Chemical Communications, Год журнала: 2023, Номер 59(77), С. 11484 - 11499
Опубликована: Янв. 1, 2023
This feature article discusses mass spectrometry-based strategies for the characterisation, localisation and differentiation of phosphorylation sulfation on proteins, considers future directions in field.
Язык: Английский
Nature Reviews Methods Primers, Год журнала: 2024, Номер 4(1)
Опубликована: Июнь 13, 2024
Язык: Английский
Процитировано
22
Nature Chemistry, Год журнала: 2025, Номер unknown
Опубликована: Янв. 13, 2025
Abstract Understanding the dynamics of membrane protein–ligand interactions within a native lipid bilayer is major goal for drug discovery. Typically, cell-based assays are used, however, they often blind to effects protein modifications. In this study, using archetypal G protein-coupled receptor rhodopsin, we found that and its effectors can be released directly from retina rod disc membranes infrared irradiation in mass spectrometer. Subsequent isolation dissociation by multiphoton enabled sequencing individual proteoforms. Specifically, categorized distinct proteoforms localized labile palmitoylations, discovered Gβγ proteoform abolishes association defined modifications on proteins influence their assembly. Given reports undesirable side-effects involving vision, characterized off-target binding two phosphodiesterase 5 inhibitors, vardenafil sildenafil, 6 (PDE6). The results demonstrate differential reactivity with PDE6 an interaction preference lipidated proteins. summary, study highlights opportunities probing proteoform–ligand natural environments.
Язык: Английский
Процитировано
3
Journal of Proteome Research, Год журнала: 2025, Номер unknown
Опубликована: Фев. 17, 2025
Top-down proteomics is the study of intact proteins and their post-translational modifications with mass spectrometry. Historically, this field more challenging than its bottom-up counterpart because species are much bigger have a larger number possible combinations sequences modifications; thus, there great need for technological development. With improvements in instrumentation multiplicity fragmentation modes available, top-down quickly gaining popularity renewed attention on increasing confidence identification quantification. Here, we systematically evaluated Sciex ZenoTOF 7600 system proteomics, applying standards to validate platform further experimenting capabilities electron-activated dissociation modification site localization. The instrument demonstrated robustness standard QC, as aided by zeno trapping. We were also able apply histone modifications, achieving high sequence coverage that allowed PTM's localization across protein optimized EAD fragmentation. ability analyze spanning range included analysis glycosylated proteins. This reference point future experiments be conducted system.
Язык: Английский
Процитировано
3
Journal of Proteome Research, Год журнала: 2025, Номер unknown
Опубликована: Янв. 30, 2025
The quantification of proteoforms, i.e., all molecular forms in which proteins can be present, by top-down proteomics provides essential insights into biological processes at the level. Isobaric labeling-based strategies are suitable for multidimensional separation and allow multiplexing samples. Here, we investigated cysteine-directed isobaric labeling iodoTMT combination with a gel- gas-phase fractionation (GeLC-FAIMS-MS) in-depth quantitative proteoform analysis. We optimized acquisition workflow (i.e., FAIMS compensation voltages, isolation windows, strategy, fragmentation method) using two-proteome mix to increase number quantified proteoforms reduce ratio compression. Additionally, implemented mass feature-based strategy widely used deconvolution algorithm FLASHDeconv, improves facilitates data GeLC-FAIMS-MS was applied quantitatively analyze proteome Escherichia coli grown under glucose or acetate as sole carbon source, resulting identification 726 differentially abundant proteoforms.
