Russell J Mortishire-Smith, Benjamin J Mortishire-Smith, John S Smith
We describe a workshop that prompts chemistry students in the final 2 years of secondary school to apply their understanding of modern analytical chemistry techniques to a 'real world' example. The scenario used is that of a forensic science laboratory that has been asked to determine the structure of an illicit compound, Revisomed (methamphetamine) being sold as a revision aid, and seized by police. Over the course of an hour, the students use a combination of infrared (IR) spectroscopy, liquid chromatography (LC), high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy data to determine the structure of Revisomed from first principles. The bulk of the workshop is spent reviewing MS data and using m/z, the isotope pattern, elemental composition and product ion data to reach three plausible isomeric structures for Revisomed, which are then distinguished by NMR spectroscopy. More broadly, the workshop focusses on the use of the scientific method and the concept that 'no ideas are bad' when exploring hypotheses. We describe the structure of the workshop, and our experience delivering it to a local academy over the last 9 years.
{"title":"From Breaking Bad to Breaking Bonds-Mass Spectrometry in the Classroom.","authors":"Russell J Mortishire-Smith, Benjamin J Mortishire-Smith, John S Smith","doi":"10.1002/rcm.9970","DOIUrl":"https://doi.org/10.1002/rcm.9970","url":null,"abstract":"<p><p>We describe a workshop that prompts chemistry students in the final 2 years of secondary school to apply their understanding of modern analytical chemistry techniques to a 'real world' example. The scenario used is that of a forensic science laboratory that has been asked to determine the structure of an illicit compound, Revisomed (methamphetamine) being sold as a revision aid, and seized by police. Over the course of an hour, the students use a combination of infrared (IR) spectroscopy, liquid chromatography (LC), high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy data to determine the structure of Revisomed from first principles. The bulk of the workshop is spent reviewing MS data and using m/z, the isotope pattern, elemental composition and product ion data to reach three plausible isomeric structures for Revisomed, which are then distinguished by NMR spectroscopy. More broadly, the workshop focusses on the use of the scientific method and the concept that 'no ideas are bad' when exploring hypotheses. We describe the structure of the workshop, and our experience delivering it to a local academy over the last 9 years.</p>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":" ","pages":"e9970"},"PeriodicalIF":1.8,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143021517","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Rationale: Teaching mass spectrometry essentials is usually connected with one of the basic courses for undergrads. Thus, specific previous knowledge is required from students. However, the necessity of teaching mass spectrometry essentials to students of different academic specializations and multidisciplinary groups can arise in every academic group. A specific workshop is needed to address such a demand.
Methods: The presented workshop consisted of several thematic parts: assembling an ambient ionization ion source using improvised materials, preparing biological samples for analysis, data acquisition, and interpretation of data to solve a simple problem from the real world.
Results: The first part of the work consisted of assembling an ambient ionization setup and obtaining mass spectra of substances from standard solutions, natural mixtures, and biological fluids such as saliva. The second half of the workshop consists of analyzing the composition of fatty acids of natural and artificial fats using the same ion source. The identification of oils is a simple model problem that makes the workshop attractive for attendees with different backgrounds.
Conclusions: The workshop provides students with practical skills that are highly valuable in fundamental and applied mass spectrometry. Students familiarize themselves with the basic concepts, instrument use, and mass spectra interpretation. They achieve basic hands-on experience in experimentation procedures and the practice of using mass spectrometry to solve problems related to real life.
