Methods in enzymology最新文献_第8页

Palmitoylation-dependent probes for labeling the Golgi apparatus. 棕榈酰化依赖性探针标记高尔基体。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-02-03 DOI: 10.1016/bs.mie.2026.01.005

Shunsuke Sawada, Keita Tsutsui, Shinya Tsukiji

引用次数: 0

De novo chemoenzymatic construction of lipid membranes. 脂质膜的从头化学酶构建。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-20 DOI: 10.1016/bs.mie.2026.01.013

Satyam Khanal, Alexander Harjung, Alessandro Fracassi

引用次数: 0

Detection, imaging and quantification of phosphoinositides using ion chromatography suppressed conductivity and mass spectrometry imaging. 利用离子色谱抑制电导率和质谱成像技术检测、成像和定量磷酸肌苷。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-16 DOI: 10.1016/bs.mie.2025.11.018

Artur Lazarian, Krista M Wartchow, Nicholas Bartelo, Laura Beth McIntire

{"title":"Detection, imaging and quantification of phosphoinositides using ion chromatography suppressed conductivity and mass spectrometry imaging.","authors":"Artur Lazarian, Krista M Wartchow, Nicholas Bartelo, Laura Beth McIntire","doi":"10.1016/bs.mie.2025.11.018","DOIUrl":"10.1016/bs.mie.2025.11.018","url":null,"abstract":"Phosphoinositide lipids have been implicated in aging, health and disease. Prior studies have identified enzymes critical for lipid metabolism, such as synthetic and degrading enzymes, which have different specificities based on the composition of the inositol ring as well as specific sub-cellular localization and multiple regulatory pathways. In addition to understanding the regulation of enzyme expression, activity and localization, it is critical to investigate levels of the specific lipids of interest. Current methods for detection and quantification of lipids include high performance liquid chromatography/ion chromatography (HPLC/HPIC) suppressed conductivity detection and mass spectrometry. Isobaric polyphosphorylated lipid species can be differentially separated using HPIC methods based on the differential chemical properties of the phosphorylation sites on the inositol ring, which are not able to be detected by mass spectrometry. However, mass spectrometry has the advantage of detecting the acyl chain composition for the individual species which has been shown to affect the signaling properties of the lipids. Emerging methods using mass spectrometry imaging offer in situ localization of the specific lipids within tissue but do not yet achieve single cell or intracellular spatial resolution. We describe methods for detection of polyphosphorylated phosphoinositides using HPIC and both untargeted and targeted imaging mass spectrometry using matrix-free desorption electrospray ionization. We also provide considerations for optimization of the methods as well as quantification of the resulting high dimensional data. Based on the central role phosphoinositides play in neuronal function and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Down Syndrome, and Traumatic Brain Injury, sensitive and accurate detection and quantification is paramount for mechanistic studies as well as biomarker and therapeutic development.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"726 ","pages":"217-252"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146257944","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Quantitative analysis of H-Ras localization using confocal microscopy. 用共聚焦显微镜定量分析H-Ras定位。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-11 DOI: 10.1016/bs.mie.2025.11.009

Anjana P Sundaresan, Mary E Brown, Mark D Distefano

引用次数: 0

High-throughput engineering of glycolaldehyde synthase for enhanced formaldehyde conversion to C2 and C3 products via mass spectrometry-based screening. 乙醇醛合成酶的高通量工程，以提高甲醛转化为C2和C3产品，通过质谱为基础的筛选。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-08 DOI: 10.1016/bs.mie.2026.05.049

Yizhou Luo, Lihao Fu, Peikai Lin, Tong Si, Jianzhi Zhang

{"title":"High-throughput engineering of glycolaldehyde synthase for enhanced formaldehyde conversion to C2 and C3 products via mass spectrometry-based screening.","authors":"Yizhou Luo, Lihao Fu, Peikai Lin, Tong Si, Jianzhi Zhang","doi":"10.1016/bs.mie.2026.05.049","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.049","url":null,"abstract":"One-carbon (C1) biomanufacturing offers a sustainable route for producing value-added chemicals. Glycolaldehyde synthase (GALS), a thiamine pyrophosphate (TPP)-dependent enzyme engineered from benzoylformate decarboxylase of Pseudomonas putida, catalyzes the condensation of formaldehyde (FALD) to yield the C2 and C3 platform chemicals glycolaldehyde (GALD) and dihydroxyacetone (DHA). However, its industrial application is constrained by low catalytic efficiency and the lack of high-throughput screening (HTS) methods. This chapter presents a comprehensive HTS workflow for engineering GALS variants. We first detail the construction of an optimized Escherichia coli chassis (BL21(DE3)-Δ6) via CRISPR/Cas9-mediated deletion of six endogenous reductase genes to minimize competing FALD consumption. Next, we present the development of a mass spectrometry (MS)-based assay using 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) derivatization coupled with RapidFire-MS/MS for simultaneous quantification of GALD and DHA. Integrating this MS assay with a robotic biofoundry enables automated screening of site-saturation mutagenesis (SSM) libraries at a throughput of ∼10 s per sample. Finally, we establish validation protocols for candidate variants, including enzyme purification and kinetic characterization using microplate-based colorimetric assays. The established methodology is readily adaptable to the engineering of diverse aldehyde-producing TPP-dependent enzymes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"736 ","pages":"399-431"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149075314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Ex vivo imaging and enzymatic analysis of intestinal mucus. 肠黏液的离体成像和酶分析。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-12 DOI: 10.1016/bs.mie.2026.04.004

Elisabeth E L Nyström, Brendan Dolan, George M H Birchenough, Malin E V Johansson

