configuring and calibrating the tims software interface for proteomic analysis

Analyse protéomique des histones et caractérisation de la modification post-traductionnelle

In eukaryotic cells, DNA is packaged as chromatin into functional units called nucleosomes. These units are composed of an octamer of four core histones (two each of H2A, H2B, H3, and H4)1,2,3,4. Histones are amongst the most abundant and highly conserved proteins in eukaryotes, which are largely responsible for gene regulation5. Histone posttranslational modifications (PTMs) play a large role in the regulation of chromatin dynamics and rigger various biological processes such as DNA transcription, replication, and repair6. PTMs occur primarily on the accessible surface of the N-terminal regions of histones that are in contact with DNA3,7. However, tail and core modifications influence chromatin structure, altering inter-nucleosome interactions and recruiting specific proteins3,8.
A current challenge during liquid chromatography-mass spectrometry (LC-MS)-based proteomics is the potential co-elution of analytes of interest. In the case of data-dependent analyses (DDA), this translates into the potential loss of several precursor ions during the MS/MS acquisition process9. Time-of-flight (ToF) instruments acquire spectra at very high frequency9,10 (up to tens of kHz)11; this makes them capable of rapidly scanning the total precursor ions within a complex sample (MS1), thus promising optimal sensitivity and MS/MS sequencing rates (up to 100 Hz)9 and making them ideal for biological sample analysis10. Nevertheless, the sensitivity available at these high scan rates is limited by the MS/MS rate9. The addition of trapped ion mobility spectrometry (TIMS) in combination with an orthogonal quadrupole time-of-flight (qToF) mass spectrometer was used to mitigate these limitations. In TIMS, all precursor ions are accumulated in tandem and eluted as a function of their mobility, rather than selecting single precursor masses with a quadrupole9. Parallel accumulation-serial fragmentation (PASEF) allows for hundreds of MS/MS events per second without any loss of sensitivity9.
The principal aim of this work was to show the recent developments of DDA using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation (CID). Histone PTMs were confidently assigned based on their retention times (RTs), mobilities, and fragmentation patterns.

Pleins feux sur l’auteur : Identification améliorée des isomères d’histones PTM grâce à LC-TIMS-ToF MS/MS et PASEF

Criblage de modification des histones par chromatographie liquide, spectrométrie de mobilité des ions piégés et spectrométrie de masse à temps de vol

A method is proposed using histone extraction protocol with 95%efficiency. Together with LC-TIMS-ToF MS/MS, in a short time, it is possible to obtain a screening for those PTMs present in the histone. And above all, the possibility of separating isomers.The separation of isomeric peptides though possible using tandem mass spectrometry alone is often difficult. By incorporating TIMS, we introduce an extra dimension of separation, simplifying many positional isomer identifications. In addition, passive technology increases the sensitivity of these experiments, further improving our ability to analyze epigenetic variations.With the increased ability to identify PTM positions, there comes the ability to further determine the correlation between various environmental or biological factors, and their effects on the epigenome. Methods that improve and facilitate analysis of histones can be applied to several research areas, including medical and environmental chemistry. The dynamics of protein confirmation in native or damaged states will be established from the implementation of TIMS MS/MS with HDX.Through LC-TIMS-ToF MS/MS and Nano LC-TIMS-ToF MS/MS, the differences in PTM existing in various biological sample will be charted.

Configuration et étalonnage de l’interface logicielle TIMS pour l’analyse protéomique

Ouvrez le logiciel de contrôle TIMS. Cliquez sur l’onglet Instrument et passez à Fonctionner. Vérifiez les paramètres TIMS, puis vérifiez les paramètres MS, y compris le début du balayage, la fin du balayage, la polarité ionique et le mode de balayage.Vérifiez les paramètres TIMS, y compris le mode, le démarrage de la mobilité, la fin de la mobilité, le temps de rampe, le temps d’accumulation, le cycle de service, le taux de rampe, le taux MS, la moyenne MS et l’étalonnage automatique. Ensuite, changez le mode de balayage en MS à des fins d’étalonnage. Après vous être assuré que l’instrument est en mode de fonctionnement, à partir de l’onglet Source, activez la seringue Hamilton 500 Microliter contenant le mélange d’accordage.Passez à l’onglet Calibrage. Cliquez sur la masse à charger, et sous le mode d’étalonnage, choisissez le mode quadratique. Contrôlez le zoom à plus ou moins 0,01 %Ensuite, cliquez sur Calibrer.Et lorsqu’un score de 100 % est atteint, cliquez sur Accepter. Ensuite, allez dans l’onglet Mobilité, et sous Mode d’étalonnage, sélectionnez Mode linéaire. Réglez la plage de détection sur plus ou moins 5 % et la largeur sur 0,1 dalton.Cliquez ensuite sur Calibrer et, lorsque vous recevez un score supérieur à 98,5 %, cliquez sur Accepter.

Research

JoVE Journal

Chemistry

Configuring and Calibrating the TIMS Software Interface for Proteomic Analysis

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

Recherche

Chimie

LC-TIMS-PASEF-ToF MS/MS Method for Proteolytic Histone Peptide Analysis

Méthode LC-TIMS-PASEF-ToF MS/MS pour l’analyse des peptides d’histones protéolytiques