{"title":"Ambient Ionization Mass Spectrometry for Fatty Acids Composition Investigation of Natural Lipids: A Multidisciplinary Workshop.","authors":"Stanislav I Pekov","doi":"10.1002/rcm.9985","DOIUrl":"https://doi.org/10.1002/rcm.9985","url":null,"abstract":"<p><strong>Rationale: </strong>Teaching mass spectrometry essentials is usually connected with one of the basic courses for undergrads. Thus, specific previous knowledge is required from students. However, the necessity of teaching mass spectrometry essentials to students of different academic specializations and multidisciplinary groups can arise in every academic group. A specific workshop is needed to address such a demand.</p><p><strong>Methods: </strong>The presented workshop consisted of several thematic parts: assembling an ambient ionization ion source using improvised materials, preparing biological samples for analysis, data acquisition, and interpretation of data to solve a simple problem from the real world.</p><p><strong>Results: </strong>The first part of the work consisted of assembling an ambient ionization setup and obtaining mass spectra of substances from standard solutions, natural mixtures, and biological fluids such as saliva. The second half of the workshop consists of analyzing the composition of fatty acids of natural and artificial fats using the same ion source. The identification of oils is a simple model problem that makes the workshop attractive for attendees with different backgrounds.</p><p><strong>Conclusions: </strong>The workshop provides students with practical skills that are highly valuable in fundamental and applied mass spectrometry. Students familiarize themselves with the basic concepts, instrument use, and mass spectra interpretation. They achieve basic hands-on experience in experimentation procedures and the practice of using mass spectrometry to solve problems related to real life.</p>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":" ","pages":"e9985"},"PeriodicalIF":1.8,"publicationDate":"2025-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143031827","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Leonardo Parasecolo, Ivan M. Monsalvo, Nikola Kovinich, Demian R. Ifa
Rationale
Understanding plant defense mechanisms against pathogens is essential for enhancing agricultural productivity and crop protection. This study focuses on the quantification of camalexin and scopoletin, two critical phytoalexins in Arabidopsis thaliana, using mass spectrometry techniques. Precise measurement of these compounds provides insights into plant resistance and supports agricultural research.
Methods
Camalexin and scopoletin were quantified using matrix-assisted laser desorption ionization high-resolution mass spectrometry (MALDI-HRMS). The matrix and solvent conditions were optimized to maximize sensitivity and accuracy. MS/MS experiments confirmed compound identification with high mass accuracy (mass error < 5 ppm). The method was validated through comparative analysis of wild-type (WT) and mutant Arabidopsis lines, using internal standards and multiple replicates to ensure precision and reliability.
Results
The method exhibited high linearity for scopoletin (R2 = 0.9992) and camalexin (R2 = 0.9987) across concentration ranges of 0.16–5 and 0.31–5 μM, respectively. Limits of detection (LOD) were 0.16 μM for camalexin and 0.04 μM for scopoletin, with limits of quantification (LOQ) at 0.2 μM and 0.08 μM, respectively. Samples analysis demonstrated reliable quantification in WT and mutant lines, with significant reductions in camalexin and scopoletin levels observed in the atwrky33-2 and atmyb15-1 mutants, respectively. Additionally, the method detected sub-physiological concentrations, confirming its sensitivity and robustness for low-level detection.
Conclusions
This study presents a validated, precise, and accurate MALDI-HRMS method for the quantification of camalexin and scopoletin in Arabidopsis thaliana. The approach not only enhances understanding of plant defense mechanisms but also offers potential applications for biotechnological and agricultural research, especially for investigating genetic variations and stress-induced phytoalexin production.