Язык: Английский
Процитировано
1
Journal of Proteome Research, Год журнала: 2025, Номер unknown
Опубликована: Фев. 28, 2025
Histone proteoforms, often presenting multiple co-occurring post-translational modifications (PTMs), are central to chromatin regulation and gene expression. A proteoform is a specific form of protein that includes variations arising from genetic changes, alternative RNA splicing, proteolytic processing, PTMs. Genome-indexed histone proteoforms define the code, influencing cellular phenotype by dictating DNA interacting partners. Understanding dynamics essential for elucidating chromatin-based regulatory mechanisms. Advances in middle-down top-down proteomics enable accurate identification quantitation thousands single run. However, resulting data complexity presents significant challenges analysis visualization. Here, we introduce two new computational methods analyze PTMs demonstrate their use mouse organs during aging. The score term "normalized interplay" addresses limitations original crosstalk "interplay" providing more complete measure PTM crosstalk. second score, ΔI or "directional an asymmetric quantifying magnitude directionality between Applying our two-stage scoring approach CrosstalkDB reveals H3
Язык: Английский
Процитировано
1
Journal of Proteome Research, Год журнала: 2025, Номер unknown
Опубликована: Март 10, 2025
Proteoforms are distinct molecular forms of proteins that act as building blocks organisms, with post-translational modifications (PTMs) being one the key changes generate these variations. Mass spectrometry (MS)-based top-down proteomics (TDP) is leading technology for proteoform identification due to its preservation intact proteoforms analysis, making it well-suited comprehensive PTM characterization. A crucial step in TDP searching MS data against a database candidate proteoforms. To extend reach organisms limited annotations, we developed Proteoform-predictor, an open-source tool integrates homology-based site prediction into creation. The new creates databases candidates after registration homologous sequences, transferring sites from well-characterized species those less proteomic data. Our features user-friendly interface and intuitive workflow, accessible wide range researchers. We demonstrate Proteoform-predictor expands tens thousands three bacterial strains by comparing them reference proteome Escherichia coli (E. coli) K12. Subsequent analysis Serratia marcescens (S. marcescens) Salmonella typhimurium typhimurium) demonstrated significant improvement protein identification, even variant sequences. As advances, will become important expanding applicability biology more diverse across phylogenetic tree life.
Язык: Английский
Процитировано
1
Nature Biotechnology, Год журнала: 2025, Номер unknown
Опубликована: Март 17, 2025
Язык: Английский
Процитировано
1
Cell chemical biology, Год журнала: 2024, Номер 31(9), С. 1665 - 1687
Опубликована: Сен. 1, 2024
Язык: Английский
Процитировано
8
Nature Methods, Год журнала: 2024, Номер unknown
Опубликована: Окт. 22, 2024
Abstract Top-down proteomics using mass spectrometry facilitates the identification of intact proteoforms, that is, all molecular forms proteins. Multiple past advances have lead to development numerous sample preparation workflows. Here we systematically investigated influence different steps on proteoform and protein identifications, including cell lysis, reduction alkylation, enrichment, purification fractionation. We found in subset proteoforms identified (for example, their number, confidence, physicochemical properties artificially generated modifications). The various strategies resulted complementary substantially increasing proteome coverage. Overall, 13,975 from 2,720 proteins human Caco-2 cells. results presented can serve as suggestions for designing adapting top-down particular research questions. Moreover, expect sampling bias modifications at level will also be useful improving bottom-up approaches.
Язык: Английский
Процитировано
8
Proteomes, Год журнала: 2024, Номер 12(2), С. 14 - 14
Опубликована: Апрель 19, 2024
With growing recognition and acknowledgement of the genuine complexity proteomes, we are finally entering post-proteogenomic era. Routine assessment proteomes as inferred correlates gene sequences (i.e., canonical ‘proteins’) cannot provide necessary critical analysis systems-level biology that is needed to understand underlying molecular mechanisms pathways or identify most selective biomarkers therapeutic targets. These requirements demand at level proteoforms/protein species, actual active players. Currently, only highly refined integrated integrative top-down proteomics (iTDP) enables analytical depth routine, comprehensive, quantitative proteome assessments across widest range proteoforms inherent native systems. Here a broad perspective field, taking in historical current realities, establish more balanced understanding where field has come from (in particular during ten years since Proteomes was launched), issues, how things likely need proceed if deep analyses succeed. We base this our firm belief best proteomic reflect, closely possible, sample moment sampling. also seek emphasise future approaches based on exploitation complementarity currently successful approaches. This emphasises continuously evaluate further optimize established approaches, avoid complacency thinking expectations but promote careful development introduction new notably those address proteoforms. Above all, wish rigorous focus quality must override largely values speed; latter would certainly be nice, could thus effectively, routinely, quantitatively assessed. Alas, composed proteoforms, not species can amplified directly mirror genes ‘canonical’). The problem hard, accept it such, payoff playing longer game promise far biomarkers, drug targets, truly personalised even individualised medicine.
Язык: Английский
Процитировано
6