{"title":"Ex vivo imaging and enzymatic analysis of intestinal mucus.","authors":"Elisabeth E L Nyström, Brendan Dolan, George M H Birchenough, Malin E V Johansson","doi":"10.1016/bs.mie.2026.04.004","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.04.004","url":null,"abstract":"The intestinal mucus layer is a highly hydrated hydrogel that forms a critical barrier separating the gut microbiota from the epithelial surface while permitting nutrient exchange and luminal transport. Despite its central role in intestinal homeostasis, native mucus remains difficult to study due to its transparency, fragility, and poor preservation by conventional fixation methods, which often distort its structure or detach it from the epithelium. These limitations have restricted investigation of mucus organization, barrier properties, and enzymatic processes under physiological conditions. Here, we describe experimental approaches for the visualization and functional analysis of intestinal mucus using live ex vivo explant preparations combined with confocal microscopy. This system preserves the native architecture, cellular heterogeneity, and mucus attachment of intact epithelial tissue while allowing controlled experimental manipulation. Fluorescent lectin-based labeling enables resolution of spatially distinct mucus domains, including crypt-associated plumes and intercrypt mucus, which differ in glycosylation patterns and penetrability. Functional barrier properties are assessed using bead penetration assays, providing quantitative measures of mucus thickness and resistance to particle infiltration. In addition, the use of fluorescent probes and peptide substrates permits detection and spatial mapping of endogenous and microbial enzymatic activity within the mucus layer. These methods enable investigation of how enzymatic remodeling influences mucus structure and barrier integrity. Together, these protocols provide a versatile platform for studying mucus dynamics, organization, and function in a physiologically relevant context, bridging the gap between in vitro systems and in vivo studies.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"732 ","pages":"541-568"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148382287","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Glyco-TRAPP: A real-time glycocalyx permeability assay for assessing transmembrane mucin barrier function in live and fixed tissues. Glyco-TRAPP：实时糖萼通透性测定，用于评估活组织和固定组织的跨膜粘蛋白屏障功能。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-04-11 DOI: 10.1016/bs.mie.2026.03.008

Fleur Claire van de Koolwijk, Thaher Pelaseyed

引用次数: 0

Cryo-electron microscopy of mucins. 粘蛋白的低温电子显微镜。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-16 DOI: 10.1016/bs.mie.2026.04.002

Meital Haberman-Seror, Roman Kamyshinsky, Deborah Fass

{"title":"Cryo-electron microscopy of mucins.","authors":"Meital Haberman-Seror, Roman Kamyshinsky, Deborah Fass","doi":"10.1016/bs.mie.2026.04.002","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.04.002","url":null,"abstract":"Mucins, the major polymeric glycoproteins constituting mucus hydrogels, are produced in a complex biosynthetic pathway during which numerous disulfide bonds and glycan modifications are introduced. High-resolution structures have been obtained for over-expressed mucin segments under conditions that mimic the Golgi apparatus, an important station for mucin modification in the secretory pathway. Golgi-like solution conditions promote the self-assembly of the mucin segments into compact, ordered states, which have the dual advantage of illuminating the mechanism for mucin polymerization and being amenable to analysis by single-particle cryo-electron microscopy (cryo-EM). While the structures of these ordered intermediate states may differ from the arrangement and properties of secreted, hydrated, full-length extracellular mucins in gels on epithelial surfaces, they provide insight into the organization of mucin globular domains, and in some cases \"disordered\" regions, and reveal fundamental aspects of mucin bioassembly. Extending cryo-EM studies to longer mucin fragments and additional mucins may expand our understanding of these remarkable macromolecules.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"732 ","pages":"159-176"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148382367","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Imaging oxidative and nitrative stress in membranes. 膜中的氧化和硝化应激成像。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-04 DOI: 10.1016/bs.mie.2025.11.006

Liming Wang, Enver Cagri Izgu

引用次数: 0

Strategies to minimize artificial lipid oxidation in mass spectrometry based epilipidomics analysis. 在基于质谱法的卵磷脂组学分析中减少人工脂质氧化的策略。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-03 DOI: 10.1016/bs.mie.2025.11.019

Sider Penkov, Gabriele Lombardi Bendoula, Maria Fedorova

{"title":"Strategies to minimize artificial lipid oxidation in mass spectrometry based epilipidomics analysis.","authors":"Sider Penkov, Gabriele Lombardi Bendoula, Maria Fedorova","doi":"10.1016/bs.mie.2025.11.019","DOIUrl":"10.1016/bs.mie.2025.11.019","url":null,"abstract":"(Per)oxidized lipids represent a well-studied component of the epilipidome, a subset of the native lipidome formed through both enzymatic and non-enzymatic lipid oxidation. Given their diverse biological roles, including cellular signalling, regulation of immune responses, and modulation of cell death pathways, accurate detection of lipid peroxidation products is essential. Mass spectrometry-based approaches have become the method of choice for the sensitive, multiplexed detection and structural characterization of oxidized lipids across a variety of biological samples. However, the structural features of lipids, particularly the presence of acyl chains containing multiple double bonds, render them susceptible not only to endogenous oxidation but also to artificial oxidation during sample preparation prior to analysis. Consequently, special care must be taken throughout sample collection, storage, and lipid extraction to minimize artefacts arising from lipid autoxidation. Here, we describe protocols developed in our laboratory over recent years aimed at preventing artificial lipid oxidation during sample preparation, with examples spanning biological materials derived from cell culture, animal and human tissue biopsies, and biofluids. Finally, we propose internal quality control procedures to assess the effectiveness of these measures in preventing lipid autoxidation.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"726 ","pages":"253-267"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146258535","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0