{"title":"Development of a Matrix-Assisted Laser Desorption Ionization High Resolution Mass Spectrometry Method for the Quantification of Camalexin and Scopoletin in Arabidopsis thaliana","authors":"Leonardo Parasecolo, Ivan M. Monsalvo, Nikola Kovinich, Demian R. Ifa","doi":"10.1002/rcm.9973","DOIUrl":"10.1002/rcm.9973","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Rationale</h3>\u0000 \u0000 <p>Understanding plant defense mechanisms against pathogens is essential for enhancing agricultural productivity and crop protection. This study focuses on the quantification of camalexin and scopoletin, two critical phytoalexins in <i>Arabidopsis thaliana</i>, using mass spectrometry techniques. Precise measurement of these compounds provides insights into plant resistance and supports agricultural research.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Camalexin and scopoletin were quantified using matrix-assisted laser desorption ionization high-resolution mass spectrometry (MALDI-HRMS). The matrix and solvent conditions were optimized to maximize sensitivity and accuracy. MS/MS experiments confirmed compound identification with high mass accuracy (mass error < 5 ppm). The method was validated through comparative analysis of wild-type (WT) and mutant <i>Arabidopsis</i> lines, using internal standards and multiple replicates to ensure precision and reliability.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The method exhibited high linearity for scopoletin (<i>R</i><sup>2</sup> = 0.9992) and camalexin (<i>R</i><sup>2</sup> = 0.9987) across concentration ranges of 0.16–5 and 0.31–5 μM, respectively. Limits of detection (LOD) were 0.16 μM for camalexin and 0.04 μM for scopoletin, with limits of quantification (LOQ) at 0.2 μM and 0.08 μM, respectively. Samples analysis demonstrated reliable quantification in WT and mutant lines, with significant reductions in camalexin and scopoletin levels observed in the <i>atwrky33-2</i> and <i>atmyb15-1</i> mutants, respectively. Additionally, the method detected sub-physiological concentrations, confirming its sensitivity and robustness for low-level detection.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>This study presents a validated, precise, and accurate MALDI-HRMS method for the quantification of camalexin and scopoletin in <i>Arabidopsis thaliana</i>. The approach not only enhances understanding of plant defense mechanisms but also offers potential applications for biotechnological and agricultural research, especially for investigating genetic variations and stress-induced phytoalexin production.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"39 6","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/rcm.9973","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142851689","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Radwa Mahmoud, Amir Khajavinia, Sedigheh Barzegar, Randy W. Purves, Robert B. Laprairie, Anas El-Aneed
Background
Analysis of the phytocannabinoids holds significant importance because of their various pharmacological properties and potential therapeutic applications. Tandem mass spectrometry (MS/MS) coupled with electrospray ionization in positive ion mode is employed in this study to describe the collision-induced dissociation (CID) behavior of a series of common phytocannabinoids with the aim of establishing a generalized MS/MS fingerprint.
Materials and Methods
Eight phytocannabinoids, namely, ∆9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), cannabigerol (CBG), tetrahydrocannabivarin (THCV), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 6-hydroxy-cannabidiol (6-OH-CBD), and 7-hydroxy-cannabidiol (7-OH-CBD), were studied. A Quadrupole-Orbitrap mass spectrometer equipped with a heated electrospray ionization (HESI-Q Orbitrap) is used to provide accurate mass measurement data for single-stage and MS/MS analysis. In addition, a triple quadrupole-linear ion trap mass spectrometer was used to perform MS/MS and second-generation MS/MS (MS3) analyses.
Results
An abundant, singly charged [M + H]+ species during single-stage MS analysis was observed for all phytocannabinoids, with mass accuracies less than 5 ppm. Because of their structural similarities, all compounds showed some common fragmentation behavior in their MS/MS analysis. By comparing the fragmentation patterns and identifying diagnostic ions, a universal MS/MS fragmentation pattern was established. The structures of the various product ions proposed in the fragmentation pathway were confirmed with exact mass measurements and MS3 experiments.
Conclusions
The evaluated compounds contain varying functional groups, resulting in unique product ions, specific to each structure. The MS/MS fingerprints will be utilized in the future for the identification of new structures as well as the development of targeted quantification methods.
