate analysis of multimodal spectra have yet to be established. Here we outline an unrestrained binomial distribution fitting approach with the corresponding statistical tests to accurately detect and, when possible, deconvolute isotopic distributions that contain multiple subpopulations. The algorithms have been incorporated into an updated version of the freely available software, HX-Express, and validated using known mixtures of peptides deuterated to varying degrees. This approach presents a readily accessible tool to fit and interpret bimodal and trimodal behavior in HDX-MS data for mixed populations, EX1 kinetics, and pulse labeling data.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"416–423 416–423"},"PeriodicalIF":3.1,"publicationDate":"2025-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143126846","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
Gas-Phase Fragmentation of Coenzyme Q10 Radical Anion Generated by APCI: A Study by High/Low-Resolution Tandem/Sequential Mass Spectrometry
IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-20 DOI: 10.1021/jasms.4c0039910.1021/jasms.4c00399
Mariachiara Bianco, Ilario Losito*, Giovanni Ventura, Beniamino Leoni, Onofrio Davide Palmitessa, Massimiliano Renna, Pietro Santamaria, Cosima Damiana Calvano and Tommaso R.I. Cataldi, 

Coenzyme Q10 (CoQ10) and closely related compounds with varying isoprenoid tail lengths (CoQn, n = 6–9) are biochemical cofactors involved in many physiological processes, playing important roles in cellular respiration and energy production. Liquid chromatography (LC) coupled with single or tandem mass spectrometry (MS) using electrospray (ESI) or atmospheric pressure chemical ionization (APCI) is considered the gold standard for the identification and quantification of CoQ10 in food and biological samples. However, the characteristic fragmentation exhibited by the CoQ10 radical anion ([M], m/z 862.684), the prevailing ion generated by APCI in negative polarity, has not been studied in detail. In this work, a systematic study was carried out to clarify this issue, using higher collisional energy dissociation (HCD) with high-resolution tandem FTMS and collision-induced dissociation-low-resolution sequential mass spectrometry (CID-MSn, n = 2–4). Various fragmentation pathways were successfully interpreted, with some structures proposed for product ions checked using density functional theory (DFT) calculations. Besides the already-known detachments of methyl radicals occurring directly from the CoQ10 radical anion and leading to ions like [M – CH3] and [M – 2CH3]•–, the homolytic cleavage of C–C bonds along the oligo-isoprenoid side chain was tentatively proposed to explain some of the observed fragmentations. As a result, the generation of uncommon yet potentially stable distonic biradical anions was hypothesized, with some of them likely undergoing intramolecular cyclization to generate ions without unpaired electrons. Diagnostic product ions emerged from the fragmentation processes of CoQ10 and were found to be common also to the radical anions of other CoQn derivatives (n = 7–9), facilitating their identification in extracts of edible Brassicaceae plant microgreens by reversed-phase liquid chromatography (RPLC)-APCI-FTMS.

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Journal of the American Society for Mass Spectrometry
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