{"title":"Establishment of a Mass Spectrometric Fingerprint of the Most Common Phytocannabinoids in Electrospray Ionization in Positive Ion Mode","authors":"Radwa Mahmoud, Amir Khajavinia, Sedigheh Barzegar, Randy W. Purves, Robert B. Laprairie, Anas El-Aneed","doi":"10.1002/rcm.9952","DOIUrl":"10.1002/rcm.9952","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>Analysis of the phytocannabinoids holds significant importance because of their various pharmacological properties and potential therapeutic applications. Tandem mass spectrometry (MS/MS) coupled with electrospray ionization in positive ion mode is employed in this study to describe the collision-induced dissociation (CID) behavior of a series of common phytocannabinoids with the aim of establishing a generalized MS/MS fingerprint.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Materials and Methods</h3>\u0000 \u0000 <p>Eight phytocannabinoids, namely, ∆<sup>9</sup>-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), cannabigerol (CBG), tetrahydrocannabivarin (THCV), 11-hydroxy-Δ<sup>9</sup>-tetrahydrocannabinol (11-OH-THC), 6-hydroxy-cannabidiol (6-OH-CBD), and 7-hydroxy-cannabidiol (7-OH-CBD), were studied. A Quadrupole-Orbitrap mass spectrometer equipped with a heated electrospray ionization (HESI-Q Orbitrap) is used to provide accurate mass measurement data for single-stage and MS/MS analysis. In addition, a triple quadrupole-linear ion trap mass spectrometer was used to perform MS/MS and second-generation MS/MS (MS<sup>3</sup>) analyses.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>An abundant, singly charged [M + H]<sup>+</sup> species during single-stage MS analysis was observed for all phytocannabinoids, with mass accuracies less than 5 ppm. Because of their structural similarities, all compounds showed some common fragmentation behavior in their MS/MS analysis. By comparing the fragmentation patterns and identifying diagnostic ions, a universal MS/MS fragmentation pattern was established. The structures of the various product ions proposed in the fragmentation pathway were confirmed with exact mass measurements and MS<sup>3</sup> experiments.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>The evaluated compounds contain varying functional groups, resulting in unique product ions, specific to each structure. The MS/MS fingerprints will be utilized in the future for the identification of new structures as well as the development of targeted quantification methods.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"39 4","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-12-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142823475","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Plasmids can play a major role in the survival of pathogenic bacteria. Plasmids are acquired through horizontal gene transfer resulting in their spread across various strains, species and genera of bacteria. Colicins are bacterial protein toxins expressed by plasmid genes and released against co-located bacterial competitors.
Methods:
Three Shiga toxin-producing E. coli (STEC), whose genomes were sequenced previously, were analyzed using a combination of antibiotic induction, MALDI-TOF-TOF mass spectrometry, top-down proteomic analysis, and small plasmid sequencing. Protein biomarkers were identified using in-house software that matches protein mass and fragment ions of backbone cleavage by the aspartic acid effect. Predicted in silico protein structures assisted in the interpretation of protein ion fragmentation.
Results:
In addition to proteomic identification of phage-encoded Shiga toxin, we were able to identify plasmid-encoded immunity proteins for colicin D and E3. The genes for these plasmid-encoded proteins were not found in the previous genomic sequencing. However, resequencing of these strains for small plasmids revealed the genes to be present on 7–8 kb sized plasmids. Upstream of the colicin/immunity genes was an inverted repeat of the SOS/LexA box that represses gene expression until antibiotic challenge.
Conclusions:
Our top-down proteomic method demonstrates that it is possible to screen putative pathogenic bacteria (whose genomes have been sequenced in full, in part or not at all) for the presence of phage- and plasmid-encoded toxin and colicin genes under SOS control. Small plasmid sequencing confirmed the presence of colicin/immunity genes (and their regulatory control) suggested from induction and top-down proteomic analysis.
{"title":"Colicin Immunity Proteins of Pathogenic Bacteria Detected by Antibiotic-Induced SOS Response, Plasmid Sequencing, MALDI-TOF-TOF Mass Spectrometry, and Top-Down Proteomic Analysis","authors":"Clifton K. Fagerquist, Yanlin Shi, Jihyun Park","doi":"10.1002/rcm.9964","DOIUrl":"10.1002/rcm.9964","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Rationale:</h3>\u0000 \u0000 <p>Plasmids can play a major role in the survival of pathogenic bacteria. Plasmids are acquired through horizontal gene transfer resulting in their spread across various strains, species and genera of bacteria. Colicins are bacterial protein toxins expressed by plasmid genes and released against co-located bacterial competitors.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods:</h3>\u0000 \u0000 <p>Three Shiga toxin-producing <i>E. coli</i> (STEC), whose genomes were sequenced previously, were analyzed using a combination of antibiotic induction, MALDI-TOF-TOF mass spectrometry, top-down proteomic analysis, and small plasmid sequencing. Protein biomarkers were identified using in-house software that matches protein mass and fragment ions of backbone cleavage by the <i>aspartic acid effect</i>. Predicted in silico protein structures assisted in the interpretation of protein ion fragmentation.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results:</h3>\u0000 \u0000 <p>In addition to proteomic identification of phage-encoded Shiga toxin, we were able to identify plasmid-encoded immunity proteins for colicin D and E3. The genes for these plasmid-encoded proteins were not found in the previous genomic sequencing. However, resequencing of these strains for small plasmids revealed the genes to be present on 7–8 kb sized plasmids. Upstream of the colicin/immunity genes was an inverted repeat of the SOS/LexA box that represses gene expression until antibiotic challenge.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions:</h3>\u0000 \u0000 <p>Our top-down proteomic method demonstrates that it is possible to screen putative pathogenic bacteria (whose genomes have been sequenced in full, in part or not at all) for the presence of phage- and plasmid-encoded toxin and colicin genes under SOS control. Small plasmid sequencing confirmed the presence of colicin/immunity genes (and their regulatory control) suggested from induction and top-down proteomic analysis.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"39 5","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142816786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ulcerative colitis (UC) is a chronic inflammatory gastrointestinal disease typically coexisting with intestinal microbiota dysbiosis, oxidative stress, and an inflammatory response. Although its underlying mechanism of action is unclear, Ma-Mu-Ran Antidiarrheal Capsules (MMRAC) have demonstrated significant therapeutic efficacy for UC.
Methods
The mechanism of action of MMRAC in the treatment of UC model was investigated by combining metabolomics, transcriptomics, and intestinal microbiota detection techniques.
Results
The high-dose group of MMRAC was determined as the best therapeutic dose by pathological changes and biochemical indexes. Transcriptome analysis revealed that 360 genes were differentially altered after MMRAC treatment. Metabolomic analysis using colon tissue yielded 14 colon tissue metabolites with significant differences. Intestinal flora analysis showed that 26 major microorganisms were identified at the genus level.
Conclusions
Based on a thorough multi-omics analysis of transcriptomics, metabolomics, and gut flora, it was determined that MMRAC regulated cysteine and methionine metabolism, arginine biosynthesis, and sphingolipid metabolism and their respective genes BHMT, PHGDH, iNOS, and SPHK1, which in turn served to inhibit UC-generated inflammatory responses and oxidative stress. Additionally, MMRAC regulated the abundance of Coprococcus, Helicobacter, Sutterella, Paraprevotella, and Roseburia in the intestinal tracts of UC mice, which was regulated toward normal levels, thereby restoring normal intestinal function.
{"title":"Integrative multi-omics reveals the mechanism of ulcerative colitis treated with Ma-Mu-Ran antidiarrheal capsules","authors":"Hailing Huang, Bailu Duan, Zhuang Huang, Shanshan Wang, Yuxin Wen, Qi Jiang, Pengyu Chen, Ping Huang, Jiajing Liu, Sili Zheng, Yan Ye, Dongning Zhang, Qiong Wang, Fang Huang, Jingjing Li, Lintao Han","doi":"10.1002/rcm.9939","DOIUrl":"10.1002/rcm.9939","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> Rationale</h3>\u0000 \u0000 <p>Ulcerative colitis (UC) is a chronic inflammatory gastrointestinal disease typically coexisting with intestinal microbiota dysbiosis, oxidative stress, and an inflammatory response. Although its underlying mechanism of action is unclear, Ma-Mu-Ran Antidiarrheal Capsules (MMRAC) have demonstrated significant therapeutic efficacy for UC.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>The mechanism of action of MMRAC in the treatment of UC model was investigated by combining metabolomics, transcriptomics, and intestinal microbiota detection techniques.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The high-dose group of MMRAC was determined as the best therapeutic dose by pathological changes and biochemical indexes. Transcriptome analysis revealed that 360 genes were differentially altered after MMRAC treatment. Metabolomic analysis using colon tissue yielded 14 colon tissue metabolites with significant differences. Intestinal flora analysis showed that 26 major microorganisms were identified at the genus level.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>Based on a thorough multi-omics analysis of transcriptomics, metabolomics, and gut flora, it was determined that MMRAC regulated cysteine and methionine metabolism, arginine biosynthesis, and sphingolipid metabolism and their respective genes BHMT, PHGDH, iNOS, and SPHK1, which in turn served to inhibit UC-generated inflammatory responses and oxidative stress. Additionally, MMRAC regulated the abundance of Coprococcus, Helicobacter, Sutterella, Paraprevotella, and Roseburia in the intestinal tracts of UC mice, which was regulated toward normal levels, thereby restoring normal intestinal function.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"39 5","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142811419","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dariusz J. Janecki, Chi-Ya Kao-Scharf, Andreas Hoffmann
Background
Consensus is that immunoglobulin IgG4 contains only N-linked glycosylation. The analysis of several batches of commercial biopharmaceutical product Dupixent using top-down intact mass spectrometry revealed that this IgG4 features a small amount of O-linked glycosylation in the Fab region. This is the first report of an O-linked glycosylation in an IgG4 antibody.
Methods
Monoclonal antibody solutions were subjected to cation exchange (CEX) and reverse phase (RP) chromatography and/or additional preconcentration/fractionation methods to prepare samples for subsequent analysis. Advanced MS analysis and fragmentation techniques (HCD, ETD, and EThcD) were employed to localize the O-linked glycosylation as well as elucidate the structure of the glycan(s).
Results
O-linked glycosylation in the IgG4 dupilumab was discovered by intact-MS. The probable location was narrowed down to four sites in the CH1 domain, and the structure of the O-linked glycan was determined to be of Core 1 type. The relative quantities of the modifications were low, but the glycosylation was consistently detected in several batches of Dupixent.
Conclusions
We discovered a rare glycosylation modification on dupilumab, an IgG4 antibody. The O-linked glycosylation was characterized and localized in the Fab region.
{"title":"Discovery and Characterization of Unusual O-Linked Glycosylation of IgG4 Antibody Using LC-MS","authors":"Dariusz J. Janecki, Chi-Ya Kao-Scharf, Andreas Hoffmann","doi":"10.1002/rcm.9969","DOIUrl":"10.1002/rcm.9969","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>Consensus is that immunoglobulin IgG4 contains only N-linked glycosylation. The analysis of several batches of commercial biopharmaceutical product Dupixent using top-down intact mass spectrometry revealed that this IgG4 features a small amount of O-linked glycosylation in the Fab region. This is the first report of an O-linked glycosylation in an IgG4 antibody.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Monoclonal antibody solutions were subjected to cation exchange (CEX) and reverse phase (RP) chromatography and/or additional preconcentration/fractionation methods to prepare samples for subsequent analysis. Advanced MS analysis and fragmentation techniques (HCD, ETD, and EThcD) were employed to localize the O-linked glycosylation as well as elucidate the structure of the glycan(s).</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>O-linked glycosylation in the IgG4 dupilumab was discovered by intact-MS. The probable location was narrowed down to four sites in the CH1 domain, and the structure of the O-linked glycan was determined to be of Core 1 type. The relative quantities of the modifications were low, but the glycosylation was consistently detected in several batches of Dupixent.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>We discovered a rare glycosylation modification on dupilumab, an IgG4 antibody. The O-linked glycosylation was characterized and localized in the Fab region.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"39 5","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11635057/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142811415","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
With the increasing application scenarios and detection needs of high-field asymmetric waveform ion mobility spectrometry (FAIMS) analysis, deep learning–assisted spectral analysis has become an important method to improve the analytical effect and work efficiency. However, a single model has limitations in generalizing to different types of tasks, and a model trained from one batch of spectral data is difficult to achieve good results on another task with large differences. To address this problem, this study proposes an adaptive multicore dual-path fusion multimodel extraction of heterogeneous features for FAIMS spectral analysis model in conjunction with FAIMS small-sample data analysis scenarios. Multinetwork complementarity is achieved through multimodel feature extraction, adaptive feature fusion module adjusts feature size and dimension fusion to heterogeneous features, and multicore dual-path fusion can capture and integrate information at all scales and levels. The model's performance improves dramatically when performing complex mixture multiclassification tasks: accuracy, precision, recall, f1-score, and micro-AUC reach 98.11%, 98.66%, 98.33%, 98.30%, and 98.98%. The metrics for the generalization test using the untrained xylene isomer data were 96.42%, 96.66%, 96.96%, 96.65%, and 97.60%. The model not only exhibits excellent analytical results on preexisting data but also demonstrates good generalization ability on untrained data.
{"title":"Adaptive Multicore Dual-Path Fusion Multimodel Extraction of Heterogeneous Features for FAIMS Spectral Analysis","authors":"Ruilong Zhang, Xiaoxia Du, Wenxiang Xiao, Hua Li","doi":"10.1002/rcm.9967","DOIUrl":"10.1002/rcm.9967","url":null,"abstract":"<div>\u0000 \u0000 <p>With the increasing application scenarios and detection needs of high-field asymmetric waveform ion mobility spectrometry (FAIMS) analysis, deep learning–assisted spectral analysis has become an important method to improve the analytical effect and work efficiency. However, a single model has limitations in generalizing to different types of tasks, and a model trained from one batch of spectral data is difficult to achieve good results on another task with large differences. To address this problem, this study proposes an adaptive multicore dual-path fusion multimodel extraction of heterogeneous features for FAIMS spectral analysis model in conjunction with FAIMS small-sample data analysis scenarios. Multinetwork complementarity is achieved through multimodel feature extraction, adaptive feature fusion module adjusts feature size and dimension fusion to heterogeneous features, and multicore dual-path fusion can capture and integrate information at all scales and levels. The model's performance improves dramatically when performing complex mixture multiclassification tasks: accuracy, precision, recall, f1-score, and micro-AUC reach 98.11%, 98.66%, 98.33%, 98.30%, and 98.98%. The metrics for the generalization test using the untrained xylene isomer data were 96.42%, 96.66%, 96.96%, 96.65%, and 97.60%. The model not only exhibits excellent analytical results on preexisting data but also demonstrates good generalization ability on untrained data.</p>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"39 5","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142805725","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pierre Thibault, Dietrich Volmer, David Goodlett, John Monaghan
{"title":"Remembering Robert Kinnear Boyd (1938-2024).","authors":"Pierre Thibault, Dietrich Volmer, David Goodlett, John Monaghan","doi":"10.1002/rcm.9963","DOIUrl":"https://doi.org/10.1002/rcm.9963","url":null,"abstract":"","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":" ","pages":"e9963"},"PeriodicalIF":1.8,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142805724","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shiva Kumar Gogikar, Sibu Sen, Shivashankar Pathinti, Gananadhamu Samanthula, Amol G. Dikundwar
Rationale
The present study aims to establish structures of the degradation products of an anti-diabetic drug, Imeglimin (IMG) approved for the treatment of type 2 diabetes mellitus in the year 2021. Degradation pathways are proposed along with in silico toxicity assessments of the observed degradation products (DPs) of the drug.
Methods
A reversed-phase high-performance liquid chromatography (RP-HPLC), equipped with a photodiode array detector, was used to separate the observed DPs with a Phenomenex Luna PFP (250 × 4.6 mm, 5 μm) column, using 10 mM ammonium formate (pH 4.5) and methanol as mobile phase. Liquid chromatography quadrupole time of flight mass spectrometry (LC-Q-ToF-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy were employed for structural elucidation. Zeneth and Derek suites were used for in silico assessments.
Results
A total of four degradation products were observed, which were successfully separated on an RP-HPLC. The structural characterization of three of the four DPs was achieved using LC-Q-TOF-MS/MS by employing electro spray ionization as well as atmospheric pressure chemical ionization. Additionally, DP-3 was isolated using a preparative HPLC and was characterized by NMR. Computationally predicted structures were compared with the experimental observations.
Conclusion
An HPLC method, capable of separating the Imeglimin and its four DPs, was developed and validated as per the ICH Q2(R1) guideline. Structure elucidation reveals a variety of products with metformin as one of the identified DPs along with a metabolite. The toxicity potential of DPs was assessed through docking studies.
理由：本研究旨在建立2021年被批准用于治疗2型糖尿病的抗糖尿病药物依米明（IMG）的降解产物结构。提出了降解途径以及观察到的药物降解产物（DPs）的硅毒性评估。方法：采用反相高效液相色谱法（RP-HPLC），采用光电二极管阵列检测器，Phenomenex Luna PFP （250 × 4.6 mm, 5 μm）柱，以10 mm甲酸铵（pH 4.5）和甲醇为流动相分离所观察到的dp。采用液相色谱-四极杆飞行时间质谱（LC-Q-ToF-MS/MS）和核磁共振（NMR）谱法进行结构解析。Zeneth和Derek套件用于计算机评估。结果：共观察到4种降解产物，并在反相高效液相色谱上成功分离。利用LC-Q-TOF-MS/MS，利用电喷雾电离和常压化学电离对四种DPs中的三种进行了结构表征。此外，利用制备型高效液相色谱分离得到了DP-3，并对其进行了核磁共振表征。计算预测的结构与实验观察结果进行了比较。结论：根据ICH Q2（R1）指南，建立了一种高效液相色谱（HPLC）分离依米明及其4种DPs的方法，并进行了验证。结构解析揭示了以二甲双胍作为鉴定的DPs之一的多种产物以及代谢物。通过对接研究评估DPs的毒性潜力。
{"title":"Forced Degradation Study of an Anti-Diabetic Drug Imeglimin: Impurity Profiling and Structure Elucidation Using LC-Q-ToF-MS/MS and NMR","authors":"Shiva Kumar Gogikar, Sibu Sen, Shivashankar Pathinti, Gananadhamu Samanthula, Amol G. Dikundwar","doi":"10.1002/rcm.9960","DOIUrl":"10.1002/rcm.9960","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Rationale</h3>\u0000 \u0000 <p>The present study aims to establish structures of the degradation products of an anti-diabetic drug, Imeglimin (IMG) approved for the treatment of type 2 diabetes mellitus in the year 2021. Degradation pathways are proposed along with in silico toxicity assessments of the observed degradation products (DPs) of the drug.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>A reversed-phase high-performance liquid chromatography (RP-HPLC), equipped with a photodiode array detector, was used to separate the observed DPs with a Phenomenex Luna PFP (250 × 4.6 mm, 5 μm) column, using 10 mM ammonium formate (pH 4.5) and methanol as mobile phase. Liquid chromatography quadrupole time of flight mass spectrometry (LC-Q-ToF-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy were employed for structural elucidation. Zeneth and Derek suites were used for in silico assessments.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>A total of four degradation products were observed, which were successfully separated on an RP-HPLC. The structural characterization of three of the four DPs was achieved using LC-Q-TOF-MS/MS by employing electro spray ionization as well as atmospheric pressure chemical ionization. Additionally, DP-3 was isolated using a preparative HPLC and was characterized by NMR. Computationally predicted structures were compared with the experimental observations.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusion</h3>\u0000 \u0000 <p>An HPLC method, capable of separating the Imeglimin and its four DPs, was developed and validated as per the ICH Q2(R1) guideline. Structure elucidation reveals a variety of products with metformin as one of the identified DPs along with a metabolite. The toxicity potential of DPs was assessed through docking studies.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"39 5","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142805726","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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